Guillaume Cornelis

Mathis Funk

Cécile Vernochet

Francisca Leal

Oscar Ale- Jandro Tarazona

Guillaume Meurice

Odile Heidmann
email: heidmann@igr.fr

Anne Dupressoir

Aurélien Miralles

Patricia Ramirez-Pinilla

Thierry Heidmann
email: heidmann@igr.fr.

T H Wrote

Alan Conley

Stephen Glickman

A syncytin in a placental lizard 1.2 A syncytin in a placental lizard An endogenous retroviral envelope syncytin and its cognate receptor identified in the viviparous placental Mabuya lizard

Keywords: endogenous retrovirus, envelope protein, placenta, haemochorial, endotheliochorial, hyena, Carnivora endogenous retrovirus, envelope protein, placenta, hemochorial, endotheliochorial, hyena, Carnivora

Mabuya MPZL1, syncytin-Mab1, and Mab-Env2-Mab-Env4) have been deposited in the GenBank database (accession nos. MG254887-MG254891, respectively).

I would first like to thank the members of the jury for taking the time to read the present manuscript, which turned out a bit longer than I had planned. I would like to thank Uriel Hazan for accepting to be the president of this jury, book-ending his involvement in my studies. What had started at the ENS Cachan and continued during my Master's degree at the Institut Pasteur, finally reaches its culmination with the present work, on a topic that Uriel suggested I look into. I would like to sincerely thank Jean-Luc Battini and Olivier Schwartz for their critical reading and evaluation of the present manuscript and their positive feedback. Finally, I want to thank Pascale Chavatte-Palmer and François Mallet, for taking the time to read my work and for accepting to be a part of the jury.

I would, of course, also like to thank Thierry Heidmann for giving me the opportunity to work in his laboratory for the past four years, for being always available when I was in need of a guiding hand and present when I needed some prodding. Thank you for not only helping me to get these results, but also for the countless hours we spent to turn them into actual scientific articles.

I am grateful to Anne Dupressoir and Gérard Pierron for spending time reading through my manuscript and their words of encouragement and advice.

I also want to thank Christa Kuhn for her enthusiasm and encouragement, which played an important part in getting me to where I am today.

Je souhaite remercier l'intégralité de l'unité 9196, en particulier Marie et Anne pour avoir partagé leurs savoirs, leurs conseils ainsi que leurs bureaux, Cécile Vernochet pour son aide inestimable, Seila pour son soutien administratif et émotionnel, Grégoire pour son optimisme inébranlable et infectieux, Caroline, Cécile Lemaître et Anthony pour les bon moments passés ensemble, et ce-dernier tout particulièrement pour nos nombreuses discussions (profondes et/ou inutiles) et son énergie aussi inépuisable qu'insupportable. Un grand merci également à Guillaume, ex-membre de l'unité, pour m'avoir encadré pendant mes débuts et avoir toujours été disponible pour mes questions.

J'aimerais remercier le gang des pasteuriens, premiers parmi eux mes ex-colocataires, Matthieu et Thomas, merci de m'avoir supporté pendant ces trois ans et quelques de vie commune au 20 avenue d'Ivry, qui s'est transformé en haut-lieu de la science, accueillant maints et maints congrès réunissant la fine fleur de la virologie et de l'immunologie parisienne. I would iii like to thank Oksana, one of the kindest and most generous persons I have had the pleasure to know and someone you can always count on. Merci à Simon l'invisible, ton rétro nous a apporté de nombreuses heures de bonheur. Merci à Vincent le sale cylon pour les côtes de boeuf et à Essia pour ses couscous, toujours aussi bons que son humeur. Merci aux deux Juju pour la bonne ambiance, et je vous souhaite bien du courage, à Juju pour supporter Matthieu et à Juju pour finir ta thèse. Merci aussi à Anaïs, Laura, Cécile, Alix, Marion et tous les autres qui ont rendu la vie parisienne tellement plus agréable.

J'aimerais également remercier mes nouveaux ex-colocataires, Raphaël, Ferretti et Viking, pour m'avoir permis de garder l'appartement quelques mois de plus, puis pour m'avoir accueilli sur notre canapé qui, entretemps, était devenu le leur.

On a somewhat stranger note I would like to thank all of the friends I have never met, but who have been there to play, talk and just hang out in the virtual parts of this world. Thank you to all sigmas, especially 48, Mori Kaal and koreamax, but also willemd, :goatdrügs:, Cal, and even Thuneral, Slap Chop and Koahi.

A great thank you to the Aqueduct Acrobats who have been able to observe my slow and steady descent into placenta and retrovirus induced madness over the past few months, especially Topaz with his awful K/D, expensive haircuts and shared disgust of terrible book adaptations, mrcompson for our academia themed diversions and unmasking the terrible truth behind omicsconferences, and Malloot, everyone's favorite tactical espionage action ground team leader and platforming liability. I also want to thank Norns and Tarth, our resident Americans, as well as Charisma and Waka.

Ich möchte natürlich auch meiner Familie danken, meiner Mutter, meiner Schwester und meinen beiden Brüdern, von deren Hilfe ich immer ausgehen konnte und deren Gesellschaft und Vertrauen mein Leben bereichert haben. Ich danke meiner Oma und meinem Opa für ihre Ermutigungen und Liebe. J'aimerais remercier Patrice, qui a été pour moi pendant ces 25 dernières années le meilleur père que j'aurais pu souhaiter, et qui m'a poussé à être curieux et intéressé en espérant de peut-être, un jour, être aussi instruit et intéressant que lui. Ich möchte hier auch ganz besonders meiner Tante Jutta danken, die innerhalb von ein paar Tagen die ganze Einführung durchgelesen und korrigiert hat.

Last but not least, I want to thank all of the people that I have shamefully forgotten to mention here, please forgive me for this oversight.

I am still unsure of how I made it this far, but one thing I know: I would not have made it without you. The words above pale in comparison to what I owe you all, I have written and rewritten them many times and I know I'll never be able to express the feeling appropriately, so all I can do is apologize and say it once more, thank you all so very much. The field of virology is a relatively new one: less than two centuries ago the notion of a pathogen smaller than even bacteria was proposed by Pasteur, but he lacked the means to prove its existence. Years after these speculations, Chamberland invented his famous filter, able to remove even bacteria from a solution passed through it. It was this discovery that enabled Beijerinck in 1898 to show that filtered sap from plants infected with what is now known as tobacco mosaic virus was able to infect other plants. He concluded from this that the causative pathogen was smaller than a bacteria and called it a virus. From there virology advanced in leaps and bounds and in the following decades a great number of viruses were identified and linked to diverse diseases.

In 1911 Rous published his discovery of what would later become known as Rous sarcoma virus (RSV, [START_REF] Rous | A sarcoma of the fowl transmissible by an agent separable from the tumor cells[END_REF]. While this was not the first retrovirus to be discovered, and while Rous himself did not know about the true nature of the infectious agent he described, this discovery would lead to the creation of both the fields of tumor viruses and retrovirology. In the 1960s, cancers caused by RSV in egg-laying hens were becoming an ever more important problem, leading to many new investigations into the characteristics and behavior of the virus. In 1961 [START_REF] Crawford | The Properties of Rous Sarcoma Virus Purified by Density Gradient Centrifugation[END_REF] isolated the virus on a density gradient and determined that it had an RNA genome. Further research in the following years, making use of Mendelian genetics, both in live chicken and in infected cells, suggested that the viral genes could be stably inherited, which would imply that they had become part of the genome. This posed a major problem since at the time the "central dogma of biology" postulated that DNA could be transcribed into RNA, but that the opposite was impossible. How then could an RNA virus integrate its genes into a DNA genome? Nevertheless, in 1964 Temin introduced the provirus hypothesis, coining this term in the context of eukaryote viruses, and postulating that RSV could convert its RNA genome into a DNA genome which could then be inserted into the host genome [START_REF] Temin | Nature of the provirus of Rous sarcoma[END_REF]. This hypothesis proved unpopular until the discovery 6 years later of reverse transciptase (RT, [START_REF] Baltimore | Viral RNA-dependent DNA Polymerase: RNA-dependent DNA Polymerase in Virions of RNA Tumour Viruses[END_REF][START_REF] Temin | RNA-dependent DNA polymerase in virions of Rous sarcoma virus[END_REF], which finally delivered the key element: an enzyme able to convert RNA information into DNA information, and which led to the naming and creation of the Retroviridae family by the International Committee on Taxonomy of Viruses in 1975(ICTV, 1975).

Since viral replication of members of this family necessitates integration into the host genome, endogenous retroviruses (ERV) were discovered in parallel with the family itself, first in chicken and mice and later in humans and other vertebrates. The identification of retroviral sequences that were an integral part of host genomes (sequencing of the human genome later revealed that ERVs represent 8% of the genome Lander et al., 2001) led to the search for the function of these elements, and a number of them have been identified as protecting their host from infection by exogenous retroviruses. A role not linked to viral infection, and probably the most remarkable example of ERV function, is the exaptation of syncytins in mammals. The first such gene was described in 2000 as participating in the fusion of trophoblastic cells in the human placenta [START_REF] Blond | An Envelope Glycoprotein of the Human Endogenous Retrovirus HERV-W Is Expressed in the Human Placenta and Fuses Cells Expressing the Type D Mammalian Retrovirus Receptor[END_REF][START_REF] Mi | Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis[END_REF]. Since then numerous other syncytins have been identified in other mammalian placentas and their role in trophoblast fusion has been confirmed using KO mice.

To delve further into these topics, I will first discuss the characteristics of mammalian and non-mammalian placentas, a pre-requisite for discussion of syncytin role and acquisition. I will then present retroviruses in greater detail and finally I will talk specifically about captured envelope genes, especially syncytins.

Placenta

The transition from oviparity (egg-laying) to viviparity (live-bearing) occured on numerous occasions during vertebrate evolution (Blackburn, 2015), most famously in Eutherians. In viviparous species, by definition, the embryo is not exposed to the external environment and the exchanges necessary for embryonic development must take place between the embryo and the mother. These exchanges are rendered possible by the placenta. The placenta is a highly variable organ, both in structure and functionality. Its simplest definition was given by Mossman in his seminal 1937 work on mammalian placentas: "an apposition or fusion of the fetal membranes to the uterine mucosa for physiological exchange" [START_REF] Mossman | Comparative morphogenesis of the fetal membranes and accessory structures[END_REF]. This definition encompasses two related notions: the placenta joins maternal and fetal tissues, and it forms an exchange interface between mother and fetus.

Challenges of viviparity 1.Maternofetal exchanges

The first major challenge facing viviparous species is for the mother to act as the go-between of the embryo and the exterior. This means that the mother must not only provide the substances required for embryo development but also that all embryonic waste products must be taken up by the mother and eliminated. The importance of these exchanges during development is very variable depending on the species (Blackburn, 2015). In some cases, the embryo obtains most nutrients from its own yolk and is reliant on the mother mostly for gas and water exchanges, in others the embryo also needs to acquire almost all nutrients necessary for development. These two extremes are called lecithotrophy and matrotrophy, respectively, and form the two ends of a continuum of reproductive strategies that differ by the degree of implication of the mother and the functional capacities of the placenta (Blackburn, 2015). Of note, while almost all oviparous species are by necessity lecithotrophic, the mammalian monotremes (platypus and echidnas) have a unique matrotrophic oviparous mode of reproduction in which females ovulate small eggs with a 3 mm diameter yolk which takes up maternal nutrients before oviposition, to reach a final diameter of 17 mm [START_REF] Hughes | Monotreme development with particular reference to the extraembryonic membranes[END_REF]. While this is mentioned here for completeness, "oviparity" will refer to the much more common lecithotrophic oviparous mode of reproduction from here on. Combining the two distinctions of oviparity/viviparity and lecithotrophy/matrotrophy allows classification of vertebrate reproductive strategies into four major categories, see Table 1.

. Placenta Since most of our species of interest will be matrotrophic viviparous, the description of placental characteristics will focus on this subgroup, however even within this group placental structures show a great amount of variability (see 1.2.1.b.).

1.1

a. Nutrients

In matrotrophic placentation, nutrients that must be supplied to the embryo include carbohydrates, most amino-acids, fatty acids and metal ions. In mammals, glucose is one of the most important fetal nutrients, providing much of the energy required during fetal growth (Wooding & Burton, 2008, chap. 2). In eutherians, maternal glycemia is higher than fetal glycemia, establishing a concentration gradient that induces glucose transport from the mother to the fetus. In order to transfer the necessary amount of glucose from maternal to fetal blood, diffusion is facilitated using glucose transporters (glucose transporter type 1 in all mammalian placentas and additionally type 3 in some, like rat, horse and ewe, see Wooding & Burton, 2008, chap. 2). Depending on the species, 30-70% of the glucose provided by the mother is used in placental tissues to permit growth and functionality of the placenta itself and does not reach the embryo (Fig. 1, Wooding & Burton, 2008, chap. 2), though some of the products of placental glucose metabolism, like lactate, will enter the fetal circulation to serve as metabolites, lessening the impact of placental glucose consumption [START_REF] Fowden | Comparative aspects of fetal carbohydrate metabolism[END_REF]. Indeed, lactate is another major actor of fetal metabolism (up to 25% of total fetal energy requirements, see Wooding & Burton, 2008, chap. 2). Similarly to glucose, lactate transport makes use of transporters for facilitated diffusion, but in this case most of the lactate is produced by the placenta itself and maternal transplacental provision of this metabolite seems absent [START_REF] Fowden | Comparative aspects of fetal carbohydrate metabolism[END_REF], leading to higher concentrations of lactate in fetal than in maternal blood.

Contrarily to carbohydrates, amino acid concentrations are higher in fetal than in maternal blood in most species, indicating that there is a net uptake of amino acids by the fetus and that active transport mechanisms must be involved (Fig. 1, Vaughan & Fowden, 2016). Interestingly, placental amino acid concentrations are even more elevated than fetal concentrations and the placenta exchanges amino acids both with the maternal and fetal circulations. Most amino acids are simply imported by active transport into the placental tissues and then cross into the the fetal circulation according to their concentration gradient, this is notably the case for essential amino acids that, by definition, cannot be synthesized by the placenta or fetus and must be provided by the mother [START_REF] Vaughan | Placental metabolism: substrate requirements and the response to stress[END_REF]. For some amino acids, transit is much more complicated, this is the case for aspartate, serine and glutamate in sheep for example. The net placental uptake from maternal blood for these three amino acids is null, they are instead taken from the fetal circulation to satisfy placental metabolic needs, including ATP generation and protein synthesis [START_REF] Vaughan | Placental metabolism: substrate requirements and the response to stress[END_REF]. In addition, the sheep placenta is a net consumer of valine, leucine and isoleucine, but it provides more glutamine, methionine and glycine to the fetus than it takes up from the mother [START_REF] Vaughan | Placental metabolism: substrate requirements and the response to stress[END_REF]. The placental role in amino acid supply to the fetus reinforces the notion, that had been introduced with lactate in the previous paragraph, that the placenta is an active middleman between mother and fetus, and that its role is not simply passive (Fig. 1).

Being hydrophobic by nature, lipids such as free fatty acids, glycerol and cholesterol can be supplied by diffusion across the membranes separating mother and fetus, especially since maternal concentrations are higher than fetal concentrations for most. For others like the essential long chain fatty acids, active transporters participate in establishing a higher concentration in fetal than in maternal blood (Wooding & Burton, 2008, chap. 2).

. Placenta

1.1

The final nutrient category discussed here concerns ions. For some like calcium, the transfer from maternal blood to the placenta can simply occur by facilitated diffusion through calcium channels. Once in the placenta, it is bound to calcium binding proteins, so as to isolate it from sensitive cellular signaling mechanisms that rely on cytoplasmic Ca 2+ concentration, and then transferred to the fetus using calcium ATPase pumps (Wooding & Burton, 2008, chap. 2). Iron transport is a bit more complex since iron ions are not present in their free form in the organism. Instead they are bound to transport proteins (uteroferrin in horse and pig or transferrin) or are acquired by breakdown of red blood cells. Uteroferrin and transferrin are secreted by maternal uterine glands and enter the placenta by endocytosis. Iron ion acquisition from red blood cells concerns mainly carnivorans and ruminants in which specialized regions of the placenta (called hemophagous zones) break down these cells after phagocytosis (Wooding & Burton, 2008, chap. 2). Of note, in some placentas (human for example) a similar endocytic method is used to transfer a few specific proteins from the mother to the fetus, such as immunoglobulin G (Wooding & Burton, 2008, chap. 2).

The substances acquired directly from maternal blood are grouped under the term "hemotroph" while the substances secreted by the mother and taken up through endocytosis are termed "histotroph".

b. Oxygen

As with many other organs and organisms, both the placenta and the fetus need a constant supply of O 2 . Since biological membranes are highly permeable to gases like O 2 , there is no need for facilitated diffusion or active transport, instead diffusion occurs according to Fick's law along the concentration gradient, leading to a lower oxygenation of fetal blood than maternal blood. The two main factors influencing the efficiency of O 2 transfer from the mother to the fetus are blood flow velocity and relative disposition of the blood vessels (see Fig. 3C Wooding & Burton, 2008, chap. 2). Interestingly both of these factors are very variable across mammalian placentation, indicating that there is not necessarily a single efficient strategy for O 2 transport since all mammals are able to produce viable offspring.

The placental O 2 consumption is high, higher than that of the fetus when normalized by weight and on par with that of the fetal brain [START_REF] Fowden | Comparative aspects of fetal carbohydrate metabolism[END_REF]. In sheep, about 75% of the O 2 consumed by the placenta is used to generate ATP using a variety of substrates, including glucose and amino acids, as discussed in 1.1.1a.. This ATP is then used primarily for synthesis of placental proteins and to fuel active transport against electrochemical gradients. 15-20% of the O 2 uptake of sheep placentas is used to produce intermediaries necessary for synthesis of progesterone and other steroid hormones [START_REF] Vaughan | Placental metabolism: substrate requirements and the response to stress[END_REF]. The proportion of O 2 that actually reaches the fetal circulation is very dependent on the species and sometimes the gestational stage. In sheep it varies from 80% at the beginning to about 45% towards the end of gestation, while in horse and pig it remains at a constant 55% or 65% respectively [START_REF] Vaughan | Placental metabolism: substrate requirements and the response to stress[END_REF].

c. Waste

It is important to remember that both the placenta and the fetus are metabolically active and that it is not only necessary to provide them with the necessary substrates, but it is also necessary to eliminate some of the metabolic byproducts. This is for example the case with CO 2 and urea, both being able to return to the maternal circulation by simple diffusion, as one is a gas and the other a small uncharged molecule able to cross biological membranes. In sheep during late gestation, it is estimated that each day 7.8 g of carbon per kg of fetal weight enter the fetal circulation and 4.6 g of carbon per kg need to exit the fetal circulation in the form of CO 2 and urea [START_REF] Battaglia | Principal Substrates of Fetal Metabolism[END_REF], illustrating the need for the placenta to permit transport in both directions.

Placentation and the maternal immune system

Since during sexual reproduction the paternal and maternal genes mix and give rise to a new hybrid genome, the fetus is a foreign tissue in the eyes of the maternal immune system. In oviparity this does not pose a major problem since mother and fetus are separated by a physical barrier, the egg shell (reviewed in Wooding & Burton, 2008, chap. 9). In viviparity however, close contact between fetal and maternal tissues is absolutely necessary for transplacental exchanges (see 1.1.1). This intimate contact necessitates the establishment of a particular immunological context to avoid attack of fetal tissues by the maternal immune system, as is seen in other equivalent allograft situations (reviewed in [START_REF] Moffett | Uterine NK cells: Active regulators at the maternal-fetal interface[END_REF]. Here again, the placenta plays a crucial role as it is the point of contact between maternal and fetal tissues. The cells of the fetus itself generally do not interact with the maternal immune system, as they are shielded by placental tissues (reviewed in [START_REF] Moffett | Uterine NK cells: Active regulators at the maternal-fetal interface[END_REF]. Interactions between the placenta and maternal immune system are complex and highly dependent on the considered species, as such the following discussion will only focus on a few major aspects. An important aspect that will not be detailed here seems to be the presence of anti-inflammatory molecules, in the absence of which even syngeneic pregnancies (in which the fetus does not present foreign genetic material) result in pregnancy failure in rodent models (reviewed in [START_REF] Moffett | Immunology of placentation in eutherian mammals[END_REF].

a. Major histocompatibility complex expression

In mammals the adaptive immune system relies on the expression of major histocompatibility complex proteins (MHC). These proteins are recognized by and display antigens to B and T lymphocytes, leading to immune responses if a foreign element is detected. In humans the MHC molecules are called human leukocyte antigens (HLA) and cells usually express HLA-A, -B and -C at their surface, however in the placenta most cells do not express any HLA [START_REF] Apps | Human leucocyte antigen (HLA) expression of primary trophoblast cells and placental cell lines, determined using single antigen beads to characterize allotype specificities of anti-HLA antibodies[END_REF]. This absence of MHC expression effectively renders the placental cells invisible to the adaptive immune system, protecting them from maternal attack and especially cytotoxic T cells.

1.1

The presence of mostly MHC null cells is also observed in rodent, pig, horse and ruminant placentas for example (reviewed in Wooding & Burton, 2008, chap. 9), and as such seems to be a common mechanism of fetal tolerance.

While this is true for the bulk of placental cells, some subsets still express MHC molecules, this is mostly the case for invasive cells. In horses for example, some cells that migrate into the maternal tissue do express MHC proteins, only becoming MHC null once they have reached their destination. In cows the so-called binucleate cells (BNC) fuse with maternal cells, giving rise to heterologous trinucleate syncytia that express MHC molecules but do not seem to present allogenic antigens (reviewed in Wooding & Burton, 2008, chap. 9). The human placenta also presents invasive cells that express MHC proteins: the extravillous trophoblasts (EVT). These cells separate from the rest of the placenta and invade maternal tissues where they induce a major remodeling of blood vessels in order to increase placental blood supply by the mother (see Fig. 6, reviewed in Wooding & Burton, 2008, chap. 8). At their surface the EVT cells express the unique combination of HLA-C, -E and -G [START_REF] Apps | Human leucocyte antigen (HLA) expression of primary trophoblast cells and placental cell lines, determined using single antigen beads to characterize allotype specificities of anti-HLA antibodies[END_REF]. HLA-E and -G are non classical HLA molecules and display a very restricted expression profile, HLA-G being exclusively expressed in EVT [START_REF] Apps | Human leucocyte antigen (HLA) expression of primary trophoblast cells and placental cell lines, determined using single antigen beads to characterize allotype specificities of anti-HLA antibodies[END_REF]. The presence of these HLA molecules is crucial for the regulation of maternal vascular remodeling, as they provide both activatory and inhibitory signals to uterine natural killer (uNK) cells (see below, [START_REF] Parham | Variable NK cell receptors and their MHC class I ligands in immunity, reproduction and human evolution[END_REF].

b. Uterine natural killer cells

During pregnancy in humans up to 70% of uterine lymphocytes are uNK lymphocytes and an equivalent population of NK has been described in rodents (reviewed in Wooding & Burton, 2008, chap. 9), suggesting that they play an important role in all highly invasive (hemochorial, see 1.2.2.d.) placentas. Despite their name and unlike systemic NK cells, uNK cells are only weakly cytotoxic in vitro and do not kill MHC-null placental cells [START_REF] Ritson | Isolation and functional studies of granulated lymphocytes in first trimester human decidua[END_REF][START_REF] King | Early human decidual cells exhibit NK activity against the K562 cell line but not against first trimester trophoblast[END_REF]. Using NK specific receptors at their surface, uNK interact with the HLA expressed at the surface of EVTs, receiving both activating and inhibiting signals (reviewed in [START_REF] Parham | Variable NK cell receptors and their MHC class I ligands in immunity, reproduction and human evolution[END_REF]. When activated, uNK cells release cytokines and proteases that regulate EVT migration and assist in vascular remodeling (reviewed in [START_REF] Burton | The placenta: a multifaceted, transient organ[END_REF]. The dialog between EVT and uNK plays a critical role in pregnancy as it has been shown that a spectrum of pregnancy complications, referred to as the "great obstetrical syndromes" (preeclampsia, intrauterine growth restriction, pre-term labor and abruption placentae among others), are linked to defects in remodeling of the maternal circulation by EVT (reviewed in [START_REF] Brosens | The "Great Obstetrical Syndromes" are associated with disorders of deep placentation[END_REF]. While the non-classical HLA-E and -G show very little genetic variation, HLA-C presents several different epitopes that interact with distinct receptors on the uNK cells. Some combinations of placental HLA-C epitopes and maternal uNK receptors have been shown to increase the risk of pregnancy complications as they bias the EVT/uNK interaction, favoring uNK inhibition over activation [START_REF] Hiby | Maternal activating KIRs protect against human reproductive failure mediated by fetal HLA-C2[END_REF].

. . Challenges of viviparity c. Adaptive immunity

Adaptive immunity in mammals is based on the recognition of antigens by B or T lymphocytes, said antigens being presented either by generic cells using class I MHC or by specialized antigen presenting cells (APC) using class II MHC. As discussed above, most placental cells are MHCnull, preventing direct recognition as foreign tissues by the adaptive immune system [START_REF] Apps | Human leucocyte antigen (HLA) expression of primary trophoblast cells and placental cell lines, determined using single antigen beads to characterize allotype specificities of anti-HLA antibodies[END_REF]. Regarding MHC expressing placental cells, it has been shown that HLA-G, one of the MHC types expressed on the surface of human EVT cells, binds to inhibitory receptors on APC with very high affinity [START_REF] Shiroishi | Human inhibitory receptors Ig-like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G[END_REF] and that this binding interferes with APC maturation, resulting in lowered presentation of allogenic peptides through class II MHC as well as induction of immunosuppressive T cells in vitro [START_REF] Ristich | Tolerization of dendritic cells by HLA-G[END_REF]. Since this mechanism necessitates a direct contact between the HLA-G expressing EVT and the maternal APC, it is restricted to the placental context and does not impact APC functionality in the rest of the maternal immune system.

Apart from effector T cells, regulatory and helper T cells also play an important role in adaptive immunity. [START_REF] Tilburgs | Evidence for a Selective Migration of Fetus-Specific CD4+CD25bright Regulatory T Cells from the Peripheral Blood to the Decidua in Human Pregnancy[END_REF] show that during pregnancy fetus-specific protective regulatory T cells migrate to the placental interface and suppress effector T cells. The role and impact of T helper cells on pregnancy is less clear. It seems that during pregnancy Th1 responses (which favor cytotoxicity and are effective against viral infections) are decreased in favor of Th2 responses (antibody generation) (reviewed in [START_REF] Moffett | Maternal allo-recognition of the fetus[END_REF]. This shifted balance is the reason why pregnant women are more susceptible to some viral infections (like influenza, reviewed in [START_REF] Mertz | Pregnancy as a risk factor for severe outcomes from influenza virus infection: A systematic review and meta-analysis of observational studies[END_REF] and why during pregnancy the severity of Th1 linked autoimmune disorders decreases while that of Th2 linked disorders increases (reviewed in [START_REF] Ostensen | Sex Hormones and Pregnancy in Rheumatoid Arthritis and Systemic Lupus Erythematosus[END_REF][START_REF] Marder | Pregnancy and autoimmune connective tissue diseases[END_REF]. [START_REF] Moffett | Maternal allo-recognition of the fetus[END_REF] stress the fact that this observed shift does not seem to have any impact on reproductive success and consider it to be a side effect of the particular hormonal context established during pregnancy.

Of note, maternal B cells are rarely observed near the placental tissues, indicating that there is no substantial local generation of EVT-specific antibodies [START_REF] Moffett | Maternal allo-recognition of the fetus[END_REF].

Maternofetal communication

Establishing the link between the mother and fetus, the placenta is responsible for acting as an intermediary and communicating with the maternal side so as to establish correct conditions for fetal growth.

a. Placental hormones

Part of this communication uses classical hormonal pathways, the placenta being a major endocrine organ whose hormones have important effects on maternal physiology during pregnancy (reviewed in [START_REF] Burton | The placenta: a multifaceted, transient organ[END_REF]. As discussed in 1.1.1.a., all nutrients necessary for pla-. Placenta

1.1

cental and fetal growth have to be supplied by the mother, who also must fulfill the needs of her own metabolism. This is particularly true for glucose, the main substrate of placental and fetal metabolism (reviewed in Wooding & Burton, 2008, chap. 2) and an important metabolite in adults. In humans the main hormones of placental origin involved in these mechanisms are progesterone, human placental lactogen (hPL) and human placental growth hormone (hGH) (reviewed in [START_REF] Freemark | Regulation of maternal metabolism by pituitary and placental hormones: Roles in fetal development and metabolic programming[END_REF][START_REF] Burton | The placenta: a multifaceted, transient organ[END_REF]. Expression and impact of these hormones on the maternal metabolism can be separated into two phases: early to mid-gestation and late gestation.

During early to mid-gestation, secretion of progesterone and hPL by the placenta increase progressively (reviewed in [START_REF] Freemark | Regulation of maternal metabolism by pituitary and placental hormones: Roles in fetal development and metabolic programming[END_REF][START_REF] Burton | The placenta: a multifaceted, transient organ[END_REF]. Both of these hormones are appetite stimulants and have been associated with weight gain and fat deposition in mice and humans [START_REF] Grueso | Plasma and cerebrospinal fluid leptin levels are maintained despite enhanced food intake in progesterone-treated rats[END_REF][START_REF] Freemark | Regulation of maternal metabolism by pituitary and placental hormones: Roles in fetal development and metabolic programming[END_REF], leading to a disruption of energetic homeostasis and a buildup of reserves. In addition hPL participates in establishing leptin resistance during pregnancy, attenuating the negative feedback loop of fatty tissues on food intake normally mediated by this hormone (reviewed in [START_REF] Ladyman | Hormone interactions regulating energy balance during pregnancy[END_REF]. Finally, hPL also stimulates beta-cell proliferation in the pancreas of the mother, leading to increased insulinaemia and thus fat deposition and energy storage (reviewed in [START_REF] Burton | The placenta: a multifaceted, transient organ[END_REF]. Taken together, these effects lead to a 10-15% increase in food intake and a 60% increase in insulin secretion during this first phase, which starts even before a significant fetal energy demand arises and corresponds to a pre-preemptive increase in energy storage (reviewed in [START_REF] Freemark | Regulation of maternal metabolism by pituitary and placental hormones: Roles in fetal development and metabolic programming[END_REF].

In late pregnancy there is a significant secretion of hGH by the syncytiotrophoblast of the placenta. This growth hormone is capable of inducing insulin resistance [START_REF] Barbour | Human placental growth hormone causes severe insulin resistance in transgenic mice[END_REF] and the resulting 45-70 % decrease in maternal insulin sensitivity leads to breakdown of fat reserves and mobilization of glucose into the maternal bloodstream, from where it can then be absorbed by the placenta (see 1.1.1.a.).

In addition to the roles in nutrient provision discussed above, fetal hormones also play an important role in the establishment and maintenance of pregnancy (and interrupting the menstrual cycle) and can be involved in the process leading to parturition at the end of gestation (reviewed in Wooding & Burton, 2008).

b. Extracellular vesicles

In addition to the soluble secreted factors described above, the placenta releases a great amount of extracellular vesicles [START_REF] Salomon | A Gestational Profile of Placental Exosomes in Maternal Plasma and Their Effects on Endothelial Cell Migration[END_REF]. These particles are lipid-bilayer particles that can contain proteins, lipids, RNAs and DNA, be classified as exosomes (40-120 nm) or microvesicles (0.2-1 µm), and participate in inter-cellular communication processes (reviewed in [START_REF] Adam | Review: Fetal-maternal communication via extracellular vesicles -Implications for complications of pregnancies[END_REF]. [START_REF] Salomon | A Gestational Profile of Placental Exosomes in Maternal Plasma and Their Effects on Endothelial Cell Migration[END_REF] show that the concentration of exosomes in plasma of pregnant woman is increased 50-100 fold when compared to non-pregnant women, depending on the stage of pregnancy. The role of these particles in maternofetal communication is still poorly understood, though it has been shown that placenta generated extracellular vesicles can interact 1.2

. . Mammalian eutherian placentas with the maternal immune system and have an immunosuppressive effect [START_REF] Sabapatha | Specific isolation of placenta-derived exosomes from the circulation of pregnant women and their immunoregulatory consequences[END_REF].

Mammalian eutherian placentas 1.2.1 Origin and evolution of the mammalian placenta a. Monophyletic origin

Since the placenta is composed of soft tissue, there is no substantial fossil record of placentation in extinct species, complicating studies of placental origin and evolution. Nevertheless, in mammals the placenta is very probably of monophyletic origin and the time of emergence can be estimed using mammalian phylogeny [START_REF] Lillegraven | The origin of eutherian mammals[END_REF]. The class Mammalia can be split into three subgroups with regard to placentation: i) the monotremes that are oviparous and do not present a placenta, ii) the marsurpials which present a short-lived placenta and iii) the eutherians or placentalia (see Fig. 2). Eutherians present long gestation periods and complex placentas and regroup most of the extant mammalian species. Since both eutherians and marsupials are placental, the parsimony principle suggests that the emergence of the mammalian placenta occurred in the last common ancestor of the two clades, after their separation from monotremes. This common origin is supported by the comparison of eutherian and marsupial placentation traits, though it cannot be entirely excluded that two indendent emergences of placentation occurred [START_REF] Lillegraven | The origin of eutherian mammals[END_REF]Blackburn, 2015). In any case the similarity of involved processes and tissues in eutherian placentation clearly suggest that at least in this branch the origin of the organ is monophyletic [START_REF] Lillegraven | The origin of eutherian mammals[END_REF].

The fossil calibrated timed tree established by dos [START_REF] Dos Reis | Phylogenomic datasets provide both precision and accuracy in estimating the timescale of placental mammal phylogeny[END_REF] indicates that a placental origin common to marsupials and eutherians would have occurred about 175 Mya. In accordance with the "long fuse" model of eutherian evolution (relatively recent diversification), the radiation of this clade occurred about 90 Mya, long after emergence of a common mammalian placenta [START_REF] Dos Reis | Phylogenomic datasets provide both precision and accuracy in estimating the timescale of placental mammal phylogeny[END_REF], indicating that the different adaptations and specializations observed in modern day eutherian placentas have evolved from this common primitive placenta during the last 90 My.

b. Placental diversity

While eutherian placentas have a single shared origin, they have since evolved into the organ exhibiting the greatest amount of inter-species diversity (reviewed in [START_REF] Enders | What Can Comparative Studies of Placental Structure Tell Us?-A Review[END_REF]Wooding & Burton, 2008, chap. 1). This becomes immediately apparent when one tries to establish a system of classifying the placentas of different species, as no single system allows for easy and clear classification (Fig. 3, reviewed in [START_REF] Chavatte-Palmer | Placentation in different mammalian species[END_REF]. Classification by shape. The most straightforward way of classifying different eutherian placentas is to simply go by external appearance, but even here there are two possible approaches.

The first is based on description of the distribution of areas of high maternofetal exchange at the surface of the placenta (Fig. 3A). These areas present folds or extruding placental villi that increase the exchange area and the distribution of such regions can follow four major patterns. In diffuse placentas (e.g. pig, horse, [START_REF] Friess | The placenta of the pig[END_REF][START_REF] Samuel | Studies on the Equine Placenta[END_REF] there are no particular areas of the placenta concentrating villi or folds. Cotyledonary or placentomal placentas (e.g. ruminants, [START_REF] Björkman | Light and electron microscopic studies on cellular alterations in the normal bovine placentome[END_REF] present several spots of increased maternofetal exchange surface.

The number of these so-called cotyledons ranges from 5 in deer to 150 in giraffe (reviewed in Wooding & Burton, 2008, chap. 1), illustrating the high amount of potential variability even within a given category. In zonary placentas (e.g. carnivorans, [START_REF] Leiser | Development and characteristics of placentation in a carnivore, the domestic cat[END_REF] the folds are grouped into a single band that circumvents the placenta. Finally, in discoid placentas the exchange zone takes the form of one (e.g. human, rodents) or two (e.g. rhesus monkey) discs at the surface of the placenta (reviewed in [START_REF] Chavatte-Palmer | Placentation in different mammalian species[END_REF][START_REF] Hafez | Chapter One -Comparative Placental Anatomy[END_REF].

The second shape-based classification relies on the way the above described folds and villi interdigitate fetal and maternal tissues (Fig. 3B). The simplest structure is found in folded placentas (e.g. pig, [START_REF] Friess | The placenta of the pig[END_REF] where placental folds simply interlock with corresponding maternal endometrial folds. Carnivores present the slightly more complex lamellar form of placentation in which the folds are more extensive and branched [START_REF] Leiser | Development and characteristics of placentation in a carnivore, the domestic cat[END_REF]. Humans as well as ruminants and horses present villous placentas where placental tissues form tree-like structures with numerous primary villi that branch out into secondary and tertiary villi that fill corresponding maternal recesses [START_REF] Enders | A comparative study of the fine structure of the trophoblast in several hemochorial placentas[END_REF][START_REF] Björkman | Light and electron microscopic studies on cellular alterations in the normal bovine placentome[END_REF][START_REF] Samuel | Studies on the Equine Placenta[END_REF]. Finally the most complex form is found in labyrinthine placentas (e.g. rodents, [START_REF] Enders | A comparative study of the fine structure of the trophoblast in several hemochorial placentas[END_REF] in which the placental tissues are crossed by an elaborate assembly of maternal channels, forming a large interconnected mesh-like structure. We can already see that both types of classification establish 1.2

. . Mammalian eutherian placentas different groups, horse being grouped with pigs in the first but with humans and ruminants in the second system. Another important and recurrent factor to these classifications is that they take into account the definitive placental structures and not the placental development.

Classification by invasiveness. [START_REF] Schlafke | Cellular Trophoblast Basis of Interaction Uterus Implantation[END_REF] distinguish between four major degrees of invasiveness of eutherian placentas during early development. The lowest degree of invasion, interdigitation, is observed in pigs where maternal and fetal epithelia are simply apposed and both maintain their integrity [START_REF] Dantzer | Electron microscopy of the initial stages of placentation in the pig[END_REF]. Displacement describes an intermediate phenotype in which individual cells or groups of cells of the uterine epithelium slough off after apposition with the fetal epithelium, leading to penetration of placental cells beyond the maternal epithelial barrier. This is observed in rats and mice for example (reviewed in Wooding & Burton, 2008, chap. 8). Ruminant placentas are an example of the third type of early invasion of maternal tissues: fusion. Fetal BNC fuse with cells of the maternal epithelium, resulting in heterologuous syncytia [START_REF] Wooding | Autoradiographic evidence for migration and fusion of cells in the sheep placenta: Resolution of a problem in placental classification[END_REF], see also 1.2.2.b.). This process allows penetration of the maternal epithelium by cells of (partial) fetal origin without loss of integrity of the former. The final type of placental invasion during implantation is intrusion and can be found in horse and human, albeit to different degrees. In the horse placenta, individual binucleated cells separate from the placenta and pass through the body of uterine epithelial cells [START_REF] Allen | The origin of equine endometrial cups. II. Invasion of the endometrium by trophoblast[END_REF]. In human placentas the entire conceptus crosses the uterine epithelium (reviewed in Wooding & Burton, 2008, chap. 8).

In addition to this classification based on invasiveness during the very early implantation step, definitive placental structures can also be classified in terms of invasiveness (Fig. 4A). As stated above, the pig placenta results mostly from the apposition of maternal and placental epithelia, without loss of integrity of the former [START_REF] Friess | The placenta of the pig[END_REF][START_REF] Dantzer | Electron microscopy of the initial stages of placentation in the pig[END_REF]. This represents the least invasive type of placentation. From there on there exists a spectrum of invasiveness, the horse placenta for example displays a bit more invasion: while epithelia maintain their integrity, a subset of binucleated placental cells cross the maternal epithelium and migrate into the endometrium [START_REF] Allen | The origin of equine endometrial cups. II. Invasion of the endometrium by trophoblast[END_REF], see also 1.2.2.a.). This spectrum culminates in the so-called hemochorial placentas where maternal tissues have been largely degraded and the placenta bathes directly in maternal blood (see 1.2.2.d.).

Classification by other factors. Another possible classification of eutherian placentas is based on the amount of maternal tissue lost during birth, however this is closely linked to the previous one: in low-invasion placentation the maternal and placetal tissues are simply apposed so that during parturition they separate cleanly, while in high-invasion placentation the deeper relationship between maternal and placental tissues necessitates tearing of the former (reviewed in [START_REF] Hafez | Chapter One -Comparative Placental Anatomy[END_REF].

It is also possible to distinguish placentas with regards to the relative disposition of maternal and placental blood vessels (Fig. 3C). The disposition of blood vessels has a major impact on

1.2

the efficiency of exchanges between them. In a concurrent system (parallel blood flows) for example, the theoretical maximal efficiency is 50% while a countercurrent system allows for a theoretical maximum of 100%. Perhaps unsurprisingly, no eutherian placenta employing a fully concurrent system has been described to this day. An example of a placenta presenting an almost perfect countercurrent system can be found in guinea pigs, while other species present intermediate arrangements like cross-current (e.g. cats) and villous (e.g. humans)(Fig. 3C, reviewed in Wooding & Burton, 2008;[START_REF] Hafez | Chapter One -Comparative Placental Anatomy[END_REF].

The final major classification is based on the histological nature of the maternofetal interface and the amount of layers that separate the maternal and fetal circulations (see Fig 4A). Wooding & Burton (2008, chap. 1) suggest that this way of categorizing placental structures is more relevant to the physiological functions of eutherian placentas and as such it will be presented in more detail than the other classifications.

Placental types by maternofetal interface

This classification was first proposed by [START_REF] Grosser | Vergleichende Anatomie und Entwicklungsgeschichte der Eihäute under Placenta: mit besonderer Berücksichtigung des Menschen[END_REF] and is based on his observation that while all fetal tissue layers (endothelium, connective, epithelium called trophoblast) were always present in every eutherian placenta, the amount of maternal tissue layers (epithelium, connective, endothelium) was variable. Grosser proposed four categories of placenta: Placenta epitheliochorialis, where all maternal tissues are still present and the trophoblast is in contact with the maternal epithelium, Placenta syndesmochorialis, in which the maternal epithelium is absent and the trophoblast is in direct contact with connective tissue, Placenta endotheliochorialis, in which both the maternal epithelium and connective tissue have been destroyed and the trophoblast is in direct contact with the maternal endothelium, and Placenta haemochorialis, in which all maternal tissues have disappeared and the trophoblast is in direct contact with maternal blood.

Most of this classification proved remarkably accurate for being based only on light microscopy, but the category of syndesmochorial placentation was based on a mistake. [START_REF] Grosser | Vergleichende Anatomie und Entwicklungsgeschichte der Eihäute under Placenta: mit besonderer Berücksichtigung des Menschen[END_REF] had concluded that in ruminant placentas the maternal epithelium was destroyed and replaced by the fetal trophoblast, but closer investigation with more elaborate methods revealed that both epithelia remain present until term. This led to ruminant placentas being described as epitheliochorial for a time, however as reviewed in [START_REF] Wooding | The synepitheliochorial placenta of ruminants: Binucleate cell fusions and hormone production[END_REF], ruminant placentas present fusion events between fetal and maternal cells, separating them from all other epitheliochorial placentas and justifying their belonging to a separate category: synepitheliochorial placentation. [START_REF] Grosser | Vergleichende Anatomie und Entwicklungsgeschichte der Eihäute under Placenta: mit besonderer Berücksichtigung des Menschen[END_REF] had also suggested that this classification might represent an evolutionary progression, that epitheliochorial placentas are the most basic and least efficient, having six cell layers separating maternal and fetal circulations, while hemochorial placentas are the most evolved and efficient, having only three layers in-between both circulations. This supposed

1.2

significant difference in efficiency based on the number of layers separating maternal and fetal blood has since been disproved as Grosser did not take into account the finer details of placental structure that can have major effects on the permeability of placental layers and the efficiency of exchanges (reviewed in Wooding & Burton, 2008;[START_REF] Chavatte-Palmer | Placentation in different mammalian species[END_REF][START_REF] Hafez | Chapter One -Comparative Placental Anatomy[END_REF]. In the following parts the different types of placentation shall be described using representative species and with particular focus on the structure of the maternal interface, cell-cell fusion events and the invasiveness of fetal tissues.

a. Epitheliochorial placentation

In epitheliochorial placentation, all three layers of maternal tissue remain present and conserve their integrity during gestation (Fig. 4A). The maternal epithelium and trophoblast are apposed and their microvilli interdigitate. The trophoblast does not present cell-cell fusion events, remaining in the form of individualized trophoblast cells called cytotrophoblasts (CT). Pig and horse placentas are the most studied representatives of this type of placentation.

Pig. At first glance, the pig placenta seems like the simplest eutherian placenta, it is lowly invasive, diffuse, folded and epitheliochorial, in other words an apposition of membranes that then fold to increase the available exchange surface [START_REF] Friess | The placenta of the pig[END_REF][START_REF] Dantzer | Electron microscopy of the initial stages of placentation in the pig[END_REF]. This results in the presence of the full complement of six layers separating maternal and fetal bloodstreams, hinting at low exchange efficiency. In reality this is not the case, while all layers remain present until term, their thickness is decreased considerably at the tip and sides of the folds, and the blood vessels indent the epithelial layers reducing the interhemal distance to as little as 2 µm, instead of 20-100 µm at the base of the fold (reviewed in Wooding & Burton, 2008, chap. 5).

Comparing the weight of fetal mass supplied per gram of placental mass suggests that, according to this particular metric, even with six layers to cross the pig placenta is more efficient (9 g of fetus/g of placenta) than human placentas that present only three interhemal layers (6 g of fetus/g of placenta) (reviewed in [START_REF] Dantzer | Comparative Morphological Aspects of Placental Vascularization[END_REF].

Horse. The horse placenta is structurally very similar to that of pigs, also resulting from a simple apposition of fetal trophoblast and maternal epithelium. Its general structure however, is villous rather than folded [START_REF] Samuel | Studies on the Equine Placenta[END_REF] and its efficiency might be even higher (14 g of fetus supplied by 1 g of placenta, reviewed in [START_REF] Dantzer | Comparative Morphological Aspects of Placental Vascularization[END_REF]. A particularity of horse placentas is the presence of a subpopulation of binucleate invasive cells. These cells start as a so-called "chorionic girdle", encircling the fetus [START_REF] Allen | The origin of equine endometrial cups. II. Invasion of the endometrium by trophoblast[END_REF]. The girdle cells are binuclear, express MHC I molecules [START_REF] Donaldson | Invasive equine trophoblast expresses conventional class I Major Histocompatibility Complex antigens[END_REF] and detach from the conceptus to invade maternal tissues. They cross the uterine epithelium by traversing the body of epithelial cells, rather than by crossing at the tight inter-cellular junctions [START_REF] Allen | The origin of equine endometrial cups. II. Invasion of the endometrium by trophoblast[END_REF]. Once on the other side they aggregate to constitute what is called endometrical cups, stop expressing MHC molecules and start secreting hormones that assist placental growth, before finally being . Placenta

1.2

destroyed by cells of the maternal immune system [START_REF] Allen | The origin of the equine endometrial cups[END_REF][START_REF] Allen | The origin of equine endometrial cups. II. Invasion of the endometrium by trophoblast[END_REF][START_REF] Donaldson | Invasive equine trophoblast expresses conventional class I Major Histocompatibility Complex antigens[END_REF]. The presence of this invasive subpopulation is especially striking since apart from it epitheliochorial placentas are non-invasive.

b. Synepitheliochorial placentation

Ruminant placentas are the only synepitheliochorial placentas described to this day. At first glance they are very similar to epitheliochorial ones, presenting all six layers at term. They were in fact classified as such until [START_REF] Wooding | The synepitheliochorial placenta of ruminants: Binucleate cell fusions and hormone production[END_REF] established the synepitheliochorial category based on the presence of cell-cell fusion events at the maternofetal interface. In contrast to the epitheliochorial placentas described above, here binucleated trophoblast cells fuse with cells of the maternal epithelium, forming heterologous syncytia that directly connect the fetal and maternal sides (see Fig 4A, reviewed in Wooding (1992)). The degree of syncytialization of the interface depends on the species, it is for example low in cattle, but high in sheep, the two most studied synepitheliochorial placentas and thus the two common examples of each category.

Binucleate cells in synepitheliochorial placentation

The appearance of BNC is a characteristic of synepitheliochorial placentation (reviewed in [START_REF] Wooding | The synepitheliochorial placenta of ruminants: Binucleate cell fusions and hormone production[END_REF]Wooding & Burton, 2008, chap. 6). These cells are generated by division of trophoblastic nuclei without cytokinesis and represent about 15-20% of trophoblast cells [START_REF] Wooding | Frequency and localization of binucleate cells in the placentomes of ruminants[END_REF]. Mature BNC are very large and present a well developed translational apparatus as well as numerous microvesicles containing dense granules (reviewed in [START_REF] Wooding | The synepitheliochorial placenta of ruminants: Binucleate cell fusions and hormone production[END_REF]Wooding & Burton, 2008, chap. 6). As in horse placentas (see 1.2.2.a. above), the BNC are migratory and invade the uterine epithelium, however the employed mechanism is very different. BNC mature between two trophoblastic cells before extending a pseudopod towards the maternofetal interface. This pseudopod squeezes between the two neighboring trophoblast cells and separates their tight junctions, replacing them with its own [START_REF] Morgan | Cell migration in the ruminant placenta: A freezefracture study[END_REF]. This maintains the integrity of the placental epithelium while allowing the BNC to contact the maternal epithelium. Once the pseudopod has made contact with the maternal epithelium, a number of specialized vesicles flock to its tip to extend the membrane surface area (Wooding & Burton, 2008). This process induces a break in the microvillous surface of the maternofetal interface since it generates a flat membranous area. The flat area of the BNC membrane bulges into the flattened membrane of the maternal epithelium and both membranes fuse, allowing the BNC cell to inject its contents into what was the maternal epithelial cell [START_REF] Wooding | Autoradiographic evidence for migration and fusion of cells in the sheep placenta: Resolution of a problem in placental classification[END_REF]. Following the fusion event the membrane of the BNC that remains between trophoblast cells gets resorbed by the trophoblasts, re-establishing a tight junction contact between the trophoblast cells [START_REF] Morgan | Cell migration in the ruminant placenta: A freezefracture study[END_REF]. For a summary of these processes, see Fig. 5A. The fusion of the BNC with a maternal epithelial cell creates a heterologous syncytium in which nuclei and cell contents are both of placental and maternal origin. This also gives the contents of the BNC cell, and especially its dense vesicles, access to the basal lamina of the maternal epithelium and (A) Fetal BNC mature between two trophoblast cells before extending cytoplasmic projections towards the maternofetal interface, replacing adherent junctions with their own in the process. Following fusion, the BNC cytoplasm mixes with the maternal epithelial cell cytoplasm and the BNC granules are transferred to the basal membrane where they secrete their contents. The trophoblast absorbs the rest of the BNC membrane and re-establishes adherent junctions. (B) In sheep type epitheliochorial placentation, BNC can fused into an already existing heterologous syncytium to form syncytial plaques. The mechanism is the same as for the formation of trinuclear cells. Adapted from [START_REF] Cornelis | Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants[END_REF] exocytosis of these vesicles is observed specifically at the basal membrane of the fused cells, towards the maternal circulation [START_REF] Wango | The role of trophoblastic binucleate cells in implantation in the goat: a morphological study[END_REF]. The granules contain placental hormones (placental lactogen and pregnancy associated glycoproteins for example, reviewed in Wooding & Burton, 2008, chap. 6) and this very particular migration and fusion process allows their release close to the maternal circulation, bypassing the maternal epithelial barrier.

Syncytia in synepitheliochorial placentation

While in all ruminant placentas fusion events implying BNC occur, the fate of the fused cells depends on the species. In some like sheep or goats, large sycnytial plaques develop at the maternofetal interface of the definitive placenta through repeated fusion of new placental cells into existing syncytia (Fig. 5B, [START_REF] Wooding | Autoradiographic evidence for migration and fusion of cells in the sheep placenta: Resolution of a problem in placental classification[END_REF]. By counting the amount of nuclei present in the syncytial plaques, [START_REF] Wooding | Role of binucleate cells in fetomaternal cell fusion at implantation in the sheep[END_REF] show that the total number is always odd, indicating that BNC are the only cells that fuse into the syncytium. Every syncytial plaque starts as a trinucleate cell, as described above, and can reach up to 25 nuclei, corresponding to about a dozen more BNC fusing into the syncytium [START_REF] Wooding | Role of binucleate cells in fetomaternal cell fusion at implantation in the sheep[END_REF]. In addition to providing a more direct access to the maternal circulation for secretions of the BNC, the syncytial plaque layer also has an amplified microvillous border to increase 1.2 exchange surface and is thinner than the maternal epithelium, reducing interhemal distance to less than 1-2 µm (reviewed in Wooding & Burton, 2008, chap. 6).

In cow and deer placentas the syncytial plaques are only present during implantation after which the uterine epithelium regrows rapidly [START_REF] King | Development of the bovine placentome during the second month of gestation[END_REF]Wathes & Wooding, 1980), at any subsequent step and in the definitive placenta only transient trinucleate cells resulting from a single fusion event can be observed (Wooding & Wathes, 1980). The trinucleate cells then undergo apoptosis after having exocyted their granules, using a pathway that is also implicated in human placentation [START_REF] Ushizawa | Cloning of the bovine antiapoptotic regulator, BCL2-related protein A1, and its expression in trophoblastic binucleate cells of bovine placenta[END_REF], which explains the absence of a more extensive syncytium.

c. Endotheliochorial placentation

As the name indicates, in endotheliochorial placentation the fetal tissue is in direct contact with maternal endothelium, the maternal epithelium and connective tissue having disappeared in the definitive placenta. The most well studied family presenting endotheliochorial placentation are the carnivorans (except for the hyena which is the only species of this family presenting a hemochorial placenta, [START_REF] Morton | Placentation in the spotted hyena (Crocuta crocuta Erxleben)[END_REF][START_REF] Wynn | Placentation in the Spotted Hyena (Crocuta crocuta), with Particular References to the Circulation[END_REF][START_REF] Oduor Okelo | The chorioallantoic placenta of the spotted hyena (Crocuta crocuta Erxleben): An electron microscopic study[END_REF], and more precisely dog and cat.

In both of these species, the placenta is of the zonary type (see 1.2.1.b.). In the zone specialized for maternofetal exchanges, the outer trophoblast cells fuse together to form a large syncytium called the syncytiotrophoblast (ST) while the underlying layer remains individualized CT [START_REF] Barrau | Development of the implantation chamber in the pregnant bitch[END_REF][START_REF] Leiser | Blastocystenimplantation bei der Hauskatze Licht-und elektronen-206 mikroskopische Untersuchung[END_REF]. As the ST comes into contact with the maternal epithelium it sends out projections that wedge between uterine cells and contain phagosomes [START_REF] Barrau | Development of the implantation chamber in the pregnant bitch[END_REF][START_REF] Leiser | Blastocystenimplantation bei der Hauskatze Licht-und elektronen-206 mikroskopische Untersuchung[END_REF]. Both the uterine epithelium and the cells of the connective tissue are destroyed by the syncytial front which penetrates these layers and surrounds and isolates cells, detaching them from their basal membranes [START_REF] Barrau | Development of the implantation chamber in the pregnant bitch[END_REF][START_REF] Leiser | Blastocystenimplantation bei der Hauskatze Licht-und elektronen-206 mikroskopische Untersuchung[END_REF]. At the tip of the fetal villi the maternal tissues show clear signs of breakdown, the debris being taken up by the ST [START_REF] Barrau | Development of the implantation chamber in the pregnant bitch[END_REF][START_REF] Leiser | Blastocystenimplantation bei der Hauskatze Licht-und elektronen-206 mikroskopische Untersuchung[END_REF]. This way the ST rapidly establishes an intimate contact with the maternal blood vessel walls, which remain intact during pregnancy [START_REF] Barrau | Development of the implantation chamber in the pregnant bitch[END_REF][START_REF] Leiser | Blastocystenimplantation bei der Hauskatze Licht-und elektronen-206 mikroskopische Untersuchung[END_REF]. The two layers are only separated by the so-called interstitial lamina, which is very close in both structure and composition of the basal lamina of maternal vessels. The lamina is of variable thickness and the fetal capillaries often indent the syncytiotrophoblast, reducing the interhemal distance locally (reviewed in Wooding & Burton, 2008, chap. 7).

Outside of the specialized region of the zonary carnivoran placenta, the maternofetal interface is of the epitheliochorial type.

d. Hemochorial placentation

The hemochorial (or haemochorial) category contains the most invasive placentas. Placental invasion of maternal tissues is so strong that the definitive maternofetal interface does not contain any maternal tissue, the trophoblast is bathing directly in maternal blood. However, there is considerable variation in the structure of this trophoblast among species of this group, it can have between one and three cell layers which may be syncytial or cellular (reviewed in Wooding & Burton, 2008, chap. 8).

Hemomonochorial.

As their name indicates, hemomonochorial placentas contain a single layer of trophoblast at term, this is the case in the definitive human placenta for example. During the early stages, the human placenta is hemodichorial, consisting of an outer ST layer overlying a continuous CT layer [START_REF] Jones | A Re-appraisal of the Morphophenotype and Basal Lamina Coverage of Cytotrophoblasts in Human Term Placenta[END_REF]. As the villi expand the syncytium remains one continous layer (covering an area of about 12 m 2 at term, [START_REF] Gaunt | Microinjection of human placenta: II: Biological application[END_REF][START_REF] Burton | Sonographic, stereological and Doppler flow velocimetric assessments of placental maturity[END_REF], while the CT layer becomes discontinuous, thus giving rise to a hemomonochorial placenta [START_REF] Jones | A Re-appraisal of the Morphophenotype and Basal Lamina Coverage of Cytotrophoblasts in Human Term Placenta[END_REF]. This change seems to be due to the vast increase in villus surface area which outpaces the growth rate of CT and is accompanied by a switch from a cuboidal to an 'octopoid' phenotype in which the CTs are flattened and send out projections to connect to neighboring cells [START_REF] Jones | A Re-appraisal of the Morphophenotype and Basal Lamina Coverage of Cytotrophoblasts in Human Term Placenta[END_REF]. Additionally the syncytial layer needs to be maintained and expanded, which implies addition of more CTs to the syncytium, reducing the number of CT remaining individual cells. Between 13 and 39 weeks of gestation the number of nuclei in the ST increases exponentially from 6.8 × 10 9 to 5.8 × 10 10 [START_REF] Simpson | From 13 weeks to term, the trophoblast of human placenta grows by the continuous recruitment of new proliferative units: A study of nuclear number using the disector[END_REF]. Since no mitotic activity has ever been desribed in the human ST, this increase must be due to the inflow of CTs into the fused layer (reviewed in Benirschke et al., 2012, chap. 6). Accordingly, [START_REF] Simpson | From 13 weeks to term, the trophoblast of human placenta grows by the continuous recruitment of new proliferative units: A study of nuclear number using the disector[END_REF] describe that the amount of CT nuclei increases throughout pregnancy (0.6 × 10 9 at week 13 to 5.8 × 10 9 at week [START_REF] Gyapay | A radiation hybrid map of the human genome[END_REF], indicating that at least some cells of this population must have stem-cell capacities and are thus able to provide a constant supply of cells for the syncytium. Interestingly the numbers supplied by [START_REF] Simpson | From 13 weeks to term, the trophoblast of human placenta grows by the continuous recruitment of new proliferative units: A study of nuclear number using the disector[END_REF] suggest that the proportion of nuclei in the CT and ST remains constant during pregnancy (90% in the ST, 10% in CT), further indicating that the incorporation of cells into the syncytial layer is a continous phenomenon that operates at a constant rate during pregnancy. Similar results were obtained by [START_REF] Mayhew | Proliferative versus hypertrophic growth in tissue subcompartments of human placental villi during gestation[END_REF], confirming that the growth of the human ST is enabled by a constant supply of CTs.

The ST is an invasive tissue, during implantation it sends out projections between the uterine epithelial cells and allows the whole conceptus to traverse the epithelium and reach the endometrium. Once the conceptus is embedded in the endometrium, the ST breaches maternal capillaries to access the maternal circulation and create blood filled inter-villous spaces [START_REF] Enders | Trophoblast differentiation during the transition from trophoblastic plate to lacunar stage of implantation in the rhesus monkey and human[END_REF]. The human placenta also presents another population of invasive cells: the extravillous trophoblasts, so-called because they separate from the villi to invade maternal tissues, more specifically the maternal arteries. There are two sub-populations, one of the two invades the endometrium around the arteries while the second invades the blood vessel lumen [START_REF] Pijnenborg | Trophoblastic invasion of human decidua from 8 to 18 weeks of pregnancy[END_REF]. Together these populations are responsible for the breakdown of the muscle sheath and widening the mouth of the arteries [START_REF] Brosens | The physiological response of the vessels of the placental bed to normal pregnancy[END_REF][START_REF] Pijnenborg | Uteroplacental arterial changes related to interstitial trophoblast migration in early human pregnancy[END_REF], both increasing the volume and slowing the velocity of bloodflow, and providing enough blood supply to the placenta while also avoiding excessive shear stress from blood velocity. [START_REF] Burton | Rheological and Physiological Consequences of Conversion of the Maternal Spiral Arteries for Uteroplacental Blood Flow during Human Pregnancy[END_REF] use computer models to predict that the observed dilation of the mouth of maternal arteries from 0.4 mm to 2.5 mm of diameter slows the blood flow from 2.5 m/s to about 10 cm/s. This is important both to avoid damaging the fetal villi and to insure that the blood remains in the intervillous spaces for long enough for exchanges to take place. The degradation of the the arterial muscle sheath is probably also important to diminish vasoreactivity to maternal signals, which might decrease the blood supply and damage the placenta and fetus [START_REF] Burton | Rheological and Physiological Consequences of Conversion of the Maternal Spiral Arteries for Uteroplacental Blood Flow during Human Pregnancy[END_REF].

The remodeling process also necessitates interaction with the maternal uNK cells, see 1.1.2.b..

In addition to the blood supply increase following these changes in maternal blood flow, the fetal blood vessels bulge into the syncytiotrophoblast towards the end of gestation [START_REF] Burton | Formation of vasculo-syncytial membranes in the human placenta[END_REF], reducing the thickness of the layers separating both circulations to about 3 µm over 40% of the total placental surface (reviewed in Wooding & Burton, 2008;[START_REF] Benirschke | Pathology of the Human Placenta[END_REF].

Hemodichorial

In the rabbit placenta, implantation occurs when the conceptus sends out syncytial knobs that fuse with the maternal epithelium [START_REF] Larsen | Electron Microscopy of the Implantation Site in the Rabbit[END_REF][START_REF] Enders | Penetration of the uterine epithelium during implantation in the rabbit[END_REF]. This triggers a syncytialisation of the latter, forming large uterine syncytial plaques which subsequently are dislodged and phagocyted by the hybrid syncytium [START_REF] Enders | Penetration of the uterine epithelium during implantation in the rabbit[END_REF]. This syncytium then sends out smaller processes that invade the basal lamina and reach the maternal blood vessels, breaching them and establishing contact with the maternal circulation [START_REF] Larsen | Electron Microscopy of the Implantation Site in the Rabbit[END_REF]. Once contact is established, the ST vacuolises which gives rise to intrasyncytial lacunae that will be filled with maternal blood, first supplied by capillaries and later by maternal arteries [START_REF] Mossman | The rabbit placenta and the problem of placental transmission[END_REF][START_REF] Larsen | Electron Microscopy of the Implantation Site in the Rabbit[END_REF]. During these processes, there is always a layer of CTs underlying the ST, thus establishing a hemodichorial placenta in which two layers of trophoblast are present [START_REF] Enders | A comparative study of the fine structure of the trophoblast in several hemochorial placentas[END_REF][START_REF] Enders | Penetration of the uterine epithelium during implantation in the rabbit[END_REF].

Contrarily to what is observed for the human placenta, the major maternal arteries remain intact during gestation, running across the full length of the placenta and supplying blood on the fetal side of the lacunae, from where it flows back down towards the maternal side and drains through maternal veins [START_REF] Mossman | The rabbit placenta and the problem of placental transmission[END_REF][START_REF] Carter | An angiographic study of the structure and function of the uterine and maternal placental vasculature in the rabbit[END_REF]. This forms a countercurrent exchanger. Even if the vessels persist to term, there is invasion by trophoblastic cells that induces formation of a sheath around the arteries, as first described in rabbit by [START_REF] Mossman | The rabbit placenta and the problem of placental transmission[END_REF] and reviewed by [START_REF] Pijnenborg | Review article: Trophoblast invasion and the establishment of haemochorial placentation in man and laboratory animals[END_REF] for rabbit and other hemochorial species.

Hemotrichorial

The hemotrichorial type of placentation is found in rodents, for example rat and mice. In these placentas as soon as the conceptus establishes contact with the uterine epithelium, the latter sloughs off and is destroyed, seemingly due to the action of maternal cells rather than the trophoblast [START_REF] Welsh | Chorioallantoic placenta formation in the rat: I. Luminal epithelial cell death and extracellular matrix modifications in the mesometrial region of implantation chambers[END_REF][START_REF] Blankenship | Penetration of the uterine epithelial basement membrane during blastocyst implantation in the mouse[END_REF]. Implantation also triggers the differentiation of some trophoblast cells into invasive giant cells that penetrate the maternal tissues and form a transient structure that supplies gases and nutrients to the embryo during early gestation (reviewed in [START_REF] Hu | Development and function of trophoblast giant cells in the rodent placenta[END_REF].

The definitive placenta that is established in the second part of gestation presents villi that are in direct contact with maternal blood [START_REF] Duval | Le placenta des rongeurs[END_REF][START_REF] Grosser | Vergleichende Anatomie und Entwicklungsgeschichte der Eihäute under Placenta: mit besonderer Berücksichtigung des Menschen[END_REF], the area per unit of volume that is in contact with maternal blood increases dramatically during gestation from 267 cm 2 /cm 3 to 489 cm 2 /cm 3 , indicating that a complex network of villi and blood spaces is formed [START_REF] Coan | Developmental Dynamics of the Definitive Mouse Placenta Assessed by Stereology[END_REF]. At the surface of the villi a characteristic trilaminar trophoblast is found. By convention the layers are numbered I-III starting from the maternal side. The two inner layers of trophoblast (II and III, also called ST-I and ST-II) are syncytial. As in human, they seem to result from cell-cell fusion events, no mitotic figures having ever been observed in the syncytium [START_REF] Hernandez-Verdun | Morphogenesis of the syncytium in the mouse placenta -Ultrastructural study[END_REF][START_REF] Hernandez-Verdun | In vitro study of chorionic and ectoplacental trophoblast differentiation in the mouse[END_REF]. ST-I and ST-II are intimately connected by a series of tight and gap junctions enhancing both structural integrity and communication between the layers [START_REF] Enders | A comparative study of the fine structure of the trophoblast in several hemochorial placentas[END_REF][START_REF] Metz | Gap junctions in hemodichorial and hemotrichorial placentae[END_REF]. The trophoblast I layer meanwhile, remains cellular and continuous until term and is only loosely attached to layers II and III, leading to the formation of fluid filled spaces between I and II [START_REF] Enders | A comparative study of the fine structure of the trophoblast in several hemochorial placentas[END_REF][START_REF] Coan | Ultrastructural changes in the interhaemal membrane and junctional zone of the murine choriollantoic placenta across gestation[END_REF]. [START_REF] Enders | A comparative study of the fine structure of the trophoblast in several hemochorial placentas[END_REF] also describes perforations in the cells of the outer layer (later confirmed by, [START_REF] Coan | Ultrastructural changes in the interhaemal membrane and junctional zone of the murine choriollantoic placenta across gestation[END_REF] that allow ST-I to be in direct contact with maternal blood and suggests that this outer fenestrated epithelium might establish regions of slower blood flow and blood stasis, enhancing maternofetal exchanges. Interestingly the cells of the outer layer differentiate into giant cells by endomitosis, suggesting a potential endocrine role, as is common for these

1.3

cells in rodent placentas [START_REF] Coan | Ultrastructural changes in the interhaemal membrane and junctional zone of the murine choriollantoic placenta across gestation[END_REF][START_REF] Simmons | Diverse subtypes and developmental origins of trophoblast giant cells in the mouse placenta[END_REF]. Of note, while the presence of three layers in these placentas might suggest a huge increase in thickness compared to the single layer present in hemomonochorial placentation, stereology studies reveal that the three layers are about 4 µm thick at term in mice [START_REF] Coan | Developmental Dynamics of the Definitive Mouse Placenta Assessed by Stereology[END_REF], remarkably close to the 3 µm observed in human (see above).

Similarly to what is observed in rabbits but not in humans, the major arterial channels persist in rodent placentas, but there is significant invasion of the vessels by giant trophoblastic cells that leads to the establishment of large blood sinuses and reduces vasoreactivity to maternal signals [START_REF] Pijnenborg | Review article: Trophoblast invasion and the establishment of haemochorial placentation in man and laboratory animals[END_REF][START_REF] Simmons | Diverse subtypes and developmental origins of trophoblast giant cells in the mouse placenta[END_REF]. As in human, the participation of uNK cells in blood vessel remodeling in rodents has been established, see 1.1.b..

Viviparity in non-mammalian species

Before going into further details on non-mammalian viviparity, a quick reminder about definitions seems to be in order. As defined in Blackburn (2015) and described in 1.1.1, viviparity is defined by giving birth to live young and regroups a spectrum of strategies for embryonic nutrition ranging from lecithotrophy (the embryo is dependent on the yolk, very few nutrients provided by the mother) to matrotrophy (the mother provides almost all nutrients necessary). Viviparity can be inferred from the size of ova or the presence of well-developed fetuses in the oviducts for example (Blackburn, 1993c), while matrotrophy can be inferred from diverse criteria such as an important increase in dry mass of the conceptus during gestation or chemical composition analysis of the conceptus at different states (an important increase in mass or specific components indicates uptake of material by the fetus, [START_REF] Blackburn | Standardized criteria for the recognition of embryonic nutritional patterns in squamate reptiles[END_REF]. It is important to distinguish viviparity and matrotrophy, the latter being a subset of the former that we will focus on in more detail, since mammals are extremely matrotrophic.

Prevalence of viviparity outside of mammals a. Invertebrates

Contrary to popular belief, it has been known for a long time that viviparity, both lecithotrophic and matrotrophic, is far from restricted to mammals. Existence of both viviparous fishes and snakes was described by Aristotle (ca -350) in his pioneering "History of animals" and descriptions of live birth and placental structures in invertebrate species go back several hundred years. [START_REF] Quoy | Voyage de la corvette l'Astrolabe : exécuté par ordre du roi, pendant les années 1826-1827-1828-1829 / sous le commandement de J. Dumont d'Urville[END_REF] for example make references to the placenta of salps, as do [START_REF] Krohn | Observations sur la génération et le développement des Biphores (Salpa)[END_REF] and Huxley (1851) a few years later. A recent comprehensive review of available literature combined with their own data led [START_REF] Ostrovsky | Matrotrophy and placentation in invertebrates: A new paradigm[END_REF] to the conclusion that out of the 34 animal phyla, 21 present at least one matrotrophic viviparous species, and in 11 of the 21 matrotrophy was a widespread or common phenomenon (Fig. 7). While the authors consider this a conservative estimate due to lacking data for many species, it still represents about 145 independent origins of matrotrophy in the evolution of animals, representing a stunning example of evolutive convergence across an impressive number of distantly related clades. Since matrotrophic viviparity describes only a subset of viviparous modes of reproduction, the total number of emergences of viviparity can safely assumed to be higher (Blackburn, 2015).

While the considerable diversity in mammalian placentas was the focus of the previous part of this work, it pales in comparison to the diversity observed between all matrotrophic animals, vertebrate or not. The term matrotrophy itself, for example, regroups six diverse modes of nutrient delivery, 1) oophagy in which the embryos ingest and assimilate sibling yolks, 2) embryophagy in which the embryo cannibalizes sibling embryos, 3) histotrophy in which the embryo absorbs nutrients secreted by the mother, 4) histophagy in which the embryo ingests said nutrients, 5) matrophagy in which the embryo eats maternal tissues or organs and 6) placentotrophy in which a specialized placental structure is implicated (Blackburn, 2015;[START_REF] Ostrovsky | Matrotrophy and placentation in invertebrates: A new paradigm[END_REF]. These categories are non-exclusive and can either occur simultaneously or successively at different developmental stages (Blackburn, 2015). The dominant modes of matrotrophy in invertebrates are histotrophy and placentotrophy [START_REF] Ostrovsky | Matrotrophy and placentation in invertebrates: A new paradigm[END_REF]. Similarly, the site of matrotophy is highly dependent on the species, but while it can occur in other tissues or body cavities, it most commonly takes place in the female genital system and body cavities [START_REF] Ostrovsky | Matrotrophy and placentation in invertebrates: A new paradigm[END_REF]. Additionally, [START_REF] Ostrovsky | Matrotrophy and placentation in invertebrates: A new paradigm[END_REF] report that some phyla present very uniform modes and sites of matrotrophy, whereas others show great intra-phyla variation. While the immense number of strategies observed in these distant clades is an interesting aspect, it is also noteworthy that dominant modes and sites seem to emerge, and that they correspond mostly to those observed in mammals, the best-studied matrotrophic clade.

b. Non-mammalian vertebrates

In vertebrate animals, Blackburn (2015) distinguishes 152 origins of viviparity and 32 origins of matrotrophy (mostly placentotrophy, [START_REF] Ostrovsky | Matrotrophy and placentation in invertebrates: A new paradigm[END_REF] in addition to the singular ones in mammals (Fig. 7). According to [START_REF] Ostrovsky | Matrotrophy and placentation in invertebrates: A new paradigm[END_REF] this makes vertebrates the group presenting the most origins of matrotrophy, though arthropodes come close with 29-31 origins (Fig. 7). It also has to be kept in mind that these numbers are impacted by the uneven levels of investigation and knowledge of different clades, potentially leading to a significant underestimation. The date of origin of vertebrate viviparity varies wildly, the oldest known example being set at about 380 Mya in now-extinct placoderm fishes (Long et al., 2008), while the most recent described emergence of viviparity occurred about 5 Mya in the comephorid fishes of Lake Baikal [START_REF] Dybowski | Ueber Comephorus baicalensis Pall[END_REF][START_REF] Wourms | Viviparity: The Maternal-Fetal Relationship in Fishes[END_REF][START_REF] Hunt | Molecular Evolution of the Cottoid Fish Endemic to Lake Baikal Deduced from Nuclear DNA Evidence[END_REF]). For comparison, dos Reis et al. (2012) place the origin of viviparity in mammals at 175 Mya). Interestingly, no example of viviparity has been observed in birds (Blackburn, 2015;[START_REF] Ostrovsky | Matrotrophy and placentation in invertebrates: A new paradigm[END_REF].

Viviparity in Squamata. Squamata (scaled reptiles) include lizards and snakes. This family presents 115 independent origins of viviparity, about 75% of all vertebrate emergences, many of . Placenta

1.3

1.3

. . Viviparity in non-mammalian species them relatively recent [START_REF] Blackburn | Are viviparity and egg-guarding evolutionarily labile in squamates?[END_REF](Blackburn, , 2015)). Accordingly many emergences have occurred at subgeneric levels, where some species of a genus are viviparous while others are oviparous [START_REF] Blackburn | Are viviparity and egg-guarding evolutionarily labile in squamates?[END_REF]. In the genus Sceloporus for example, at least four independent origins of vivipariy have been identified (reviewed in Mendez-de la [START_REF] Mendez-De La Cruz | Constraints on the evolution of viviparity in the lizard genus Sceloporus[END_REF].

Most of these viviparous species are closer to the lecithotrophic end of the spectrum than the matrotrophic one, only small amounts of nutrients are generally provided by the mother, but there are six different clades exhibiting a substantial amount of matrotrophy (Flemming & Blackburn, 2003;Blackburn, 2015). This is mostly the case in Scincidae (skinks), a family that contains four of the six clades of matrotrophic lizards: the Mediterranean Chalcides chalcides (Blackburn, 1993b), the neotropical Mabuya genus (Blackburn et al., 1984;Jerez & Ramírez-Pinilla, 2001) and the African genera Eumecia anchietae (Flemming & Branch, 2001) and Lubuya ivensii (previously named Trachylepis ivensii, Blackburn & Flemming, 2010). In these species the placenta is highly specialized for nutrient transfer and structurally very similar to the placentas observed in mammalian species, often presenting syncytialized cells layers, specialized zones such as areolae and placentomes, and cellular invasion of maternal tissues by the fetus (Jerez & Ramírez-Pinilla, 2001;Vieira et al., 2007;Blackburn & Flemming, 2012, see also 1.3.2.c.).

Viviparity in fishes.

Viviparity has been observed both in bony (Teleosts, 13 origins) and cartilaginous fishes (Chondrichtyans, at least 9 origins) (Blackburn, 2015), as well as in the extinct armored fishes (Placodermi, 1 origin, Long et al., 2008). Both extant clades present a number of origins of matrotrophy, sometimes even several following a single emergence of viviparity, as is the case in Poeciliopsis (teleosts, [START_REF] Reznick | Independent Origins and Rapid Fish Genus Poeciliopsis[END_REF] or carcharhiniforms (Chondrichtyans, Gilmore, 1993;Blackburn, 2015). In fact matrotrophy seems to be particularly common in fishes. While Squamates represent 75% of vertebrate origins of viviparity, only 5% of these viviparous clades contain matrotrophic species, whereas bony and cartilaginous fishes only represent about 15% of origins of viviparity, but about 75% of viviparous clades have evolved matrotrophy, suggesting that the latter are predisposed to the emergence of matrotrophy (Blackburn, 2015).

In teleosts, most matrotrophic species employ a form of histotrophy or histophagy where the embryo absorbs or ingests nutrients that are released by specialized ovarian tissue. This pattern has arisen multiple times in different species and in some, like Poeciliidae, the absorptive surfaces of the embryo are closely apposed to maternal tissues, forming a placenta [START_REF] Wourms | Viviparity: The Maternal-Fetal Relationship in Fishes[END_REF]Blackburn, 2015). In Chondrichtyans, matrotrophy is often associated with oophagy in which the embryo assimilates resources provided by other unfertilized ova that are either released continually over the course of gestation, as is the case in Lamnoids, or alongside the fertilized ovum, as is the case in Pseudotriakidae [START_REF] Yano | Comments on the Reproductive Mode of the False Cat Shark Pseudotriakis microdon[END_REF][START_REF] Gilmore | Reproductive biology of lamnoid sharks[END_REF].

1.3.2 Examples of non-mammalian placentas

In this section selected non-mammalian placentas will be briefly described and compared to mammalian examples. Higher emphasis will be put on the description of the Mabuya placenta as it is of particular relevance later.

a. Non-vertebrates: the placenta of salps

The most complex invertebrate placenta is found in Salpidae, tunicate chordates that live in the ocean [START_REF] Bone | The placenta of the salp (Tunicata: Thaliacea)[END_REF][START_REF] Ostrovsky | Matrotrophy and placentation in invertebrates: A new paradigm[END_REF]. As was first pointed out by von Chamisso (1819), salps have a very particular mode of reproduction involving the alternation of two distinct generations: the asexual oozooid and the protogynous hermaphrodite (first female then male) blastozooid. The oozooid results from sexual reproduction but reproduces asexually by growing a long stolon that differentiates into a chain of linked blastozooids [START_REF] Bone | The placenta of the salp (Tunicata: Thaliacea)[END_REF]. The chain then breaks into several pieces and the linked blastozooids swim around in smaller chains. As protogynous hermaphrodites, the blastozooid first develops a single egg and afterwards mature testis and spermatozoids, this leads to younger blastozooids being fertilized by older blastozooids and avoids auto-fertilization [START_REF] Bone | The placenta of the salp (Tunicata: Thaliacea)[END_REF]. The fertilized egg then grows within the blastozooid, establishing a round placenta, and develops into an oozooid which then gets released into the sea alongside the placenta. After separation, the blastozooid starts producing sperm to fecundate younger still female blastozooids and the released oozoid produces a stolon generating the next generation of blastozooids [START_REF] Bone | The placenta of the salp (Tunicata: Thaliacea)[END_REF]. In smaller salp species, this cycle might be as fast as 46 hours under favorable conditions [START_REF] Heron | Population ecology of a colonizing species: The pelagic tunicate Thalia democratica -I. Individual growth rate and generation time[END_REF], while in larger species it is thought to take 36 hours to grow the stolon and less than a week for gestation of the oozooid within the blastozooid [START_REF] Bone | The placenta of the salp (Tunicata: Thaliacea)[END_REF].

After fertilization of the egg in the blastozooid ovary, the conceptus breaks through the ovary wall into the jet chamber of the mother, where it implants and establishes the salp placenta [START_REF] Sutton | The Sexual Development of Salpa fusiformis (Cuvier): Part I[END_REF][START_REF] Bone | The placenta of the salp (Tunicata: Thaliacea)[END_REF]. The mature placenta is composed of two syncytial, highly interdigitated cell layers separating the maternal and fetal blood spaces, reminiscent of mammalian hemodichorial placentation except for the absence of fetal endothelium (see 1.2.2.d., Bone et al., 1985). A major difference is the origin of the cell layers, both syncytia are initially of maternal origin and can therefore not be termed syncytiotrophoblast. The inner (mother-facing) syncytial layer presents additional fusion events of maternal blood cells into the syncytium while fetal leukocytes seem to add to the outer (fetus-facing), resulting in a heterologous syncytium as is observed in some cases of mammalian placentation, even though in this case the fetal contribution does not implicate specialized placental trophoblasts [START_REF] Bone | The placenta of the salp (Tunicata: Thaliacea)[END_REF]. At term the thickness of each of the two layers is between 50 and 100 µm, the outer layer being the thicker of the two. This represents a significantly higher distance to cross when compared to mammals, where the interhemal distance can be as low as 2 µm in pigs, 3 µm in humans or 4 µm in mice (see 1.2.2). It is important to note however that the salp placenta is probably not involved in gas exchanges and respiratory functions, as salp blood does not contain respiratory pigments. It is probable that the major functions accomplished by this placenta are transfer of nutrients and attachment of the embryo in the maternal jet-chamber through which water is pumped constantly [START_REF] Bone | The placenta of the salp (Tunicata: Thaliacea)[END_REF]. While gases cross placental layers using simple diffusion (which, according to Fick's law, is strongly impacted by an increase in diffusion distance), many nutrients use forms of facilitated or active transport, for which the impact of layer thickness might be less important (see 1.1.1.a. and b.) b. Fish: placentation in sharks Sharks are part of the Chondrychtians, the vertebrate group with the second highest percentage of viviparous species among vertebrates (55% according to Blackburn, 2015). Most of the matrotrophic species in this group employ oophagy or histotrophy/phagy but about 30% of sharks, like the spadenose shark (Scoliodon laticaudus), are placentotrophic and develop complex placentas [START_REF] Wourms | Maximization of evolutionary trends for placental viviparity in the spadenose shark, Scoliodon taticaudus[END_REF][START_REF] Haines | Histology and Immunology of the Placenta in the Atlantic Sharpnose Shark, Rhizoprionodon terraenovae[END_REF]. In this shark the embryo grows from a 1 mm egg to a size of 150 mm over the course of 6 months, this growth is accompanied by an important increase in mass of about 58000 times, a strong indicator of extreme matrotrophy [START_REF] Wourms | Maximization of evolutionary trends for placental viviparity in the spadenose shark, Scoliodon taticaudus[END_REF][START_REF] Blackburn | Standardized criteria for the recognition of embryonic nutritional patterns in squamate reptiles[END_REF]. After implantation, the extra-embryonic parts of the 1 mm egg develop into a 12 mm sphere that remains in contact with maternal tissues and is connected to the embryo by the umbilical stalk. The sphere is highly vascularized and covered by an extremely thin epithelium, allowing a decrease in interhemal distance, but contrarily to what is commonly seen in mammalian placentas, the epithelium presents a smooth border without microvilli [START_REF] Wourms | Maximization of evolutionary trends for placental viviparity in the spadenose shark, Scoliodon taticaudus[END_REF]. The histological nature of the maternal side of the interface seems unclear, [START_REF] Wourms | Maximization of evolutionary trends for placental viviparity in the spadenose shark, Scoliodon taticaudus[END_REF] being of the opinion that maternal blood is in direct contact with the placental epithelium, suggesting a hemochorial structure, but there is no definite proof that it is not epitheliochorial, with completely intact maternal layers.

In addition to the placenta, the spadenose shark embryo develops so-called appendiculae, long well-vascularized projections with a thin epithelium, that originate on the stalk connecting the placenta to the embryo [START_REF] Wourms | Maximization of evolutionary trends for placental viviparity in the spadenose shark, Scoliodon taticaudus[END_REF]. Since these projections are not in direct contact with maternal tissues, they cannot be considered placental tissues sensu [START_REF] Mossman | Comparative morphogenesis of the fetal membranes and accessory structures[END_REF], but they seem nevertheless to be implicated in maternofetal exchanges, probably gaseous exchanges in particular [START_REF] Wourms | Maximization of evolutionary trends for placental viviparity in the spadenose shark, Scoliodon taticaudus[END_REF]. Interestingly, these appendiculae can not only be observed in other shark species, but they also present a high structural similarity with the trophonemata of rays, another family of Chondrichtyans that presents highly matrotrophic species [START_REF] Wourms | Viviparity: The Maternal-Fetal Relationship in Fishes[END_REF]Blackburn, 2015). Trophonemata are projections of the maternal epithelium that secrete histotrophe to feed the embryo, as such they are also specialized for maternofetal exchanges, even though in this case the direction is opposite than for appendiculae [START_REF] Wourms | Viviparity: The Maternal-Fetal Relationship in Fishes[END_REF]Blackburn, 2015).

A more recent study on the Atlantic sharpnose shark (Rhizoprionodon terranovae, another placental shark part of the sister group to Scoliodon) investigated the immune context of shark

1.3

placentation and found cells that resemble mammalian uNK and are associated with maternal blood vessel [START_REF] Haines | Histology and Immunology of the Placenta in the Atlantic Sharpnose Shark, Rhizoprionodon terraenovae[END_REF]. The presence of such cells with a potential similar role in maternal blood supply would be a striking example of evolutive convergence in an already very convergent context since the shark placenta has emerged independently of the mammalian one.

c. Lizards: placentation in Mabuya and other Scincidae

As stated above, while the squamates present an astonishing 115 independent origins of viviparity, most of the viviparous species are lecithotrophic (Flemming & Blackburn, 2003;Blackburn, 2015). Of the six known examples of highly matrotrophic squamates, four belong to the Scincidae family of lizards (commonly called skinks) and at least two of these four are associated with invasive and highly complex placentas.

Viviparity and placentation in the Neotropical skinks Mabuya. Matrotrophic viviparity in the Mabuya genus was first described in Mabuya heathi, a Brazilian representative of the genus. [START_REF] Vitt | Reproduction in the lizard Mabuya heathi (Scincidae): a commentary on viviparity in New World Mabuya[END_REF]; Blackburn et al. (1984) describe that this species ovulates very small eggs (1 mm, 0.4 mg) that show a large increase in dry mass over a long period of gestation (neonate: 31 mm, 154 mg). Fertilization occurs in September/October and parturition in October of the following year, this one year gestation period is much longer than the three month period observed in most viviparous reptiles (Flemming & Blackburn, 2003). About 90% of the total dry mass increase of the embryo occurs during the last three months of gestation, once the definitive Mabuya placenta is formed [START_REF] Vitt | Reproduction in the lizard Mabuya heathi (Scincidae): a commentary on viviparity in New World Mabuya[END_REF]Blackburn et al., 1984). As Flemming & Blackburn (2003) put it: "In fact, in terms of the total amount of nutrients provided by the placental membranes, these lizards lie within the range of placental mammals". A similar reproduction pattern was then described for many other species of the Mabuya genus, including: M.agilis, M.brachypoda, M. caissara, M. frenata, M. macrorhyncha, M. nigropunctata and the as of yet unnamed but highly studied Mabuya sp. IV, and assumed for M. carvalhoi, M. dorsivittata and M. guaporicola (reviewed in Flemming & Blackburn, 2003). It therefore seems highly likely that the matrotrophic viviparous mode of reproduction is common to the entire genus and results from a single emergence, about 25-30 Mya during the radiation of this clade (Karin et al., 2016;Pereira & Schrago, 2017).

The detailed structure and function of the Mabuya placenta has been studied in several Mabuya species(see for example Blackburn & Vitt, 2002;Leal & Ramírez-Pinilla, 2008), but most thoroughly in a population of Mabuya sp. IV from Curití, Colombia (Jerez & Ramírez-Pinilla, 2001, 2003;Ramírez-Pinilla et al., 2006;Vieira et al., 2007;Wooding et al., 2010;Ramírez-Pinilla et al., 2011;[START_REF] Hernández-Díaz | Proteomic Profile of Mabuya sp. (Squamata: Scincidae) Ovary and Placenta During Gestation[END_REF]. This species was originally referred to as Mabuya mabouya, however following redefinitions of Mabuya phylogeny, this Andean species is now called Mabuya sp. IV (Pinto-Sánchez et al., 2015). It is also of relevance here to mention that Mabuya phylogeny, as well as Scincidae phylogeny in general, still presents a number of uncertainties and older publications often used now deprecated names as some 1.3

. . Viviparity in non-mammalian species species have since been sorted into other genera (see for example [START_REF] Mausfeld | Phylogenetic affinities of Mabuya atlantica Schmidt, 1945, endemic to the Atlantic ocean archipelago of Fernando de Noronha (Brazil): necessity of partitioning the genus Mabuya Fitzinger, 1826 (Scincidae: Lygosominae)[END_REF]Karin et al., 2016, for publications re-distributing Scincidae species).

The definitive Mabuya placenta presents four types of regions specialized for maternofetal exchanges (Fig. 8A). The areolas are small invaginations of the placental tissue that form during the last stages of gestation, accompanied by a slight evagination of the uterine epithelium in the corresponding region. The created cavity is filled with secretions of maternal origin as well as with fetal cell debris, some of the epithelial cells having degenerated to form the invagination and both are taken up by the placental epithelium (Jerez & Ramírez-Pinilla, 2001;Ramírez-Pinilla et al., 2006). In mammalian placentas, notably in epitheliochorial or synepitheliochorial placentas, areolas similarly designate areas of highly active maternal secretion and corresponding fetal uptake (reviewed in Wooding & Burton, 2008, chap. 1), though it is probable that the nature of secreted substrates is different (Ramírez-Pinilla et al., 2006). Another region of exchange can be found on the abembryonic pole of the Mabuya placenta where the placental epithelium presents small regions of tall hypertrophic cells forming the so-called absorptive plaques (Jerez & Ramírez-Pinilla, 2001). There seem to be approximately 60 absorptive plaques at the surface of a late-term placenta and in each of them the maternal and fetal epithelia are closely apposed and do not present microvillous junctions but instead a complex system of interwoven cytoplasmic projections (Ramírez-Pinilla et al., 2006). In mammalian placentas, absorptive plaques are usually involved in absorbing secretions of uterine glands but in the Mabuya there are no significant glands in the corresponding regions, it is therefore likely that here again the exchanged material is of a different nature. Ramírez-Pinilla et al. (2006) describe numerous lipid droplets present on both sides of the absorptive plaques, suggesting that lipid transfer might be an important function occurring in these regions. Between the absorptive plaques are areas specialized for gaseous exchanges, called respiratory segments, in which the fetal and maternal epithelia are closely apposed and very thin to reduce interhemal distance (to about 8 µm) and the subepithelial vascularization in increased to favor gas transfer (Jerez & Ramírez-Pinilla, 2001;Ramírez-Pinilla et al., 2006). Contrary to what is observed in many mammalian placentas, there is no loss of maternal cell layers to reduce the interhemal distance in these gas-exchange regions.

The placentome is the area located at the embryonic pole, dorsally to the embryo, and to which the umbilical cord is attached. In Mabuya sp. IV, the placentome is a small disc of 6 mm of diameter in which the maternal and fetal tissues are highly folded and interdigitated (Fig. 8B, Jerez & Ramírez-Pinilla, 2001). In the placentome, the fetal epithelium contains two distinct cell types, large wide binucleated cells (giant cells) and tall thin mono-or binucleated cells wedged between the former (interstitial cells) (Jerez & Ramírez-Pinilla, 2001;Ramírez-Pinilla et al., 2006). While these two populations also constitute the placental epithelium outside of the placentome, their differences are exacerbated in this region since the giant cells are extremely hypertrophied and always present two large nuclei. On the maternal side the epithelial cells have fused to form a large syncytium covering the entirety of the placentomal interface, this fusion seems to occur before contact between the two epithelia, precluding participation of fetal

1.3

cells in the establishment of the syncytium (Jerez & Ramírez-Pinilla, 2001, 2003;Ramírez-Pinilla et al., 2006). The interface of the two epithelia is microvillous, with both the maternal syncytium and the fetal epithelial giant cells sending out interdigitating microvilli, increasing the exchange surface (Ramírez-Pinilla et al., 2006). Periodically this microvillous border get interrupted by the presence of small cells at the apex of a placental giant cell (Ramírez-Pinilla et al., 2006;Vieira et al., 2007). These small cells occur in groups of variable number and shape and send out thin and very long projections that cross the maternal syncytium and establish an intimate contact with maternal capillaries (Vieira et al., 2007). The projections surround the capillaries and form an extremely complex system of double membranes in direct contact with the maternal syncytial cytoplasm (Fig. 8C, Vieira et al., 2007). The presence of these invasive cells suggests that the relationship between maternal and fetal tissues is much closer than expected at first glance. In these regions the maternofetal interface is closer to the endotheliochorial than the epitheliochorial type since there is direct contact between placental tissues and maternal endothelium. The paraplacentome is a small region of non-folded tissues (less than 1 mm) that surrounds the placentome (Fig. 8A,B) and is delimited on the outer side by an invagination of the placental epithelium (Jerez & Ramírez-Pinilla, 2001). At the edge of the placentome and paraplacentome the uterine syncytium stops abruptly and the maternal epithelium is unicellular in the paraplacentome. The paraplacentomal epithelium is virtually identical to that in the placentome, being composed of giant and interstitial cells (Jerez & Ramírez-Pinilla, 2001). There is no microvillous border in between maternal and fetal tissues in the paraplacentome, though at the border to the placentome there is some interdigitation of the two (Ramírez-Pinilla et al., 2006). The maternal side of the paraplacentome presents secreting glands and the presence of lipid droplets in both maternal and fetal cells suggests that this region might also be involved in lipid transfer (Ramírez-Pinilla et al., 2006).

All things considered, the placenta of Mabuya sp. IV is remarkably similar to the ones found in mammalians. It presents specialized structures of homologous design (areolas, absorptive plaques, placentome) that seem to accomplish similar functions, even though the nature of the nutrients that are exchanged in these regions seems to differ (perhaps not surprisingly since in reptiles the main source of fetal energy is lipids, as opposed to glucose for mammals, see [START_REF] Thompson | A review of the evolution of viviparity in lizards: structure, function and physiology of the placenta[END_REF]. While folding and interdigitation is a phenomenon seen in many tissues where an increase of the exchange surface can be advantageous, the Mabuya placenta also presents a syncytial layer, as is the case in many mammalian placentas, as well as mechanisms of fetal invasiveness to establish a more initimate contact with the maternal bloodstream. All these characteristics are remarkable examples of convergence, seeing as these two placentas emerged 150 My apart and since the last common ancestor of mammals and Mabuya would have lived 300 Mya (Kumazawa, 2007;[START_REF] Dos Reis | Phylogenomic datasets provide both precision and accuracy in estimating the timescale of placental mammal phylogeny[END_REF]Pereira & Schrago, 2017).

A second origin of extreme matrotrophy in African Scincidae. In addition to the Mabuya genus, the Scincidae family contains the two other lizard species presenting a similarly complex (A) Overview of a gravid uterus displaying the apposed maternal and fetal tissues, as well as regions specialized for maternofetal exchanges: the placentome, absorptive plaques and areola. The paraplacentome forms a band around the placentome and is therefore present on both sides. (B) Detailed representation of the maternofetal interface in the placentome and paraplacentome region. In the placentome, maternal and fetal tissues are highly interdigitated, with numerous microvilli between fetal and uterine cells. The fetal epithelium is formed primarily by giant binucleated chorionic cells. The uterine epithelium is replaced by a large syncytial structure formed by the fusion of the uterine cells. In the paraplacentome the syncytium is abruptly replaced by uterine epithelium. Cells still present microvilli, but the tissues are no longer interdigitated. (C) Detail of cellular invasion in the mabuya placenta. (left) Electron micrograph of the complex double membrane system originating from fetal invasive cell projections. (right) Representation of the double membrane projections formed around maternal blood vessels by the fetal invasive cells, using the same color scheme as in (A) and (B). Adapted from Vieira et al. (2007); Wooding et al. (2010).

1.3

placenta: Eumecia anchietae and Lubuya ivensii (formerly known as Mabuya ivensii or Trachylepis ivensii, [START_REF] Metallinou | A single origin of extreme matrotrophy in African mabuyine skinks[END_REF]. Both of these rare species belong to a single clade of African Scincidae and are the sole members of their respective genus [START_REF] Metallinou | A single origin of extreme matrotrophy in African mabuyine skinks[END_REF]Pereira & Schrago, 2017). It has now been established that the emergence of viviparity and matrotrophy in this African clade is independent of that observed in the Mabuya genus, though it remains unclear on whether it represents a single common, or two separate origins [START_REF] Metallinou | A single origin of extreme matrotrophy in African mabuyine skinks[END_REF].

The only detailed description of the Eumecia anchietae placenta available presents it as a simple folded epitheliochorial placenta, without the presence of a placentome (Flemming & Branch, 2001). While this study did have access to one late-term embryo (stage 40 of the Dufaure & Hubert (1961) development table), its conservation state was too poor to allow study of the placenta (AF Flemming, personal communication) and since in Mabuya sp. IV the highly interdigitated placentome forms only during stages 35-40 (Jerez & Ramírez-Pinilla, 2003), it is possible that the structure of the Eumecia anchietae placenta becomes more complex and invasive later on.

The placenta of Lubuya ivensii on the other hand is much more complex and highly invasive, even more so than that of Mabuya, though its study is complicated by the rarity of the species. During implantation the Lubuya placenta sends out knobs of cells that traverse the uterine epithelium and reach through to the basement membrane (Blackburn & Flemming, 2010, 2012). The nature of these knobs seems to be syncytial even though this has not been clearly demonstrated so far. Once these placental knobs reach the basement membrane, they begins to send out projections under the uterine epithelium and start to grow between it and the basement membrane, forming an outer placental epithelium that also seems to be syncytial in nature (Blackburn & Flemming, 2012). The growth of placental epithelium beneath the uterine cell layer leads to the shedding of the latter and its replacement by a placental epithelial, possibly syncytial, layer. Near term, most of the uterine epithelium has been destroyed and the outer placental epithelium is in direct contact with the basement membrane and the endothelium of maternal blood vessels, giving rise to an endotheliochorial placenta (Blackburn & Flemming, 2012). The inner placental epithelium from which the knobs originate is in close but loose apposition with the outer layer (Blackburn & Flemming, 2012). The Lubuya invensii placenta does not seem to present distinct regions of histotrophic and respiratory exchanges. In most squamates (as is the case in Mabuya), there is a distinction between regions of nutrient exchange which present large secretory and absorptive cells (areolas, absorptive plaques, placentome) and regions of gas exchange where the cell layers are thinned to permit more efficient diffusion (respiratory segments), the absence of this distinction indicates that the Lubuya ivensii placenta absorbs both gases and nutrients directly from the maternal blood circulation (Blackburn, 1993a;Jerez & Ramírez-Pinilla, 2001;Blackburn & Flemming, 2012). In the end, while they both present invasive fetal cells, the Mabuya and Lubuya placentas are quite different, having taken different paths of ensuring appropriate provision of nutrients and gases to the fetus. This would be in accordance with the

Retroviruses

Retroviruses are the members of the Retroviridae family of viruses, officially created in 1975 (ICTV, 1975) and named after the process of retrotranscription, synthesis of a DNA molecule using an RNA matrix. This concept had long been believed impossible, going against a deeply held belief that the DNA to RNA to protein pathway was strictly one-way, but in 1970 [START_REF] Temin | RNA-dependent DNA polymerase in virions of Rous sarcoma virus[END_REF] and [START_REF] Baltimore | Viral RNA-dependent DNA Polymerase: RNA-dependent DNA Polymerase in Virions of RNA Tumour Viruses[END_REF] demonstrated the existence of enzymes able to synthesize complementary DNA (cDNA) from an RNA molecule. These enzymes have been called reverse transcriptases (RT) and are crucial to the retroviral life cycle, allowing them to convert their RNA genome into a DNA genome that can then insert itself into the genome of the host cell. The particularities of the retroviral replication cycle give rise to two great categories: exogenous (or infectious) retroviruses that exist as viral particles (virions) and infect cells, and endogenous retroviruses (ERV) that exist as integrated DNA genomes. Retroviral virions are spherical particles of about 80-100 nm diameter composed of a lipid envelope that contains a protein core (Fig. 9A). The envelope is of cellular origin but incorporates the viral envelope glycoproteins. The core is of variable shape depending on the retroviral family and contains the RNA genome as well as the proteins necessary to start the viral cycle following entry in a new host cell (reviewed in Coffin et al., 1997, chap. 1 and 2).

As for viruses in general, the origin of retroviruses is unknown. Investigation into endogenous retroviruses, which can act as a sort of fossil trace, show that there is a vast amount of retroviral lineages that infect or have infected hosts all across vertebrate phylogeny, with numerous hostswitching events [START_REF] Hayward | Pan-vertebrate comparative genomics unmasks retrovirus macroevolution[END_REF]. Retroviruses are a very ancient family of viruses, Aiewsakun & Katzourakis (2017) describe a group of retroviruses that are over 450 My old, by comparison the oldest non-retro viruses described to date are 310 My old insect viruses [START_REF] Theze | Paleozoic origin of insect large dsDNA viruses[END_REF]. An emergence about 450 Mya would put the apparition of retroviruses at about the same time as the apparition of jawed vertebrates during the Palaeozoic [START_REF] Dos Reis | Uncertainty in the Timing of Origin of Animals and the Limits of Precision in Molecular Timescales[END_REF]. Since the ICTV meeting in Paris in 2002, the Retroviridae family is split into two subfamilies, Orthoretrovirinae and Spumaretrovirinae [START_REF] Mayo | ICTV at the Paris ICV: Results of the plenary session and the binomial ballot[END_REF]. The former subfamily contains most known retroviruses and unless noted otherwise will be the subject of the following section. For some details about Spumaretrovirinae see 2.1.1.b.

Exogenous Retroviruses

Genomic orgarnisation

The first published sequenced retroviral genome was that of the murine leukemia virus (MLV, [START_REF] Shinnick | Nucleotide Sequence of Moloney Murine Leukemia Virus[END_REF]. It revealed a genome of about 8 kb presenting three major open reading frames (ORF) flanked by a 600 bp long repeated sequence. This general genomic organisation has since been described in most retroviruses, with genome sizes ranging from about 7 to 12 kb (Fig. 9B, reviewed in Coffin et al., 1997, chap. 1 and 2). A variable factor in retroviral genome organisation is the amount of proteins encoded, while all retroviruses present the three major ORFs (group-specific antigen, gag; polymerase, pol; envelope, env), some genera present additional ORFs encoding so-called accessory proteins, in this case they are referred to as complex retroviruses (reviewed in Coffin et al., 1997, chap. 1 and 2). Additionally it is important to note that the relative arrangement of the major ORFs is dependent on the virus, often the different ORFs are in different frames relating to each other and thus can overlap (see Fig. 9 and 2. 1.3.d.). Due to the retrotranscription step, all retroviral genomes can exist as either single stranded positive sense RNA or double stranded DNA, with minor differences between the extremities of both forms.

a. Non-coding features

In addition to the ORFs that code for viral proteins, retroviral genomes present conserved non-coding domains that are critical to the replication cycle.

Long terminal repeats. Probably the most well-known non-coding domains, the long terminal repeats (LTR) are identical sequences, starting in TG and ending in CA in the integrated DNA form, present at both ends of the DNA genome, and ranging in size from 300 to 1000 nts (reviewed in [START_REF] Mager | Mammalian Endogenous Retroviruses[END_REF]. The length and sequence of the LTR is specific to a given retrovirus, but they can all be split into three parts: U3 (100-1000 nts), R (10-100 nts) and U5 (100-200 nts), as illustrated in Fig. 9B. In the DNA genome form, both LTRs present all three regions, while in the RNA form, U3 is unique to the 3' LTR and U5 to the 5' LTR, due to the way retrotranscription works (see Fig. 13 2.1.3.b.). U3, the segment that somewhat confusingly is unique to the 3' LTR of the RNA form, but is situated 5' to the whole genome in the DNA form, contains enhancer and promoter elements driving RNA transcription in eukaryotic cells. The presence of these elements was first described in the MLV genome [START_REF] Laimins | Characterization of enhancer elements in the long terminal repeat of Moloney murine sarcoma virus[END_REF][START_REF] Graves | Delineation of transcriptional control signals within the Moloney murine sarcoma virus long terminal repeat[END_REF] and they allow the binding of a variety of cellular vertebrate transcription factors (rewieved in [START_REF] Hurst | Epigenetic control of human endogenous retrovirus expression: Focus on regulation of long-terminal repeats (LTRs)[END_REF]. In recent years there have been a number of examples of cellular genes of diverse tissue types whose expression can be driven by the presence of an integrated LTR nearby, illustrating the transcript initiation potency of these elements [START_REF] Thompson | Long Terminal Repeats: From Parasitic Elements to Building Blocks of the Transcriptional Regulatory Repertoire[END_REF], see also 2.2.2.a.). The [START_REF] Konvalinka | Retroviral proteases and their roles in virion maturation[END_REF]. (B) Structural organization of retroviral genomes. For each family the organization of the LTRs and the canonical retroviral ORFs is represented to scale (see bottom), using the indicated type species. The order of subunits is indicated on the first genome and conserved in all others, faded boxes represent spacer or non-canonical subunits, accessory and regulatory genes are not displayed. Arrows with FS represents synthesis of Pro or Pol by frameshift, with R-T by read-through (see 2.1.3.d.). Adapted from viralzone.expasy.org transcript initiation site is by definition the border between U3 and R. The R segment, which is the only part of the LTR present at both extremities of the RNA genome, is essential for the template switching step during retrotranscription, see 2. 1.3.b. and Fig. 13. It also contains a polyadenylation signal which forms the border between R and U5 (reviewed in Coffin et al., 1997, chap. 6).

Primer binding site. The primer binding site (PBS) is a generally 18 nts long sequence situated a few nucleotides after the end of U5 [START_REF] Shine | Nucleotide sequence at the 5' terminus of the avian sarcoma virus genome[END_REF][START_REF] Peters | RNA-directed DNA synthesis in Moloney murine leukemia virus: interaction between the primer tRNA and the genome RNA[END_REF]. Binding of the 3' end of a tRNA to this site is necessary to act as a primer for synthesis of the genomic DNA minus strand during retrotranscription (for an early review, see [START_REF] Taylor | An analysis of the role of tRNA species as primers for the transcription into DNA of RNA tumor virus genomes[END_REF], since like all DNA polymerases, RT needs a double stranded template. The exact sequence of the PBS is highly dependent on the studied retrovirus, only the first three nucleotides are fixed (TGG) since the tRNA binds using its 3' end which always finishes in ACC (reviewed in [START_REF] Coffin | Retroviruses. Cold Spring Harbor[END_REF]. Since the sequence of the PBS determines the nature of the bound tRNA, different retroviruses will need tRNAs corresponding to different amino-acids, this was used originally to name and classify ERVs. Human endogenous retrovirus W (HERV-W) for example presents a PBS able to bind tryptophan (W) associated tRNAs [START_REF] Blond | Molecular characterization and placental expression of HERV-W, a new human endogenous retrovirus family[END_REF].

Polypurine tract. The polypurine tract is a short stretch of 6-19 purines immediately upstream of U3, in the 3' part of the genome. [START_REF] Sorge | Polypurine tract adjacent to the U3 region of the Rous sarcoma virus genome provides a cis-acting function[END_REF] showed that this domain is absolutely critical for the replication of RSV and it was later shown that this domain's resistance to RNAse cleavage by the viral RT generates the primer necessary for synthesis of the viral DNA genome plus strand (see 2.1.3.b. and Fig. 13, reviewed in Hughes, 2015). Some retroviruses like human immunodeficiency virus (HIV) present more than one PPT [START_REF] Charneau | A single-stranded gap in human immunodeficiency virus unintegrated linear DNA defined by a central copy of the polypurine tract[END_REF]. In the case of HIV the second PPT is located in the middle of the genome and is necessary for optimal viral replication [START_REF] Charneau | A second origin of DNA plus-strand synthesis is required for optimal human A Second Origin of DNA Plus-Strand Synthesis Is Required for Optimal Human Immunodeficiency Virus Replication[END_REF].

b. Gag

The Gag polyprotein is coded by the 5' most ORF and always contains the structural matrix (MA), capsid (CA) and nucleocapsid (NC) proteins in that order [START_REF] Heidmann | Identification of an endogenous retroviral envelope gene with fusogenic activity and placenta-specific expression in the rabbit: A new "syncytin" in a third order of mammals[END_REF]. It can contain additional peptides and domains between the three canonical proteins in a virus specific arrangement (reviewed in [START_REF] Mattei | Retrovirus maturation -An extraordinary structural transformation[END_REF][START_REF] Perilla | Molecular Architecture of the Retroviral Capsid[END_REF]. Gag contains all the necessary domains for formation of a viral capsid surrounded by a lipid envelope, as shown by the fact that its expression is sufficient to induce formation of viral-like particles (VLP, particles that are composed only of the structural components of a virion) in vitro [START_REF] Klikova | Efficient in vivo and in vitro assembly of retroviral capsids from Gag precursor proteins expressed in bacteria[END_REF]. During the maturation step of the viral cycle (see Fig. 12 and 2.1.3) the Gag polyprotein is cleaved, releasing MA, CA and NC as independent proteins and triggering a structural rearrangement marking the apparition of the mature virion (see Fig. 9, reviewed in [START_REF] Mattei | Retrovirus maturation -An extraordinary structural transformation[END_REF].

2.1

MA tends to assemble in a homotrimer of alpha helical folds that creates a flat surface presenting numerous basic residues, thereby allowing binding to lipid membranes [START_REF] Hill | Crystal structures of the trimeric human immunodeficiency virus type 1 matrix protein: implications for membrane association and assembly[END_REF]. In some retroviruses such as HIV, the N-terminal glycine residue is myristoylated and the presense of the myristic acid is necessary for membrane association [START_REF] Bryant | Myristoylation-dependent replication and assembly of human immunodeficiency virus 1[END_REF]. CA is composed of two independent domains joined by a flexible hinge [START_REF] Campos-Olivas | Solution structure and dynamics of the Rous sarcoma virus capsid protein and comparison with capsid proteins of other retroviruses 1 1Edited by P. E. Wright[END_REF], the N-terminal domain (NTD) and C-terminal domain (CTD). The NTD contains six to seven alpha helices and seems to play a moderate role in immature, but a critical role in mature capsid assembly (reviewed in [START_REF] Mattei | Retrovirus maturation -An extraordinary structural transformation[END_REF]. The CTD contains four alpha helices as well as the major homology region (MHR), a stretch of 20 amino acids that is highly conserved between retroviruses and even present in the distantly related Ty3 yeast retrotransposon [START_REF] Orlinsky | Mutations in the Ty3 major homology region affect multiple steps in Ty3 retrotransposition[END_REF]. The high degree of conservation of this sequence is particularly striking as Gag sequences are highly divergent, only the 3D structure seems to be conserved among retroviruses. Deletion and mutation experiments of the MHR region suggest that it is implicated in Gag oligomerization during formation of the immature capsid, though its exact function is poorly understood [START_REF] Chang | Mutations in Capsid Major Homology Region Affect Assembly and Membrane Affinity of HIV-1 Gag[END_REF][START_REF] Tanaka | Mutations of Conserved Residues in the Major Homology Region Arrest Assembling HIV-1 Gag as a Membrane-Targeted Intermediate Containing Genomic RNA and Cellular Proteins[END_REF]. Structure analysis of immature Gag conformation supports the theory that the MHR is involved in interaction between different Gag molecules [START_REF] Bharat | Structure of the immature retroviral capsid at 8Å resolution by cryo-electron microscopy[END_REF][START_REF] Bharat | Cryo-electron microscopy of tubular arrays of HIV-1 Gag resolves structures essential for immature virus assembly[END_REF][START_REF] Schur | Structure of the immature HIV-1 capsid in intact virus particles at 8.8 Å resolution[END_REF]. Finally NC contains positively charged regions and zincfinger like motifs that allow it to bind to nucleic acids. This binding is necessary to induce Gag oligomerization as in absence of nucleic acids Gag assumes a folded conformation in which NC interacts with MA [START_REF] Datta | Conformation of the HIV-1 Gag Protein in Solution[END_REF].

c. Pro and Pol

The pol ORF codes for several enzymes needed in retroviral replication. It always codes for the reverse transcriptase (RT) and the integrase (IN), and in most retroviruses for the protease (PR or Pro). The pro ORF is always found between gag and pol, and most of the time it is attached 5' to the latter, but in some retroviruses this coding sequence is instead located in the gag ORF or forms its own independent ORF (see 9, reviewed in Coffin et al., 1997, chap. 2 and 7). The location of pol relative to gag is also variable, in most viruses pol is situated in an overlapping -1 frame when compared to gag (HIV and RSV for example, [START_REF] Jacks | Expression of the Rous sarcoma virus pol gene by ribosomal frameshifting[END_REF][START_REF] Jacks | Characterization of ribosomal frameshifting in HIV-1 gag-pol expression[END_REF], while in others it is simply located downstream of gag in the same frame after a stop codon (murine leukemia virus, MLV, for example, [START_REF] Philipson | Translation of MuLV and MSV RNAs in nuclease-treated reticulocyte extracts: Enhancement of the gag-pol polypeptide with yeast suppressor tRNA[END_REF].

Expression of polyproteins is a strategy observed in many different virus groups, including retroviruses. Since most retroviral proteins are expressed as part of precursor polyproteins (Gag, Gag-Pro and Gag-Pro-Pol), PR plays an essential role in separating the different proteins (MA, CA, NC, PR, RT, IN) as was first described by von der [START_REF] Helm | Cleavage of Rous sarcoma viral polypeptide precursor into internal structural proteins in vitro involves viral protein p15[END_REF] and [START_REF] Yoshinaka | Properties of a P70 proteolytic factor of murine leukemia viruses[END_REF]. PR is a small autocatalytic aspartic protease that is active at lower pH values only [START_REF] Hansen | Partial purification and substrate analysis of bacterially expressed HIV protease by means of monoclonal antibody[END_REF][START_REF] Billich | Synthetic Peptides as Substrates and Inhibitors of Human Immune Deficiency Virus-1 Protease[END_REF]. Its active conformation is a homodimer in which each monomer contains a catalytic Asp, Thr/Ser, Gly triad (reviewed in [START_REF] Konvalinka | Retroviral proteases and their roles in virion maturation[END_REF]. The enzymatic activity of different PR from different retroviruses is variable and probably correlated with its expression level in vivo during infection. Alteration of enzymatic activity level can lead to formation of morphologically abnormal viral particles [START_REF] Sedlacek | Myeloblastosis Associated Virus (MAV) Proteinase Site-Mutated to Be HIV-like Has a Higher Activity and Allows Production of Infectious but Morphologically Altered Virus[END_REF]. Substrate specificity of PR is complex as there does not seem to be a strictly defined sequence that acts as cleaving site, only preferences for aromatic or large aliphatic residues (especially Leu) in +1 and -1 of the cleavage site, as well as for large hydrophobic amino acids (especially Glu) in +2 and -2 has been observed (reviewed in [START_REF] Konvalinka | Retroviral proteases and their roles in virion maturation[END_REF]. Additionally, the structure of the substrate seems to play an important role as PR seems to be able to recognize the asymetrical structures of its natural substrates [START_REF] Prabu-Jeyabalan | Substrate shape determines specificity of recognition for HIV-1 protease: Analysis of crystal structures of six substrate complexes[END_REF].

RT, the iconic enzyme that gives the family its name, contains two major domains needed for viral replication: the DNA polymerase and the RNase H domain. The retroviral DNA polymerase domain is capable of both RNA-directed DNA-synthesis and of DNA-directed DNA-synthesis, exhibiting a wider template range than most cellular polymerases, even though they might share a common ancestor (reviewed in [START_REF] Hughes | Reverse Transcription of Retroviruses and LTR Retrotransposons[END_REF]. As most DNA polymerases, RT needs to recognize a primer-template duplex to initiate DNA synthesis. Analysis of HIV RT DNA polymerisation ex vivo indicates that it is a rather slow enzyme, adding about 70 nucleotides per minute [START_REF] Thomas | Determination of the ex vivo rates of human immunodeficiency virus type 1 reverse transcription by using novel strand-specific amplification analysis[END_REF]. Additionally viral RTs seem to be relatively error-prone polymerases, lacking proof-reading activity and retroviral mutation rates being on average about 1.5 × 10 -5 mutations/bp/cycle in vivo (reviewed in [START_REF] Svarovskaia | Retroviral mutation rates and reverse transcriptase fidelity[END_REF]. The RNase H domain of RT is responsible for the degradation of the RNA-DNA duplexes that are created during reverse transcription, it is an endonuclease that is able to cleave RNA specifically when it is associated to DNA [START_REF] Mölling | Association of viral reverse transcriptase with an enzyme degrading the rna moiety of RNA-DNA hybrids[END_REF]. Indeed, RNase H does not seem to have any sequence specificity for its cleavage, instead recognizing the particular 3D structure of RNA/DNA duplexes [START_REF] Nowotny | Crystal structures of RNase H bound to an RNA/DNA hybrid: Substrate specificity and metal-dependent catalysis[END_REF].

Once the retroviral genome has been reverse-transcribed into a double-stranded DNA form, it must insert into the genome of the host cell to allow viral replication. This process is allowed by the IN protein and known as integration (see Fig. 14 and 2.1.3.c.). IN is composed of three separate domains, the NTD which contains an invariant HHCC motif and is implicated in LTR recognition, the catalytic core domain (CCD) which contains a DX 35 D catalytic site, and the CTD which shows a conserved structure with low sequence identity and is involved in DNA binding [START_REF] Engelman | Identification of conserved amino acid residues critical for human immunodeficiency virus type 1 integrase function in vitro[END_REF][START_REF] Dyda | Crystal structure of the catalytic domain of HIV-1 integrase: similarity to other polynucleotidyl transferases[END_REF][START_REF] Lesbats | Retroviral DNA Integration[END_REF]. The CCD catalyzes the two reactions needed for integration, during 3'-processing it cleaves a di-or trinucleotide off the ends of the viral non-inserted dsDNA genome, generating invariable CA dinucleotides with an exposed reactive -OH group, then during strand transfer it uses these reactive hydroxyl groups to ligate the 5' and 3' ends of the viral genome with the host genome [START_REF] Brown | Retroviral integration: structure of the initial covalent product and its precursor, and a role for the viral IN protein[END_REF][START_REF] Craigie | The IN protein of Moloney murine leukemia virus processes the viral DNA ends and accomplishes their integration in vitro[END_REF]. The two ligations occur at a distance of four to six nucleotides from each other, depending on the viral species. The strand transfer step only ligates one of the two strands of the viral genome to one strand of host genome, resulting in a single strand break that is then repaired by the host cell DNA repair mechanism and resulting in the characteristic target site duplication (TSD, see Fig. 14 and 2.1.3.c., reviewed in Lesbats et al., 2016).

d. Env

Retroviral envelope proteins are transmembrane glycoproteins that are embedded in the lipid envelope of the virion. Since they are of special interest in the present study, a detailed description will be provided in 3.1, additionally the following does not apply to the Env of epsilonretroviruses, which seem to display significant differences to all other orthoretroviral Env (see Rovnak et al., 2007, as well as 2.1.2.a.). The env ORF is the most 3' of the three major retroviral ORFs and differs from gag and pol as it is not expressed as part of the retroviral polyproteins generated from a genomic RNA. Env is expressed from a subgenomic transcript that is initiated in the 5' LTR and spliced between a donor site shortly after the LTR and an acceptor site shortly before env. The transcript terminates in the 3' LTR at the polyadenylation site, as is usual for retroviral transcripts. Env is composed of two domains that are cleaved by cellular furin-like proteases but remain associated. The surface subunit (SU) is bound to the transmembrane subunit (TM) which contains a helix domain anchoring Env in the viral membrane. They are type 1 viral fusion proteins with the SU being implicated in receptor recognition and binding, and the TM containing a hydrophobic fusion peptide that, alongside a number of conformational changes, allows it to induce fusion of the viral and cellular membranes (see 3.1.2, reviewed in Coffin et al., 1997;Henzy & Johnson, 2013;[START_REF] Podbilewicz | Virus and cell fusion mechanisms[END_REF][START_REF] Harrison | Viral membrane fusion[END_REF]. The association between the SU and TM subunits can be either covalent or non-covalent, which splits Env into gamma-type and beta-type respectively (reviewed in Henzy & Johnson, 2013).

e. Regulatory and accessory proteins

Regulatory and accessory (or auxiliary) proteins are found in complex retroviruses and are expressed in addition to the main proteins described above. They are present in lentiviruses such as HIV and deltaviruses such as human T-lymphotropic virus (HTLV, which is now officially called primate T-lymphotropic virus, PTLV), as well as some in some Spumaretrovirinae (reviewed in [START_REF] Coffin | Retroviruses. Cold Spring Harbor[END_REF][START_REF] Faust | Making Sense of Multifunctional Proteins: Human Immunodeficiency Virus Type 1 Accessory and Regulatory Proteins and Connections to Transcription[END_REF]. HIV-1 is considered the model complex retrovirus and is by far the most studied. Its genome encodes two regulatory proteins: trans-activator (Tat) and regulation of expression of viral proteins (Rev), and four accessory proteins: virion infectivity factor (Vif), viral protein r (Vpr), viral protein u (Vpu) and negative factor (Nef). As all regulatory and accessory proteins, they are small ORFs expressed by alternative splicing and/or initiation (Fig. 9 and 10). Many functions have been linked to these proteins, but we will limit the description to their probable primary functions (for a more complete description see [START_REF] Faust | Making Sense of Multifunctional Proteins: Human Immunodeficiency Virus Type 1 Accessory and Regulatory Proteins and Connections to Transcription[END_REF]. (top) Arrangement of canonical and non-canonical ORFs in the HIV-1 genome, using the same color code as Fig. 9, with the addition of accessory and regulatory ORFs in light blue. (bottom) Canonical (a-b) and non-canonical (c-g) retroviral transcripts expressed by the HIV-1 genome, to scale with the latter. Dotted lines represent splicing events and transcripts are sorted into unspliced (a), partially spliced (b-d) or fully spliced (e-g) categories. Adapted from viralzone.expasy.org transcription machinery to allow synthesis of full length viral RNAs [START_REF] Berkhout | Tat trans-activates the human immunodeficiency virus through a nascent RNA target[END_REF][START_REF] Feinberg | The role of Tat in the human immunodeficiency virus life cycle indicates a primary effect on transcriptional elongation[END_REF]. Expression of the full complement of viral genes necessitates export of differentially spliced viral RNA from the nucleus. In HIV-1 for example, Gag and Pol are expressed from an unspliced RNA, Env, Vif, Vpr and Vpu from a spliced trancript missing gag and pol and Rev, Tat and Nef from multiply spliced transcripts missing gag, pol and most of env (Fig. 10, reviewed in [START_REF] Pollard | The HIV-1 Rev protein[END_REF]. Since the cellular machinery only exports fully spliced transcripts, only Rev, Tat and Nef should be expressed. Rev contains a nuclear localization sequence triggering its re-import into the nucleus, where it is able to bind viral RNAs by interacting with the Rev response element contained in env. After binding of viral RNA and oligomerisation, Rev can mediate export of the unspliced or partially spliced viral RNA, as it also contains a nuclear export sequence [START_REF] Malim | The HIV-1 rev transactivator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA[END_REF].

Regulatory proteins of

Accessory proteins of HIV-1. As their name indicates, accessory proteins are not always needed for viral replication, whereas expression of the regulatory proteins is necessary even in highly permissive cells [START_REF] Balliet | Distinct Effects in Primary Macrophages and Lymphocytes of the Human Immunodeficiency Virus Type 1 Accessory Genes vpr, vpu, and nef: Mutational Analysis of a Primary HIV-1 Isolate[END_REF]. The primary function of most accessory proteins is to counter specific aspects of host resistance to viral infection, thus if the cell is already permissive, no action of the accessory proteins is needed. Vif is found not only in HIV-1 but in all lentiviruses except one (equine infectious anemia virus, EIAV) and is necessary to counteract the host cell APOBEC3 restriction factors [START_REF] Sheehy | Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein[END_REF]. These cellular components are cytidine deaminases participating in the innate immune defense against retroviral infections by inducing genome wide C to U mutations [START_REF] Sheehy | Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein[END_REF][START_REF] Harris | DNA deamination mediates innate immunity to retroviral infection[END_REF]. They are counteracted by Vif which can trigger ubiquitination and proteasome degradation of APOBEC3 [START_REF] Sheehy | The antiretroviral enzyme APOBEC3G is degraded by the proteasome in response to HIV-1 Vif[END_REF]. Vpu counteracts the host restriction factor Tetherin, which binds egressing viral particles and prevents their release and dissemination in the host [START_REF] Neil | Tetherin inhibits retrovirus release and is antagonized by HIV-1 Vpu[END_REF]. It has been shown that Vpu can both bind to Tetherin and prevent its recycling to the membrane once internalized, as well as trigger its ubiquitination and lysosomal degradation [START_REF] Douglas | Vpu Directs the Degradation of the Human Immunodeficiency Virus Restriction Factor BST-2/Tetherin via a TrCP-Dependent Mechanism[END_REF][START_REF] Iwabu | HIV-1 accessory protein Vpu internalizes cell-surface BST-2/tetherin through transmembrane interactions leading to lysosomes[END_REF]. Vpr is a conserved protein among all human and primate lentiviruses and of the many reported functions, [START_REF] Faust | Making Sense of Multifunctional Proteins: Human Immunodeficiency Virus Type 1 Accessory and Regulatory Proteins and Connections to Transcription[END_REF] consider its conserved ability to stop the cell cycle in G2 phase [START_REF] Planelles | Vpr-Induced Cell Cycle Arrest Is Conserved among Primate Lentiviruses[END_REF] the primary function. This cell cycle arrest seems to be due to the interaction with and sequestration of the CRL4 DCAF1 ubiquitin ligase complex [START_REF] Hrecka | Lentiviral Vpr usurps Cul4-DDB1[VprBP] E3 ubiquitin ligase to modulate cell cycle[END_REF][START_REF] Belzile | HIV-1 Vprmediated G2 arrest involves the DDB1-CUL4AVPRBP E3 ubiquitin ligase[END_REF] and seems to allow increased LTR driven expression of viral RNA [START_REF] Goh | HIV-1 Vpr increases viral expression by manipulation of the cell cycle: A mechanism for selection of Vpr in vivo[END_REF]. Similarly to Vpr, the function of Nef during HIV-1 infection is not well-known at present, but it has repeatedly been implicated in regulating the amount of several cellular proteins (Tetherin, CD4, MHC-I) at the cell membrane (reviewed in [START_REF] Faust | Making Sense of Multifunctional Proteins: Human Immunodeficiency Virus Type 1 Accessory and Regulatory Proteins and Connections to Transcription[END_REF]. In absence of Nef the incidence of acquired immunodeficiency syndrome (AIDS) after HIV-1 infection seems delayed or absent [START_REF] Kestier | Importance of the nef gene for maintenance of high virus loads and for development of AIDS[END_REF][START_REF] Kirchhoff | Absence of Intact nef Sequences in a Long-Term Survivor with Nonprogressive HIV-1 Infection[END_REF].

Regulatory and Accessory proteins in other orthoretroviruses. Interestingly, the presence or absence as well as nature of retroviral regulatory and accessory proteins is very variable even at the intragenus level. As stated above, some, like Vif, are common to almost all lentiviruses, others, like Vpr, seem to be conserved in the primate subset of lentiviruses, but most are HIV-1 specific. Even when accessory proteins are present across the genus, it does not necessarily mean that their function is identical. Simian immunodeficiency virus (SIV) does not present the Vpu gene, instead Nef is the major actor of Tetherin counteraction here [START_REF] Jia | Species-Specific Activity of SIV Nef and HIV-1 Vpu in Overcoming Restriction by Tetherin/BST2[END_REF]. Similarly HIV-2 has no Vpu and while it does express Nef, the anti-tetherin action is carried by Env, one of the canonical retroviral proteins that displays an additional role in this virus (Le [START_REF] Tortorec | Antagonism to and intracellular sequestration of human tetherin by the human immunodeficiency virus type 2 envelope glycoprotein[END_REF]. In both SIV and HIV-2, the mechanism of Tetherin escape also differs from the one employed by HIV-1 Vpu, being limited to intracellular sequestration instead of degradation (reviewed in [START_REF] Sauter | Counteraction of the multifunctional restriction factor tetherin[END_REF]. While the organization and function of major retroviral genes is rather similar, obviously deriving from a very old common ancestor, the variability of accessory proteins indicates a more recent origin of these genes, denoting a particular adaptation to host defenses and physiology.

The other major orthoretroviral genus containing additional proteins are the deltaretroviruses, represented by HTLV-1. Here again, the full spectrum of functions ascribed to these proteins is too large to be in the scope of this work, and only the basics will be discussed (for a more complete description see [START_REF] Edwards | Orf-I and Orf-II-encoded proteins in HTLV-1 infection and persistence[END_REF][START_REF] Giam | HTLV-1 Infection and Adult T-Cell Leukemia/Lymphoma-A Tale of Two Proteins: Tax and HBZ[END_REF]. The genome of HTLV-1 contains three regulatory genes: Trans-activating transcriptional regulator (Tax), Rev homolog (Rex) and HTLV-1 basic zipper factor (HBZ), and three accessory genes: p12, p13 and p30. Tax and Rex are expressed by alternative double splicing events, similarly to Tat and Rev in HIV-1 (Fig. 10) and their functions are similar. Tax is a strong trans-activator of viral gene expression (similarly to Tat), but also hyper-activates the NF-κB signaling pathway, induces genome instability and promotes host cell-cycle progression. In fact the effects of Tax on host cell physiology are so wide-reaching that its expression alone is sufficient to trigger cellular transformation ex vivo (reviewed in [START_REF] Matsuoka | Human T-cell leukaemia virus type 1 (HTLV-1) infectivity and cellular transformation[END_REF][START_REF] Giam | HTLV-1 Infection and Adult T-Cell Leukemia/Lymphoma-A Tale of Two Proteins: Tax and HBZ[END_REF].Rex, as its name indicates, plays a similar role to Rev, allowing export of unspliced or partially spliced viral transcripts from the nucleus after recognition of a specific Rex response element (reviewed in [START_REF] Younis | The human T-cell leukemia virus Rex protein[END_REF]. The HBZ gene of HTLV-1 is very particular, being located on the antisense strand of the viral genome and expressed under action of the 3' LTR [START_REF] Gaudray | The complementary strand of the human T-cell leukemia virus type 1 RNA genome encodes a bZIP transcription factor that down-regulates viral transcription[END_REF]. HBZ counteracts many of the activities of Tax and Rex, inhibiting viral transcription. While this may seem counterproductive, it is probably implicated in the establishment of viral latency and the persistence of HTLV-1 infections [START_REF] Matsuoka | Human T-cell leukaemia virus type 1 (HTLV-1) infectivity and cellular transformation[END_REF]Giam & Semmes, 2016, reviewed in). p12 and its protease-cleaved form p8 seem to provide protection from the innate immune system, for example by downregulating expression of MHC-I and T-cell activation. p13 and p30 seem to dampen the effects of Tax on the host cell, p30 can for example prevent export of tax/rex mRNA from the nucleus while p13 prevents interaction of Tax with host cell transcription factors. Additionally, all of the accessory proteins of HTLV-1 seem to be able to induce proliferation of resting T-cells, the target of HTLV-1 infection (reviewed in [START_REF] Edwards | Orf-I and Orf-II-encoded proteins in HTLV-1 infection and persistence[END_REF].

Retroviral phylogeny

The study of retroviral phylogeny is primarily based on the comparison of conserved domains of the pol gene, as RT contains a number of highly conserved motifs [START_REF] Mcclure | Sequence comparisons 209 of retroviral proteins: relative rates of change and general phylogeny[END_REF][START_REF] Poch | Identification of four conserved motifs among the RNA-dependent polymerase encoding elements[END_REF]. Alignments of these RT domains sort retroviruses into two subfamilies and several genera (Fig. 11A). In addition to RT based classification, the TM subunit of Env contains conserved motifs and can also be used for phylogeny purposes (Fig. 11B). The trees established using RT or TM do not overlap, as recombination events have led to associations of Pol and Env from different retroviral genera (see MMTV and SMRV in Fig. 11 and Bénit et al., 2001, for examples). A potent tool for investigating the evolution of the retroviral lineage are endogenous retroviruses, as they can be likened to fossil remains of ancient retroviruses which can be compared to the genomes of extant retroviruses, most of which are very young [START_REF] Jern | Use of endogenous retroviral sequences (ERVs) and structural markers for retroviral phylogenetic inference and taxonomy[END_REF][START_REF] Feschotte | Endogenous viruses: Insights into viral evolution and impact on host biology[END_REF]. The following section will contain a brief overview of the different retroviral clades as they are currently defined by the ICTV (accessible at https://talk.ictvonline.org/taxonomy/). For each genus the type species is named alongside the size of its genome, whether it is a simple or complex retrovirus and the type of Env it presents.

a. Subfamily Orthoretrovirinae

Alpharetrovirus Type species: avian leukosis virus (ALV): 7.4 kb, simple, avian gamma-type

Env

The genus Alpharetrovirus contains 9 species of oncogenic avian retroviruses, among which ALV and RSV. In the canonical alpharetroviral genome, pro is part of the gag ORF, situated at the 3' end but in the same reading frame (Fig. 9B). This leads to a unique case among retroviruses in which Pro is expressed at a much higher level, similar to that of Gag (reviewed in [START_REF] Konvalinka | Retroviral proteases and their roles in virion maturation[END_REF]. Compensating this higher level of expression is a mutation in the active site of RSV Pro which reduces enzymatic activity. If this mutation is reversed, the higher activity combined with the higher expression provoke aberrant virion morphology [START_REF] Konvalinka | An engineered retroviral proteinase from myeloblastosis associated virus acquires pH dependence and substrate specificity of the HIV-1 proteinase[END_REF][START_REF] Sedlacek | Myeloblastosis Associated Virus (MAV) Proteinase Site-Mutated to Be HIV-like Has a Higher Activity and Allows Production of Infectious but Morphologically Altered Virus[END_REF]. The Env protein of alpharetroviruses is a variant of the more common gamma-type. In avian gamma-type Env, rather than being located directly at the border of SU and TM, the fusion peptide is situated about nine amino acids after the cleavage site and is flanked by cysteines that form a disulfide bond [START_REF] Gallaher | Similar Structural Models of the Transmembrane Proteins of Ebola and Avian Sarcoma Viruses[END_REF].

Betaretrovirus Type species: mouse mammary tumor virus (MMTV): 8.8 kb, simple, beta-type Env There are 5 decribed species of betaretroviruses, among which MMTV, Mason-Pfizer monkey virus (MPMV) and the much studied Jaagskiete sheep retrovirus (JSRV). In betaretroviruses, pro is contained in its own ORF between gag and pol but separated from both by a frameshift (Fig. 9B). While most of the viruses in this genus present a beta-type Env, some like MPMV seem to have acquired a gamma-type Env by recombination events during their evolutionary history. These viruses cluster with other betaretroviruses on an RT tree, but very clearly with gammaretroviruses on a TM tree [START_REF] Sonigo | Nucleotide sequence of Mason-Pfizer monkey virus: An immunosuppressive D-type retrovirus[END_REF]. Betaretroviral genomes seem to display a bias towards A and T nucleotides [START_REF] Berkhout | Tat trans-activates the human immunodeficiency virus through a nascent RNA target[END_REF][START_REF] Jern | Use of endogenous retroviral sequences (ERVs) and structural markers for retroviral phylogenetic inference and taxonomy[END_REF].

0.5 C A E V P T L V -1 H I V -2 W E H V -1 SF V W E H V -2 H I V -1 M M T V F L V FF V E F V E IA V RSV M P M V A M V A L V P T L V -2 W D S V S M R V B F V R E V BLV K oR V J S R V M L V G a L V R D 1 1 4 β α α δ δ ε ε γ γ λ λ spuma spuma 0.9 H IV -2 F F V P T L V -2 Ga LV P T L V -1 J S R V B L V FLV W E H V -2 WE HV -1 ALV M M T V S M R V S F V R D 1 1 4 HIV -1 R S V W D S V BFV E I A V E F V M P M V C A E V K o R V REV M L V AMV β-type CX n C -ISD γ-type CX 6 CC +ISD β β A B TM RT
Gammaretrovirus Type species: murine leukemia virus (MLV): 8.3 kb, simple, gamma-type

Env

Gammaretroviruses are the largest genus with 18 described species, among which feline leukemia virus (FLV), gibbon ape leukemia virus (GaLV) and Koala retrovirus (KoRV). In this prolific genus, pro is attached 5' to the pol ORF and in the same frame as gag but separated by a stop codon (Fig. 9B). By definition, gammaretroviruses present gamma-type envelope genes which seem to confer a broader range of potential hosts than beta-type Env, as gammaretroviruses and their envelopes are found not only in mammals but also in birds, reptiles, amphibians and fish (Henzy & Johnson, 2013, reviewed in). It is also noteworthy in the context of this work that most syncytins are of probable gammaretroviral origin (see 3.3.2).

Deltaretrovirus Type species: bovine leukemia virus (BLV): 8.4kb, complex, gamma-type Env In addition to BLV, this genus contains the primate retroviruses HTLV-1 to 3. All deltaretroviruses have gag and pol genes that are more similar to those of betaroviruses and present pro as its own ORF (Fig. 9B), however their Env is clearly gamma-type, indicating that this genus arose from a recombination event (Fig. 11, reviewed in Henzy & Johnson, 2013). It has long been thought that no endogenous deltaretroviral elements existed, but [START_REF] Farkašová | Discovery of an endogenous Deltaretrovirus in the genome of long-fingered bats (Chiroptera: Miniopteridae)[END_REF] describe a provirus in long-fingered bats that seems to belong to this family. It is important to note that this provirus is highly incomplete, having lost NC and the entirety of pro, pol and env, and that the classification into deltaretroviruses is mostly based on the high similarity of Gag to HTLV-1 Gag, the presence of two putative regulatory protein ORFs, and the nucleotide bias that is characteristic of known deltaretroviral sequences (an over-representation of C, [START_REF] Kypr | Nucleotide composition bias and CpG dinucleotide content in the genomes of HIV and HTLV[END_REF][START_REF] Berkhout | Codon and amino acid usage in retroviral genomes is consistent with virus-specific nucleotide pressure[END_REF]. While it is frustrating to not have access to the complete genome of this deltaretroviral fossil, it suggest that features such as regulatory genes where already present about 20 Mya in this clade [START_REF] Farkašová | Discovery of an endogenous Deltaretrovirus in the genome of long-fingered bats (Chiroptera: Miniopteridae)[END_REF]. In any case, deltaretroviruses are conspicuously under-represented in endogenous elements [START_REF] Hayward | Pan-vertebrate comparative genomics unmasks retrovirus macroevolution[END_REF].

Epsilonretrovirus Type species: walleye dermal sarcoma virus (WDSV): 12.7 kb, complex, epsilon-type Env This smallest and probably least well know retroviral genus only contains three species: WDSV and walleye epidermal hyperplasia virus 1 and 2. The relative position of pro in relation to gag and env is similar to that in the more well known gammaretroviruses: gag is in the same reading frame as pro-pol but the two ORFs are separated by a stop codon. The Env of epsilonretroviruses however, displays major differences with both beta-and gamma-type Env. With about 1200 amino acids, the WDSV Env is approximately twice as large as classical orthoretroviral Env. WDSV Env seems to contain at least two transmembrane domains (as opposed to one for other retroviral genera) and presents an internal membrane addressing signal in the intra-cellular loop between the two transmembrane domains. This is coherent with the observation of assembled virions associated with intracellular membranes ex vivo [START_REF] Rovnak | Establishment of productively infected walleye dermal sarcoma explant cells[END_REF]. Additionally these viruses are complex retroviruses, displaying three additional reading frames that code for proteins implicated in viral gene expression, cell proliferation and regulation of apoptosis (reviewed in [START_REF] Rovnak | Walleye dermal sarcoma virus: Molecular biology and oncogenesis[END_REF]. While the only three known extant epsilonretroviruses are fish viruses, the analysis of endogenous elements bearing similarities to this genus suggest that this family of viruses used to infect mammals, reptiles and amphibians as well [START_REF] Brown | Multiple Groups of Endogenous Epsilon-Like Retroviruses Conserved across Primates[END_REF]. Three more fish retroviruses are considered probable members of this species (snakehead retrovirus, salmon swimbladder sarcoma virus, perch hyperplasia virus, reviewed in [START_REF] Rovnak | Walleye dermal sarcoma virus: Molecular biology and oncogenesis[END_REF].

Lentivirus Type species: human immunodeficiency virus 1 (HIV-1): 9.2 kb, complex, betatype Env Among the 10 lentiviral species is the without a doubt most well-known human retrovirus: HIV, the causative agent of AIDS. Due to its important impact on human society, HIV-1 is probably the most actively studied retrovirus, especially with regards to regulatory and accessory proteins, and 3D structures. In lentiviruses, pro-pol is in a shifted -1 reading frame compared to gag (Fig. 9B). Additionally the mature virion displays a characteristic conical arrangement of CA proteins (Fig. 16A, [START_REF] Woodward | Electron Cryotomography Studies of Maturing HIV-1 Particles Reveal the Assembly Pathway of the Viral Core[END_REF]. As with deltaretroviruses, endogenous lentiviruses have been described only recently [START_REF] Katzourakis | Discovery and analysis of the first endogenous lentivirus[END_REF] and seem to be a rare occurence [START_REF] Hayward | Pan-vertebrate comparative genomics unmasks retrovirus macroevolution[END_REF]. Lentiviral genomes show a bias towards A nucleotides [START_REF] Kypr | Nucleotide composition bias and CpG dinucleotide content in the genomes of HIV and HTLV[END_REF][START_REF] Berkhout | Codon and amino acid usage in retroviral genomes is consistent with virus-specific nucleotide pressure[END_REF] and contrary to all other retroviral genera, Lentivirus do not contain any oncogenic viruses.

b. Subfamily Spumaretrovirinae

Spumaviruses have long been know to display characteristics setting them apart from other retroviruses, and in 2002 they were officially sorted into a subfamily separate from all other retroviruses [START_REF] Mayo | ICTV at the Paris ICV: Results of the plenary session and the binomial ballot[END_REF]. The Spumaretrovirinae subfamily contains a single genus, Spumavirus. A recently accepted proposal by [START_REF] Khan | Spumaretroviruses: Updated taxonomy and nomenclature[END_REF] introduces major changes to the phylogeny of this family, splitting it into five genera (according to hosts, as spumaretroviruses co-speciate with their hosts: Simiispumavirus, Prosimiispumavirus, Bovispumavirus, Felispumavirus, Equispumavirus) and updating the naming conventions. While the ICTV accepted this proposal, it has not been ratified yet and since spumaretroviruses are of limited relevance to the present study, this section will not delve into the differences between the new genera.

The type species for the soon to be defunct Spumavirus genus is the simian foamy virus (SFV): 13.3 kb, complex. According to [START_REF] Khan | Spumaretroviruses: Updated taxonomy and nomenclature[END_REF], SFV should be renamed to eastern chimpanzee simian foamy virus and become the type species of Simiispumavirus.

2.1

The differences between spumaretroviruses and orthoretroviruses start with the genomic organisation, the LTRs are much larger (about 1700 nts), there is an additional transcription initiation site (an internal promoter in the 3' region of env) and pro-pol is expressed as a spliced transcript (reviewed in [START_REF] Rethwilm | Molecular biology of foamy viruses[END_REF]. Transcription starts at the internal promoter, which is basally active, and results in the production of the anti-APOBEC accessory protein "bel1 and bel two" (Bet, [START_REF] Löchelt | The antiretroviral activity of APOBEC3 is inhibited by the foamy virus accessory Bet protein[END_REF] and the regulatory protein trans-activator of spumaretroviruses (Tas). The latter then both increases transcription from the internal promoter and initiates 5' LTR driven transcription to allow expression of the other retroviral genes [START_REF] Keller | Characterization of the transcriptional trans activator of human foamy retrovirus[END_REF][START_REF] Löchelt | Human foamy virus genome possesses an internal, Bel-1-dependent and functional promoter[END_REF]. Even the canonical retroviral genes gag, pol and env present characteristics that set them apart from those of orthoretroviruses. Spumaretroviral Gag does not present the highly conserved MHR [START_REF] Orlinsky | Mutations in the Ty3 major homology region affect multiple steps in Ty3 retrotransposition[END_REF] and is not cleaved into three proteins, instead remaining mostly intact and conferring an immature appearance to virions, even though they are infectious (reviewed in [START_REF] Rethwilm | Molecular biology of foamy viruses[END_REF]. Additionally Gag is unable to form VLPs on its own, the presence of Env is necessary to obtain particles [START_REF] Fischer | Foamy virus particle formation[END_REF]. The pro ORF is part of the pol ORF in spumaretroviruses, but there is only a single cleavage event, resulting in a PR-RT and an IN molecule. The latter also acts differently than in orthoretroviruses during 3' processing, only cleaving the 5' part of the sequence so that once inserted the 5' LTR does start with TG, but the 3' LTR does not necessarily end in CA [START_REF] Enssle | An active foamy virus integrase is required for virus replication[END_REF]. Finally, Env is expressed in an inside-out fashion when compared to orthoretroviruses, with the N-terminal end facing the cytoplasm rather than the lumen of the endoplasmic reticulum (ER). Additionally the exceptionally long signal peptide that adresses the protein to the ER during synthesis is not degraded but instead forms an integral part of the functional, tri-partite spumaretroviral Env [START_REF] Lindemann | A particle-associated glycoprotein signal peptide essential for virus maturation and infectivity[END_REF].

Beyond the structural differences, one of the major factors distinguishing Spumaretrovirinae and Orthoretrovirinae is the timing of reverse transcription. In the latter, this step occurs just after viral entry into a new cell, the infectious genome is RNA (see Fig. 12 and2.1.3), while in spumaretroviruses the reverse transcription step occurs between assembly and entry into new cells, meaning that the infectious form of the genome is DNA [START_REF] Yu | Evidence that the human foamy virus genome is DNA[END_REF]. This timing of reverse transcription is remarkably similar to that observed in Hepadnaviridae, one of the other rare families of reverse transcribing viruses. This resemblance to a non-retrovirus is even more intriguing when taking into account the proposed basal location of spumaretroviruses in the retroviral lineage [START_REF] Jern | Use of endogenous retroviral sequences (ERVs) and structural markers for retroviral phylogenetic inference and taxonomy[END_REF][START_REF] Hayward | Pan-vertebrate comparative genomics unmasks retrovirus macroevolution[END_REF], a hypothesis that was recently strengthened by the discovery of 450 My old spumaretrovirus-like endogenous elements, by far the most ancient known retroviral sequences (Aiewsakun & Katzourakis, 2017). Considering Spumaretrovirinae as the most ancestral retroviruses would suggest that reduction of genome size, use of a single promoter and non-complex genomes are all derived characteristics in retroviral evolution.

Viral cycle a. Viral entry

As might be expected, the first essential step of retroviral infection is the entry of the viral particle into the adequate host cell (Fig. 12, and for HIV-1 it has been shown that the penetration of a single virion can be enough to trigger acute infection [START_REF] Keele | Identification and characterization of transmitted and early founder virus envelopes in primary HIV-1 infection[END_REF]. For retroviruses, viral entry is mediated by the type I fusion protein Env, and can be decomposed into two steps, which will be considered briefly here and in much more detail in 3.1.2. First comes an attachment step, in which the SU subunits of the Env protein trimers present at the surface of the lipid viral envelope recognize and bind specific receptors present on the target cells. This SU/receptor interaction frees the metastable TM trimer which extends its hydrophobic fusion peptides, inserting them into the target membrane (reviewed in [START_REF] Podbilewicz | Virus and cell fusion mechanisms[END_REF][START_REF] Harrison | Viral membrane fusion[END_REF]. After the attachment step, the TM trimer changes conformation and brings the two membranes close enough to induce merging of the lipid layers and membrane fusion. Depending on the retroviral species, this process can further necessitate acidification of the environment, which implies fusion in an endosome rather than at the plasmic cell membrane (reviewed in [START_REF] Podbilewicz | Virus and cell fusion mechanisms[END_REF][START_REF] Harrison | Viral membrane fusion[END_REF]. The SU/receptor interaction that confers its tropism and specificity to the virus is absolutely essential for infection. Viruses can bind to cells not presenting the correct receptor, and even enter the cell, but they are unable to initiate infection if viral entry is not mediated by Env [START_REF] Maréchal | Cytosolic Gag p24 as an index of productive entry of human immunodeficiency virus type 1[END_REF][START_REF] Pizzato | Initial binding of murine leukemia virus particles to cells does not require specific Env-receptor interaction[END_REF][START_REF] Pizzato | Evidence for nonspecific adsorption of targeted retrovirus vector particles to cells[END_REF]. Following fusion of the viral and cellular membranes, the capsid which contains the viral genome and enzymes is released into the cytoplasm.

b. Reverse transcription

Once the capsid enters the cytoplasm of the host cell, it does not immediately disassemble, instead it and its contents form the so-called reverse transcription complex (RTC) inside which reverse transcription occurs (Fig. 12), though parts of the capsid dissociate from the RTC during this step, in a process called uncoating [START_REF] Fassati | Characterization of intracellular reverse transcription complexes of Moloney murine leukemia virus[END_REF], 2001). The amount of dissociation and its importance for viral replication remain disputed, however the presence of a proteic shell around the RTC presents obvious theoretical advantages for the virus, such as keeping all necessary enzymes as well as the nucleic acid substrate confined in a small volume (reviewed in [START_REF] Hughes | Reverse Transcription of Retroviruses and LTR Retrotransposons[END_REF]. Additionally, preservation of the capsid might prevent recognition of viral double-stranded DNA in the cytoplasm by host cell sensors, a strong signal of viral infection.

In HIV-1 it has been show that CA proteins can interact with host cell factors to shield the viral DNA genome from the innate immune system [START_REF] Rasaiyaah | HIV-1 evades innate immune recognition through specific cofactor recruitment[END_REF]. Inside the CA shell, the RTC contains two copies of the viral RNA genome which are bound to NC proteins, RT and the other viral proteins like IN, whose potential role in reverse transcription is less well known (reviewed in [START_REF] Hughes | Reverse Transcription of Retroviruses and LTR Retrotransposons[END_REF]. The virion always contains 2 copies of the RNA genome [START_REF] Nikolaitchik | Dimeric RNA Recognition Regulates HIV-1 Genome Packaging[END_REF], even though only a single one is needed for reverse transcription. The infecting virion attaches to the host cell membrane through Env-receptor interaction which also triggers fusion and viral entry. In the cytoplasm, the RNA genome is reverse transcribed into DNA within the reverse transcription complex (RTC). The DNA genome is uncoated (dissociation of the capsid) forming the pre-integration complex (PIC), which is imported into the nucleus. In the nucleus the viral genome is integrated into the cellular genome and viral transcripts are expressed by the host machinery. Viral proteins are synthesized in the cytoplasm or the endoplasmic reticulum/Golgi (Env only) and are addressed towards the plasmic membrane. At the membrane the Gag containing polyproteins assemble and bind two copies of viral genomic RNA. The assembly buds and takes with it an Env containing fragment of the cellular membrane, releasing an immature virion. Finally, PR provokes virion maturation which yields a new infectious virion.

Colors are the same used in Fig. 9.

The presence of a second copy probably serves as a back-up in case of nicked or otherwise damaged RNA, but the important consequences of the presence of two RNA genomes in the RTC are the potential recombination events that can occur if the RT switches template during reverse transcription and the two packaged copies differ [START_REF] Hu | Genetic consequences of packaging two RNA genomes in one retroviral particle: pseudodiploidy and high rate of genetic recombination[END_REF]. The presence of NC is necessary for reverse transcription, probably as its nucleic acid binding properties allow it to assist the RT by preventing formation of secondary structures that would negatively impact reverse transcription (reviewed in [START_REF] Hughes | Reverse Transcription of Retroviruses and LTR Retrotransposons[END_REF].

Reverse transcription itself is a rather complicated process in which the RT must perform both DNA polymerization using RNA and DNA templates, and RNA/DNA hybrid degradation through its RNase H activity. The different steps are reviewed in [START_REF] Hughes | Reverse Transcription of Retroviruses and LTR Retrotransposons[END_REF] and will be summed up here as well as presented in Fig. 13.

1. A cellular, previously encapsidated, tRNA with a complementary 3' sequence binds the PBS on the viral plus-strand RNA genome, serving as primer.

2. RT uses the resulting tRNA/RNA duplex as primer to start synthesis of the minus-strand DNA genome. This results in the generation of a short 5'-U5-R-3' DNA fragment, at which point RT has reached the 5' end of the viral genome. This creates a DNA/RNA hybrid and provokes RNase H mediated degradation of the copied RNA sequence, the 5' end of the RNA genome is now the PBS. This frees the associated DNA.

3. Since the R sequence is by definition found at both ends of the genome, the synthesized 5'-U5-R-3' DNA fragment transfers to the 3' R sequence, giving rise to a new RNA/DNA hybrid that can be elongated. The RT resumes DNA minus-strand synthesis and extends this strand up to the current 5' end of the RNA genome, the PBS.

4. RNase H activity degrades the remaining RNA genome which is now completely paired to the minus-strand DNA. In the process it spares the relatively RNase H resistant PPT.

5. The PPT sequence now forms an isolated RNA/DNA duplexe which is used as primers for plus-strand synthesis. This generates a plus-strand RNA/DNA 5'-PPT-U3-R-U5-PBS-3' fragment, at which point the 3' end of the minus-strand DNA is reached. The tRNA sequence of the minus-strand is now associated with the DNA PBS sequence of the plusstrand and is thus degraded by RNase H. Similarly, by the time plus-strand synthesis has reached the 3' end of the genome, RNase H has degraded the RNA PPT sequence, generating a fully DNA 5'-U3-R-U5-PBS-3' plus-strand fragment.

2.1

R R U5 R PBS PPT U3 R PBS PPT U3 R U5 R PBS PPT U3 R PBS U5 U5 PPT U3 R PBS U5 R gag pol env PPT U3 R U5 R U5 R PBS PPT U3 PBS PPT U3 U5 R U5 R PBS PPT U3 U5 R U3 PBS PPT U3 U5 U5 R PBS PPT U3 U5 R U3 PBS PPT U3 R U5 U3 R 1.

2.

3.

4.

5.

6.

Figure 13 -Schematic representation of the reverse transcription steps. RNA is shown in green with the polypurine tract (PPT) in dark green, plus-strand DNA is shown in purple and minus-strand in red. A dotted line represent RNase H mediated RNA degradation and the position of the indicated genomic elements is not to scale. For simplicity gag, pol and env are only displayed for the first step, though they are present throughout. (1.) A complimentary tRNA (green arrow with loops) anneals to the primer binding site (PBS). ( 2.) RT initiates DNA minus-strand synthesis and copies the viral RNA genome to its R extremity. The resulting RNA/DNA duplex is degraded by RT RNase H activity. ( 3.) The minus-strand DNA dissociates and re-associated to the 3' R segment of the viral RNA. (4.) RT elongates the minus-strand DNA until reaching the 5' extremity of the RNA genome. This forms another DNA/RNA duplex that is degraded by RNase H activity, except for the more resistant PPT. ( 4.) The PPT forms an RNA/DNA duplex able to serve as primer for plus-strand synthesis, RT elongates this strand until reaching the end of the PBS-complementary sequence of the tRNA sill attached to the minus-strand. This creates an RNA/DNA duplex and the tRNA is degraded. During synthesis, the PPT is finally degraded (5.) The nascent plus-dissociates and re-associates to the minus-strand PBS using its PBS-complementary 5' end. This allows the RT to finish elongating both DNA strands and obtain a double stranded U3RU5-gag-pol-env-U3RU5 DNA genome. Adapted from [START_REF] Hughes | Reverse Transcription of Retroviruses and LTR Retrotransposons[END_REF].

Obviously, this complex reaction requires the RT to constantly discriminate between its elongation and degradation functions. Using an in vitro method, Abbondanzieri et al. (2008) show that the binding orientation of RT is highly dependent on the structure of the nucleic acid it binds to. When using a DNA/DNA primer/template complex, RT orients itself so that the DNA polymerisation site is close to the 3' end of the primer, and the RNase H domain is facing the other way. With an RNA/DNA primer/template complex, the RNase H domain is located close to the 3' end of the primer, while the polymerisation site is close to the 5' end. Additionally, when using the PPT sequence as RNA primer, RT is oriented in RNase H configuration if the nucleotides following the PPT sequence are RNA, but if there are no nucleotides 3' of the PPT or they are DNA, RT is able to quickly transition between both orientations, perhaps explaining how it is able to both use the PPT as primer for plus-strand synthesis but to then also cleave the PPT from the following DNA. Addition of complementary nucleotides (but not non-complementary) are able to stabilize the RT in polymerisation conformation (Abbondanzieri et al., 2008).

Depending on the retrovirus, the active form of RT differs. While in MLV RT acts as a simple monomer [START_REF] Roth | Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli[END_REF], in ALV it can assemble as a monomer, a heterodimer, or a homodimer, and in HIV RT forms a heterodimer. In ALV the three forms that have been decribed are an RT monomer, an RT/(RT+IN) heterodimer and an (RT+IN)/(RT+IN) dimer [START_REF] Hizi | RNA-dependent DNA polymerase of avian sarcoma virus B77. II. Comparison of the catalytic properties of the alpha, beta2, and alphabeta enzyme forms[END_REF]. While the heterodimer seems to be the predominant form in purified preparations, all three might play a role in viral replicationas they display distinct enzymatic characteristics, especially concerning RNase H activity [START_REF] Hizi | DNA-processing activities associated with the purified α, β2, and αβ molecular forms of avian sarcoma virus RNA-dependent DNA polymerase[END_REF]. The HIV RT is by far the best characterized, forming a heterodimer with a large subunit that consists of the complete RT and a smaller subunit in which a protease cleavage removed almost all the RNase H domain [START_REF] Lightfoote | Structural characterization of reverse transcriptase and endonuclease polypeptides of the acquired immunodeficiency syndrome retrovirus[END_REF][START_REF] Kohlstaedt | Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor[END_REF]. The conformation of both subunits is very different, even though most of the sequence is shared, and the large subunit is the only one to display an active polymerase and RNaseH site, indicating that the smaller incomplete subunit plays a structural role [START_REF] Kohlstaedt | Crystal structure at 3.5 A resolution of HIV-1 reverse transcriptase complexed with an inhibitor[END_REF]. Interestingly, the RT of the distantly related yeast retrotransposon Ty3 assumes an asymetric homodimeric structure in which each subunit displays a conformation similar to that of one of the subunits of HIV RT, although in this case it seems that polymerase and RNase H activities are carried by distinct subunits [START_REF] Nowak | Ty3 reverse transcriptase complexed with an RNA-DNA hybrid shows structural and functional asymmetry[END_REF].

c. Genome integration

Following reverse transcription, the RTC is converted into pre-integration complex (PIC, Fig. 12). The exact mechanism of this conversion and the composition of the PIC are not well known, neither are potential differences between different retroviruses, as there often is only a single copy of this complex per infected cell (reviewed in [START_REF] Hughes | Reverse Transcription of Retroviruses and LTR Retrotransposons[END_REF][START_REF] Lesbats | Retroviral DNA Integration[END_REF]. The intasome, a part of the PIC, on the other hand has been well characterized in several viral species. This structure arises from the structure and sequence specific interaction of IN multimers with the viral DNA genome and is essential for IN activity and retroviral genome integration (reviewed in [START_REF] Grawenhoff | Retroviral integrase protein and intasome nucleoprotein complex structures[END_REF]Engelman & Cherepanov, 2017). The

2.1

first intasome nucleoprotein complex to be characterized was that of the spumaretrovirus SFV [START_REF] Hare | Retroviral intasome assembly and inhibition of DNA strand transfer[END_REF], which turned out to be similar to the intasomes of orthoretroviruses, but less complex. In SFV, four IN proteins bind the two ends of the viral DNA in a symmetric fashion. Both genomic ends are sandwiched between two IN which use their own CCD alongside the other's NTD to bind the viral DNA, crosslinking the whole stucture. Both of these inner IN also use their CTD to engage host cell DNA during integration. This structure is stabilized by two outer IN that each bind to one inner IN using a CCD-CCD dimerization interface and seem to only have a structural role [START_REF] Hare | Retroviral intasome assembly and inhibition of DNA strand transfer[END_REF]. Characterization of the intasomes of MMTV, RSV, maedi-visna virus (MVV, a lentivirus) and HIV-1 revealed that they contained 8 IN for non-lentiviruses and up to 16 IN for lentiviruses [START_REF] Ballandras-Colas | Cryo-EM reveals a novel octameric integrase structure for betaretroviral intasome function[END_REF][START_REF] Yin | Crystal structure of the Rous sarcoma virus intasome[END_REF][START_REF] Ballandras-Colas | A supramolecular assembly mediates lentiviral DNA integration[END_REF]Passos et al., 2017, respectively). Interestingly, while the dimer of tetramer or tetramer of tetramer stuctures observed for these intasomes sounds very different from that observed for the SFV dimer of dimer, the positions of the four IN at the core of the structure are extremely similar, defining the intasome core as a highly conserved structure among retroviruses (reviewed in Engelman & Cherepanov, 2017). While there are no structures currently available for gamma-or epsilonretroviruses, Engelman & Cherepanov (2017) predict their intasomes to contain four IN proteins, based on sequence properties.

The intasome core catalyses two subsequent bimolecular nucleophilic substitutions (Fig. 14, Engelman et al., 1991).

1. 3'-processing, during which the inner IN use water as a nucleophil to cleave the ends of the viral DNA. This reaction forms invariant CA-3' ends with exposed hydroxil groups (the reason why all integrated LTR start in TG and end in CA). Since only the 3' ends are cleaved, a 5' overhang is generated at both ends of the DNA genome.

2. Strand transfer, during which the inner IN use the exposed hydroxyl groups of the recessed 3' ends of the viral genome to attack a phosphdiester bond of the host cell genome and link its 3' end to the 5' of host DNA. Being a bimolecular nucleophilic substitution, the breaking of the phosphodiester bond and ligation of the 3' end occur simultaneously.

There are several interesting aspects of strand transfer. First, the two phosphodiester bonds that are implicated in this reaction are always separated by a defined number of nucleotides, which is determined by the 3D structure of the intasome and thus specific of each retrovirus. The structure of the SFV intasome is such that the two attacks, each perfomed by one of the inner IN, are always four nucleotides apart, while for HIV-1 and MMTV this distance is of 5 and 6 respectively (reviewed in [START_REF] Lesbats | Retroviral DNA Integration[END_REF]. Second, strand transfer links the 3' end of the viral genome to the host genome by nicking the latter, but does not link the 5' overhangs and the newly nicked host genome ends, leaving a pair of single stranded gaps. These gaps are probably repaired by the host cell machinery, which degrades the 5' overhangs using a 5'-Flap endonuclease, fills in the 4-6 missing nucleotides generated by the staggered nucleophilic substitution and finally ligates (6 here). This binds the 3' ends of the viral genome strands to the host genome. Cellular enzymes then repair the remaining single-strand nicks, cutting the viral 5' flaps (the complementary nucleotides to those lost during 3' processing), and filling the 4-6 bp gap, generating the TSD (gree frames). The representations are not to scale. the 5' ends of viral DNA to the 3' ends of the host genome (reviewed in [START_REF] Lesbats | Retroviral DNA Integration[END_REF]. This filling in of the stagger is responsible for the TSD, a perfect repetition of a 4-6 nucleotide long sequence on each side of a newly inserted retroviral genome (Fig. 14).

The targeting of strand transfer is not a completely random process in retroviruses. While at the sequence level there is only weak indication of specificity [START_REF] Wu | Weak Palindromic Consensus Sequences Are a Common Feature Found at the Integration Target Sites of Many Retroviruses Weak Palindromic Consensus Sequences Are a Common Feature Found at the Integration Target Sites of Many Retroviruses[END_REF], at the genomic level some retroviral genera present a clear insertion bias [START_REF] Schröder | HIV-1 integration in the human genome favors active genes and local hotspots[END_REF]. Lentiviruses show a preference for actively transcribed and intron-rich genes, gammaretroviruses prefer to insert in strong enhancers and active gene promoter regions, whereas spumaretroviruses prefer to avoid actively transcribed loci. Alpha-, beta-and deltaretroviruses show little to no insertional bias, and the genome-wide distribution of epsilonretroviral insertion has not been well-studied (reviewed in [START_REF] Kvaratskhelia | Molecular mechanisms of retroviral integration site selection[END_REF][START_REF] Lesbats | Retroviral DNA Integration[END_REF]. The insertional bias in both lentiviruses and gammaretroviruses seems to be due to interactions between the PIC and cellular factors, namely LEDGF/p75 and BET respectively. These cellular factors are able to interpret the transcriptional state of host cell chromatin and bind preferentially to a particular state. Exploitation of this property by the viral PIC permits preferential insertion in parts of the genome that are more favorable to viral replication. Since the proteins and binding mechanisms involved (as well as the resulting insertional biases) are clearly distinct, usage of cellular factors to guide lenti-and gammaretroviral insertion is a noteworthy example of evolutive convergence (reviwed in [START_REF] Kvaratskhelia | Molecular mechanisms of retroviral integration site selection[END_REF][START_REF] Lesbats | Retroviral DNA Integration[END_REF].

d. Protein expression

Following integration of the retroviral genome into the cellular genome, forming the so called provirus, gene expression must occur to form new particles (Fig. 12). Viral transcripts are synthesized by the cellular RNA Pol II using the transcription initiation site in the 5' LTR and cell transcription factors or in the case of complex retroviruses regulatory proteins. Since the host cell machinery is responsible for transcription, viral RNAs are capped and polyadenylated. This transcription step yields a complete genomic RNA, which can then either be used directly for translation of gag, pro, and pol, or be spliced (reviewed in [START_REF] Coffin | Retroviruses. Cold Spring Harbor[END_REF]. As stated previously, in simple retroviruses the only spliced viral transcript is the one used for env expression while in complex retroviruses there exist several additional alternatively spliced forms that encode the regulatory and accessory proteins. In addition to serving as template for protein expression, the unspliced genomic RNA can also be packaged into new virions to serve as genome for the next infection (reviewed in [START_REF] Freed | HIV-1 assembly, release and maturation[END_REF]. The importance of unspliced or partially spliced viral RNA is problematic for viral replication, as the host cell machinery inhibits nuclear export of unspliced RNAs. Different strategies have been developed to force export of unspliced or partially spliced RNAs. In complex retroviruses this task is often accomplished by a regulatory protein such as Rev for HIV-1 [START_REF] Malim | The HIV-1 rev transactivator acts through a structured target sequence to activate nuclear export of unspliced viral mRNA[END_REF] or Rex for HTLV-1 (reviewed in [START_REF] Younis | The human T-cell leukemia virus Rex protein[END_REF]. In simple retroviruses, the constitutive transport element (CTE) sequence present between env and the 3' LTR allows export of non-spliced RNA [START_REF] Bray | A small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev-independent[END_REF]. This small sequence interacts directly with the nuclear RNA export factor 1 (NXF1)/NTF2-related export protein 1 (NXT1) family of proteins, which mediate nuclear export of the CTE containing transcript (reviewed in [START_REF] Cullen | Nuclear mRNA export: Insights from virology[END_REF].

Once exported from the nucleus, the unspliced genomic transcript needs to mediate expression of Gag, Pro and Pol. Since translation always starts at the 5' end of the transcript, the latter are expressed in the form of polyproteins: Gag-Pro and Gag-Pro-Pol, or just Gag-Pro-Pol, depending on the viral genome organization (Fig. 9). This has an important impact on relative protein levels. In most retroviruses the pol ORF is in an overlapping -1 frame relative to gag and contains the coding sequence for Pro in 5'. This is the case in HIV for example and expression of Gag-Pro-Pol occurs by ribosomal frameshifting after Gag synthesis, allowing circumvention of the Gag stop codon (for an early review see [START_REF] Brierley | Ribosomal frameshifting on viral RNAs[END_REF]. Since ribosomal frameshifting is a somewhat rare event, in these viruses the level of expression of Gag-Pro-Pol represents about 5% of the level of Gag. In other viruses like MLV, pro-pol is in a separate ORF in the same reading frame. The only way to translate Pro-Pol is stop codon readthrough, once more leading to a reduced amount of Gag-Pro-Pol expression when compared to Gag, readthrough occurring in about 7% of cases [START_REF] Irigoyen | Ribosome profiling of the retrovirus murine leukemia virus[END_REF]. Another disposition is found in HTLV, MMTV and MPMV for example, in which gag, pro and pol are each their own shifted reading frame, necessitating one or two sequential frameshifts for expression of the Gag-Pro or Gag-Pro-Pol precursor respectively. Finally, in RSV, pro is attached to gag, while pol is still in a shifted frame, leading to the expression of an unusually high amount of Pro, on par with the level of Gag and the usual lower amount of Gag-Pro-Pol.

While Gag-Pro-Pol is expressed in the cytoplasm from the unspliced transcript, Env expression occurs using the spliced sub-genomic RNA. The presence of a hydrophobic signal peptide at the 5' end of the ORF directs the nascent protein to the ER for co-translational translocation, as is common for transmembrane protein (reviewed in [START_REF] Nyathi | Co-translational targeting and translocation of proteins to the endoplasmic reticulum[END_REF]. Once synthesis is complete, the future extracellular domain of Env is in the ER lumen while its transmembrane helix is embedded in the ER membrane. At this stage Env proteins start grouping into the trimeric form that will be present at the surface of the virion. Env transits through the Golgi network in which cellular furin-like proteases separate SU and TM. This cleavage is necessary to free the fusogenic peptide at the N-terminal extremity of TM and enable the fusogenic potential of Env (reviewed in Coffin et al., 1997, chap. 2). The SU is additionally N-glycosylated before reaching the cell membrane [START_REF] Felkner | Mutational analysis of the N-linked glycosylation sites of the SU envelope protein of Moloney murine leukemia virus[END_REF][START_REF] Dedera | Role of asparagine-linked glycosylation in human immunodeficiency virus type 1 transmembrane envelope function[END_REF].

e. Assembly and budding

Once the necessary proteins and viral RNA genomes have been synthesized, they need to congregate and assemble into a highly organized structure, which then needs to bud off a cellular membrane, acquiring the viral lipid envelope and forming the immature virion which is released from the infected cell (Fig. 12). The Gag polyprotein and all three of its subunits (MA-CA-NC, see 2.1.1.b.) play a major role in this process. In most orthoretroviruses, assembly

2.1

occurs at the cellular plasmic membrane, the exception to this rule are betaretroviruses in which the capsid assembles in the cytoplasm before being transported to the membrane, this aspect will be discussed below. In non-betaretroviruses, Gag and Gag-Pro-Pol molecules traffic to the cell membrane. In HIV-1 it has been shown that this trafficking is dependent of MA and a particular membrane phosphoinositide (phosphatidylinositol PtdIns(4,5)P 2 ). Alteration of the MA sequence or depletion of PtdIns(4,5)P 2 leads to retargeting of Gag towards intracellular membranes (Ono & Freed, 2004;Ono et al., 2004). Structural studies show that the binding of HIV-1 Gag to PtdIns(4,5)P 2 triggers exposure of the myristric acid linked to Gag, which can insert into the cell membrane and stabilize the interaction [START_REF] Saad | Structural basis for targeting HIV-1 Gag proteins to the plasma membrane for virus assembly[END_REF]. Once at the membrane Gag induces the formation of lipid raft microdomains by recruiting cholesterol and sphingolipids, and the over-representation of these two species alongside PtdIns(4,5)P 2 in HIV-1 viral envelopes reflects their participation in particle formation (reviewed in [START_REF] Freed | HIV-1 assembly, release and maturation[END_REF]. While Gag multimerization can begin during the transfer from the cytoplasm, the major part of it occurs once Gag is anchored at the membrane [START_REF] Kutluay | Analysis of the Initiating Events in HIV-1 Particle Assembly and Genome Packaging[END_REF]. This oligomerization process implicates mostly CA-CA domain interactions and is increased when the NC domain of Gag binds to a nucleic acid. Gag proteins associate as a continuous network of hexameric rings to form a curved lattice that creates a bud at the membrane. In order to accommodate the amount of curvature necessary to form a 120 nm diameter sphere, the lattice of hexamers contains randomly distributed gaps (reviewed in [START_REF] Freed | HIV-1 assembly, release and maturation[END_REF][START_REF] Mattei | Retrovirus maturation -An extraordinary structural transformation[END_REF]. While the general hexameric arrangement of immature Gag seems conserved among retroviruses, the details and especially the organization of the CA NTD seem to vary [START_REF] Schur | Structure of the immature HIV-1 capsid in intact virus particles at 8.8 Å resolution[END_REF]. Since Gag-Pro-Pol polyproteins contain functional MA and CA domains, they behave the same as Gag with regards to membrane association and lattice assembly, ensuring encapsidation of PR, RT and IN in the virion through their association with Gag domains. RT can also bind to the cellular tRNA needed for initiation of reverse transcription and thus lead to its encapsidation (reviewed in [START_REF] Coffin | Retroviruses. Cold Spring Harbor[END_REF][START_REF] Hughes | Reverse Transcription of Retroviruses and LTR Retrotransposons[END_REF].

Cytoplasmic unspliced viral RNAs serve as genomes for new virions and thus have to be encapsidated during viral assembly. The trafficking of viral RNA to the cellular membrane probably implicates interaction with the NC domain of Gag, though it has been proposed that it occurs by simple diffusion [START_REF] Chen | Cytoplasmic HIV-1 RNA is mainly transported by diffusion in the presence or absence of Gag protein[END_REF]. Viral RNAs dimerize in the cytoplasm by using the dimerization initiation signal (DIS) and it is this dimer that is subsequently recognized by Gag NC, which binds to another RNA sequence called packaging signal, or ψ [START_REF] Nikolaitchik | Dimeric RNA Recognition Regulates HIV-1 Genome Packaging[END_REF]. In HIV-1, DIS sequences are in the 5' untranslated region (UTR) of the genome and are variable between strains, GCGCGC and GUGCAC being the most common [START_REF] Hussein | Delineation of the preferences and requirements of the human immunodeficiency virus type 1 dimerization initiation signal by using an in vivo cell-based selection approach[END_REF]. The ψ sequence is also contained in this 5' UTR and usually contains a region that is absent in spliced transcripts, thus ensuring encapsidation of unspliced genomic RNA (reviewed in [START_REF] Ali | Cross-and co-packaging of retroviral RNAs and their consequences[END_REF]. Additionally the PBS of unspliced viral RNAs can bind a matching tRNA primer, leading to its encapsidation. In the cytoplasm and during transfer, Gag is mainly bound to the 5' UTR and the RRE of HIV-1 viral RNA through its NC domain and this interaction involves a small amount of Gag molecules. At the membrane the RNA binds to a high number of anchored Gag NC domains (reviewed in [START_REF] Freed | HIV-1 assembly, release and maturation[END_REF].

The final component needed for the assembly of the virions is Env, which takes a different synthesis pathway from the previous viral components, transiting through the ER and Golgi body until it reaches the cell membrane in its definitive cleaved, glycosylated and trimeric form. It is unclear how exactly Env assembles at the membrane bud engendered by the Gag lattice. Studies in HIV-1 have suggested a direct interaction between the cytoplasmic tail of Env and Gag MA domains, but the tail in this species is particularly long, making generalization of these results problematic (reviewed in [START_REF] Freed | HIV-1 assembly, release and maturation[END_REF]. More generally, [START_REF] Freed | HIV-1 assembly, release and maturation[END_REF] offers several non-mutually exclusive mechanisms of Env packaging. Passive incorporation proposes that Env diffuses passively across the membrane and that during budding the virion takes with it all Env proteins present on the bud at that moment. Gag-Env co-targeting implies the specific addressing of both Gag and Env to the same area of cellular membrane, potentially involving the lipid raft microdomains established by Gag. Finally Gag and Env could interact, either directly or using a host protein as an intermediary, which would anchor Env to the Gag lattices of membrane buds.

As mentioned previously and illustrated by MPMV, betaretroviruses use a different assembly system. Instead of assembling at the cellular membrane, the Gag lattice forms and encapsulates viral RNA in the pericentriolar cytoplasm due to an 18 aa retention signal present on Gag [START_REF] Choi | Identification of a cytoplasmic targeting/retention signal in a retroviral Gag polyprotein[END_REF]. MPMV Env transfers through the ER and Golgi before reaching the recycling endosome, from there it reaches the assembly site at the surface of small vesicles and binds to the immature Gag and RNA assembly. Finally the vesicles use the microtubule network for anterograde transport to reach the cell membrane, where the virus buds [START_REF] Pereira | Direct evidence for intracellular anterograde co-transport of M-PMV Gag and Env on microtubules[END_REF].

Once all the components are assembled at the spherical membrane bud, the nascent virion needs to dissociate its membrane from that of the cell to be released. Retroviral Gag proteins contain so-called late domains, located at different positions of the protein. Deletion of this domain in HIV-1 abolishes virion release, the particles remaining attached to the membrane by a thin stalk that the virus is unable to cut on its own [START_REF] Gottlinger | Effect of mutations affecting the p6 gag protein on human immunodeficiency virus particle release[END_REF]. There are three main late domain sequences: P(T/S)AP, PPXY and YPXL (found for example in HIV, RSV and EIAV respectively), all three allowing interaction with the host cell ESCRT (endosomal sorting complexes required for transport) pathway. Most retroviruses also encode additional auxiliary domains, both HIV and RSV also present a YPXL domain for example (reviewed in [START_REF] Votteler | Virus budding and the ESCRT pathway[END_REF][START_REF] Freed | HIV-1 assembly, release and maturation[END_REF]. It is unclear whether or not the recruited ESCRT proteins (notably ESCRT-III) enter the nascent virion or whether they gather only in and around the stalk [START_REF] Van Engelenburg | Distribution of ESCRT Machinery at HIV Assembly Sites Reveals Virus Scaffolding of ESCRT Subunits[END_REF][START_REF] Bleck | Temporal and spatial organization of ESCRT protein recruitment during HIV-1 budding[END_REF], nor is it clear how ESCRT assembly leads to membrane scission. Several models are proposed, all involving a growing spiral dome composed of numerous ESCRT-III monomers (reviewed in [START_REF] Schöneberg | Reverse-topology membrane scission by the ESCRT proteins[END_REF]. [START_REF] Lippincott-Schwartz | A Consensus View of ESCRT-Mediated Human Immunodeficiency Virus Type 1 Abscission[END_REF] propose a model in which ESCRT recruitment starts concurrently with membrane budding and leads to incorporation of proteins of this pathway into the nascent virion, from where they can coordinate assembly of a spiral dome of ESCRT-III, provoking a progressive thinning of the membrane stalk until it is pinched off and the virion is released (Fig. 15). About 5-6 minutes elapse between initial detection of Gag and release of a virion ex vivo [START_REF] Jouvenet | Imaging the biogenesis of individual HIV-1 virions in live cells[END_REF].

f. Maturation

The final crucial step in retroviral replication is the maturation step in which the polyproteins get cleaved so as to release the individual proteins (Fig. 12). Immature virions not only have a very distinct architecture from mature virions (Fig. 16A), they are also not infectious as the viral cycle requires the viral proteins to be separated from one another. The viral polyproteins are cleaved by PR, which itself is part of a polyprotein (most commonly Gag-Pro-Pol), concomitantly or shortly after release, preventing loss of the individualized subunits into the cytoplasm (reviewed in [START_REF] Konvalinka | Retroviral proteases and their roles in virion maturation[END_REF]. This mechanism requires a tight regulation of PR activity to prevent premature cleavage. While the exact regulation mechanisms are unknown, the active form of PR is a homodimer, reducing the risk of premature cleavages as the odds of formation of a PR dimer before assembly is generally low. The exception to the rule is found in alpharetroviruses, in which PR is synthesized at the same level as Gag, and thus present at a much higher concentration. This suggests that there might also be other factors, such as interactions with cellular proteins, that could inhibit PR activity until maturation, such a reliance on particular cellular indicators would also explain why in betaretroviruses maturation occurs only once the assembled particle reaches the cell membrane (reviewed in [START_REF] Konvalinka | Retroviral proteases and their roles in virion maturation[END_REF]. Additionally, in HIV-1 PR dimers in the context of a polyprotein are less stable than those formed of individualiued PR, and tend to adopt an inactive conformation, reducing the risk of premature activation [START_REF] Louis | Autoprocessing of HIV-1 protease is tightly coupled to protein folding[END_REF].

2.2

Once maturation is triggered, a highly organized cleavage sequence is initiated (Fig. 16B). A number of studies in HIV have revealed that the first cleavages occur between CA and NC, releasing the RNA associated NC protein as well as Pro-Pol from the still membrane-bound MA-CA. The second cleavage separates MA and CA and MA remains associated to the viral membrane while CA starts to form the mature capsid. Finally, PR, RT and IN are released and small spacer peptides with a regulatory role are removed from CA and NC. Alteration of the kinetics of polyprotein cleavage leads to aberrant mature morphology or non-infectivity (reviewed in [START_REF] Mattei | Retrovirus maturation -An extraordinary structural transformation[END_REF]. Once released from the membrane, CA organizes into a fullerene structure of hexamers alongside 12 CA pentamers (which are naturally curved) to allow incurvation of the lattice [START_REF] Ganser | Assembly and analysis of conical models for the HIV-1 core[END_REF]. The exact form of the mature capsid surrounding the viral genome depends on the genus, in HIV for example, it is conical with 5 pentamers at one end and 7 on the other, in MPMV it is conical with 6 pentamers at each end, and in RSV and MLV it is polyhedral (icosahedral) with 12 evenly spaced pentamers forming the vertices (Fig. 16A, reviewed in [START_REF] Mattei | Retrovirus maturation -An extraordinary structural transformation[END_REF]. The exact mechanism of transition from the immature to the mature capsid is unknown, but it has been shown that the CA-CA interactions involved are different. While the immature assembly seems to be mainly dependent on CTD-CTD interactions, the formation of the mature assembly is reliant on NTD-NTD intrahexamer and CTD-CTD inter-hexamer interactions (reviewed in [START_REF] Mattei | Retrovirus maturation -An extraordinary structural transformation[END_REF][START_REF] Perilla | Molecular Architecture of the Retroviral Capsid[END_REF]. This new mature capsid is less stable than the immature assembly, allowing dissociation from the viral membrane after cell entry and disassembly after reverse transcription (reviewed in [START_REF] Konvalinka | Retroviral proteases and their roles in virion maturation[END_REF].

In addition to cleaving the polyproteins, PR can also be implicated in activating the fusogenic potential of Env. In MLV and MPMV, PR cleaves the cytoplasmic tail of TM late during maturation, enhancing the fusogenicity of Env [START_REF] Brody | Postassembly cleavage of a retroviral glycoprotein cytoplasmic domain removes a necessary incorporation signal and activates fusion activity[END_REF][START_REF] Rein | Function of the cytoplasmic domain of a retroviral transmembrane protein: p15E-p2E cleavage activates the membrane fusion capability of the murine leukemia virus Env protein[END_REF]. In HIV the envelope is not a substrate of PR but Gag interactions with the long cytoplasmic tail of TM inhibit fusion of immature particles [START_REF] Murakami | Regulation of human immunodeficiency virus type 1 Env-mediated membrane fusion by viral protease activity[END_REF][START_REF] Wyma | Coupling of Human Immunodeficiency Virus Type 1 Fusion to Virion Maturation : a Novel Role of the gp41 Cytoplasmic Tail Coupling of Human Immunodeficiency Virus Type 1 Fusion to Virion Maturation : a Novel Role of the gp41 Cytoplasmic Tail[END_REF]. Maturation seems to also affect the repartition of HIV Env at the surface of the virion, on immature virions Env forms several distinct clusters, but after maturation all Env are gathered in a single cluster and this concentration seems to increase fusogenicity [START_REF] Chojnacki | Maturation-dependent HIV-1 surface protein redistribution revealed by fluorescence nanoscopy[END_REF].

Endogenous Retroviruses

Endogenization is the process by which a viral genome becomes a permanent part of the genetic material of its host, so that it may be transmitted vertically to the host's descendants. While there are examples of endogenizations across many viral families (and all seven categories of the Baltimore classification), the very particular replication cycle of retroviruses, with its necessary genomic integration step, leads to their over-representation among endogenous viral elements, especially in animals (reviewed in [START_REF] Feschotte | Endogenous viruses: Insights into viral evolution and impact on host biology[END_REF]

Endogenization of retroviral sequences a. Infection of germinal cells

The nature of the vertically transmitted genome during sexual reproduction is determined by the genome present in the cells of the germline at that moment. If such a cell is infected by a retrovirus, the viral genome will be inserted into the germline cell genome during integration and will then be transmitted vertically alongside (and as a part of) the parental genetic material, resulting in endogenization (Fig. 17). The potential of retroviral infections to spread to germline cells and to result in the formation of new endogenous retroviruses (ERV), has been demonstrated in mice for a long time [START_REF] Lock | Studies of 207 the mechanism of spontaneous germline ecotropic provirus acquisition in mice[END_REF]. Following integration in the germline and vertical transmission, the ERV can then spread through the population as an allele would, though it is often lost. The integration and subsistence of an ERV is therefore a rather rare event. Endogenization is understandably a very ancient process, it probably developed simultaneously with the retroviral replication pathway and endogenizations as old as 430 My have been described (Aiewsakun & Katzourakis, 2017). This long history of animal-retrovirus interaction has led to an accumulation of rare endogenization events, so that nowadays 1-10% of the genome is composed of ERVs and ERV remnants. In humans they represent about 8% (Lander et al., 2001), in mouse about 10% of the genome (Waterston et al., 2002). While endogenization is an ancient phenomenon, it is still ongoing with recent or even current examples being found in sheep (JSRV), mice (MLV, [START_REF] Kozak | Origins of the endogenous and infectious laboratory mouse gammaretroviruses[END_REF] and most notably in koalas (KoRV, [START_REF] Tarlinton | Retroviral invasion of the koala genome[END_REF].

KoRV was first classified as an ERV as during their initial study, [START_REF] Hanger | The Nucleotide Sequence of Koala (Phascolarctos cinereus) Retrovirus: a Novel Type C Endogenous Virus Related to Gibbon Ape Leukemia Virus[END_REF] were able to amplify parts of the viral genome in all koalas tested, but subsequent studies showed that different koalas present different amounts and locations of viral insertions, which contradicts vertical transmission [START_REF] Tarlinton | Retroviral invasion of the koala genome[END_REF]. Additionally, the inserted proviruses were still functional, containing intact ORFs and hinting at a very recent insertion into the genome. On the other hand [START_REF] Tarlinton | Retroviral invasion of the koala genome[END_REF] showed that within family units, there was vertical transmission of KoRV insertions and that koala germline cells harbored viral DNA in their genome. This combination of characteristics of exogenous and endogenous viruses led to the conclusion that KoRV is a currently endogenizing retrovirus. Prevalence studies have revealed that 100% of koalas in north-east Australia are KoRV positive, while in the south-east this number progressively drops to about 25% [START_REF] Simmons | Prevalence of koala retrovirus in geographically diverse populations in Australia[END_REF][START_REF] Legione | Koala retrovirus genotyping analyses reveal a low prevalence of KoRV-A in Victorian koalas and an association with clinical disease[END_REF]. [START_REF] Simmons | Prevalence of koala retrovirus in geographically diverse populations in Australia[END_REF] also measured the number of KoRV copies in the koala genome and showed that north-eastern koalas present on average 165 copies/cell, in accordance with KoRV being endogenous in these animals, while south-eastern koalas present less than 1.53 × 10 -3 copies per genome, reflecting an exogenous infection that does not affect all cells. A single population of south-eastern koalas presented about 1.5 copies/genome, representing a potential very recent endogenization with only one or two insertions. The study of a small isolated population of koalas on Kangaroo Island might also shed some light on the current state of KoRV. This population has been isolated from the mainland since the 1920s, and in 2004, none of the 26 tested animals were positive for KoRV, whereas in 2007, 24 of the 162 tested koalas presented evidence of KoRV infection, indicating that the virus was spreading to this population and is a still active infectious retrovirus [START_REF] Tarlinton | Retroviral invasion of the koala genome[END_REF][START_REF] Simmons | Prevalence of koala retrovirus in geographically diverse populations in Australia[END_REF].

The most recently active ERVs in humans are members of the HERV-K group. This group can be further split into two subgroups: insertions common to primates and humans and insertions that are specific to humans, having occured after their radiation during evolution [START_REF] Medstrand | Human-specific integrations of the HERV-K endogenous retrovirus family[END_REF]. Recent comparative analyses of over 2500 sequenced human genomes found 36 polymorphic insertion sites, present at an allelic frequency ranging from 0.05 to over 75% and denoting very recent amplifications [START_REF] Wildschutte | Discovery of unfixed endogenous retrovirus insertions in diverse human populations[END_REF]. Age estimations on some of the unfixed loci suggest insertions as recently as 0.15 or 0.25 Mya [START_REF] Jha | Human endogenous retrovirus K106 (HERV-k106) was infectious after the emergence of anatomically modern humans[END_REF][START_REF] Marchi | Unfixed Endogenous Retroviral Insertions in the Human Population[END_REF].

b. Intra-genome spreading of ERVs

As mentioned above, in the north-eastern koalas in which KoRV has become endogenous each cellular genome contains on average 165 copies of the viral genome [START_REF] Simmons | Prevalence of koala retrovirus in geographically diverse populations in Australia[END_REF]. After endogenization, ERVs often greatly increase the number of proviral copies in the genome, either by re-infection or through intracellular spreading (Fig. 18). The re-infection pathway is the same as that described for exogenous retroviruses above, except the starting point is gene expression from an already integrated provirus. The new virions that are released can either infect new cells, among them germline cells that already present copies of the viral genome, leading to a progressive accumulation of copies. Intracellular spreading of ERVs uses a different pathway in which the virus never exits the cell, instead viral genes are expressed so as to form intracellular particles that then re-integrate into the genome, this process is also called retrotransposition (reviewed in [START_REF] Dewannieux | Endogenous retroviruses: acquisition, amplification and taming of genome invaders[END_REF][START_REF] Johnson | Endogenous Retroviruses in the Genomics Era[END_REF]. The switch from amplification by re-infection to amplification by retrotransposition is generally accompanied by loss of the env gene and necessitates a retargeting of Gag to intracellular membranes.

An example of the transition between amplification pathways can be found in mice with the intra-cisternal A-type particle (IAP) and IAP-related retroviral element containing an env gene (IAPE) ERVs. IAP sequences present about a thousand copies in the mouse genome, some 700 of which contain functional gag, pro and pol genes (but no env) that allow these sequences to retrotranspose in the genome, augmenting the number of copies [START_REF] Dewannieux | Identification of autonomous IAP LTR retrotransposons mobile in mammalian cells[END_REF][START_REF] Ribet | An infectious progenitor for the murine IAP retrotransposon: emergence of an intracellular genetic parasite from an ancient retrovirus[END_REF]. During this process, Env-less VLPs are generated by budding into cistaernae of the ER, but no particles are ever observed outside the cell [START_REF] Dewannieux | Identification of autonomous IAP LTR retrotransposons mobile in mammalian cells[END_REF]. Later studies by [START_REF] Ribet | An infectious progenitor for the murine IAP retrotransposon: emergence of an intracellular genetic parasite from an ancient retrovirus[END_REF] showed that the mouse genome also contains about 14 copies of IAP-like elements with an associated env gene, among which only 2 copies have remained fully functional. These rare functional IAPE sequences were able to produce infectious extracellular particles, IAPE Gag being addressed to the cellular membrane instead of the ER. This suggests that colonization of the mouse genome was initiated by repeated infections by IAPE elements, followed by a switch to an intracellular amplification pathway accompanied by a change in Gag addressing and a loss of Env. Comparisons of IAP and IAPE copy numbers clearly show that the loss of Env and re-targeting of Gag led to a much more efficient copy number amplification. This was later confirmed by in silico analyses that showed that in many mammalian genomes, loss of env is associated with high copy number and that about 20% of ERV lineages are responsible for about 80% of ERV loci [START_REF] Magiorkinis | Env-less endogenous retroviruses are genomic superspreaders[END_REF]. Intracellularization of ERVs might offer several clear advantages over the traditional extracellular retroviral pathway. Staying in the same cell permits a much higher concentration of viral particles (increasing the probability of insertion and possibly saturating host restriction factors) and avoidance of some factors of the immune system, but it also irremediably ties the fate of the ERV to that of its host, as loss of Env prohibits a switch back to horizontal transmission, unless subsequent recombination or complementation events occur (reviewed in [START_REF] Dewannieux | Endogenous retroviruses: acquisition, amplification and taming of genome invaders[END_REF].

In humans, analysis of gene conservation in the HERV-K elements discussed above reveals a continuous purifying selection, including of env, suggesting that HERV-K amplification occurred through re-infection [START_REF] Belshaw | Long-term reinfection of the human genome by endogenous retroviruses[END_REF]. The human-specific HERV-K copies show enough conservation that their consensus sequence allows production of an infection retrovirus (that is probably close to the progenitor of the family), and that recombination events between two unfixed loci would be sufficient to obtain a functional endogenous viral genome [START_REF] Dewannieux | Identification of an infectious progenitor for the multiple-copy HERV-K human endogenous retroelements[END_REF]. Interestingly, infection using the previously mentioned resurrected HERV-K virus discloses an insertional bias towards actively transcribed genomic regions, while the current-day fixed insertions in the human genome are preferentially found outsided of transcription units, revealing that amplification is influenced by the host which favors fixation of non-deleterious insertions only (Fig. 18, [START_REF] Brady | Integration target site selection by a resurrected human endogenous retrovirus[END_REF]. While some highly conserved and fully intact viral loci have been identified (such as HERV-K113, [START_REF] Turner | Insertional polymorphisms of full-length endogenous retroviruses in humans[END_REF], no actively replicating ERV has been reported in humans. This is probably linked to the fact that ERV integration activity has decreased fourfold in humans over the last 10 My [START_REF] Magiorkinis | The decline of human endogenous retroviruses: extinction and survival[END_REF].

c. Groups of ERVs

Due to their tendency to amplify within the host genome, ERVs give rise to numerous related sequences, all originated from one or sometimes a few original endogenization events. These groups are often called "families", however this is misleading as family designates a much higher level of phylogenetic classification (all retroviruses are part of the Retroviridae family). Nevertheless, it can be useful to sort insertions into groups reflecting their genetic proximity and to compare them to the existing exogenous retroviral genera. Assignation of a particular species or even genus to an ERV can be a delicate matter, as their sequence can often be incomplete and under a different selective pressure than those of exogenous viruses (see 2.2.3). ERVs are often assigned to a class based on their RT similarity to exogenous viruses. Class I ERVs are related to gamma-and epsilonretroviruses, II to lenti-, alpha-, beta-or deltaviruses and III to spumaviruses (Fig. 19A, reviewed in [START_REF] Johnson | Endogenous Retroviruses in the Genomics Era[END_REF]. These three classes are not equally represented in vertebrate genomes and different vertebrate clades display over-or underrepresentation of some [START_REF] Hayward | Pan-vertebrate comparative genomics unmasks retrovirus macroevolution[END_REF]. The "other" category contains unclassified retroviral species, often related to spumaviruses. (B) Detailed distribution of ERVs in different host genomes. The trend line indicates the total amount of ERV genomes identified in the species, the bars the proportion each lineage represents among the ERVs of the given genome. A cladogram of retroviral lineages and of the host species is provided as ordinate and abscissa respectively. For simplicity, ERVs not belonging to any of the seven major lineages are not represented. Adapted from [START_REF] Hayward | Pan-vertebrate comparative genomics unmasks retrovirus macroevolution[END_REF].

of them (Fig. 19B). In general class I and II ERVs are the most numerous elements in vertebrate genomes, especially gamma-and betaretrovirus derived sequences. Delta-and lentiviral ERVs are almost completely absent in all vertebrate genomes and some of the more species specific retroviral genera are present in significant numbers only in their host species: alpharetroviruses in birds and epsilonretroviruses in fish [START_REF] Hayward | Pan-vertebrate comparative genomics unmasks retrovirus macroevolution[END_REF].

Impact of retroviral integration on the host a. LTR integration can drive ectopic gene expression

While LTRs do not code for genes, they are powerful gene expression regulators, being able to initiate transcription and to terminate it by polyadenylation. As such, their presence in the genome can have a major impact on the regulation of cellular gene expression. This aspect is doubly important as the immense majority of older ERV integrations have degenerated to a solo LTR, present in the genome without any accompanying viral sequences (see 2.2.3.b.).

Insertional mutagenesis. While insertion of retroviruses can present some biases, it is still a rather random process and a source of insertional mutagenesis. While it is possible that this results simply from the insertion of a provirus into a host gene, disrupting the ORF, the more common mechanism of insertional mutagenesis following insertion implicates the LTR sequences of the provirus. The insertion of an LTR near a gene or in an intron can drive ectopic gene expression or aberrant splicing and polyadenylation events. Since recent ERV insertions are rare in humans [START_REF] Magiorkinis | The decline of human endogenous retroviruses: extinction and survival[END_REF], this phenomenon is more often investigated in mice, which present a number of very active ERV groups (reviewed in [START_REF] Maksakova | Retroviral elements and their hosts: Insertional mutagenesis in the mouse germ line[END_REF]. Nevertheless, there are a number of reports of HERV LTRs implicated in the expression of human oncogenes, though in healthy tissues they are heavily methylated to repress expression (reviewed in [START_REF] Babaian | Endogenous retroviral promoter exaptation in human cancer[END_REF]. This ability of retroviral integrations to trigger expression of oncogenes is now also used to screen for new genes that may be implicated in cancer (reviewed in [START_REF] Bii | Identifying Cancer Driver Genes Using Replication-Incompetent Retroviral Vectors[END_REF].

In mice, retroviral insertional mutagenesis accounts for at least 10% of all known de novo mutations, most of them due to the IAP and the early transposon (ETn)/MusD groups (reviewed in [START_REF] Maksakova | Retroviral elements and their hosts: Insertional mutagenesis in the mouse germ line[END_REF]. ETn are LTR elements that lost their coding ORFs and are dependent of expression of the MusD ERV for retrotransposition [START_REF] Ribet | An active murine transposon family pair: retrotransposition of "master" MusD copies and ETn trans-mobilization[END_REF]. Interestingly, the mutagenic mechanisms of IAP and ETn insertions seem to differ (Fig. 20). According to [START_REF] Maksakova | Retroviral elements and their hosts: Insertional mutagenesis in the mouse germ line[END_REF] IAP elements are mostly implicated in internal disruption (aberrant splicing, potentially also leading to exon skipping), while ETn tend to provoke aberrant transcription termination and polyadenylation. Additionally [START_REF] Maksakova | Retroviral elements and their hosts: Insertional mutagenesis in the mouse germ line[END_REF] reference a number of aberrant transcript initiations caused by IAP LTR promoter activity, but none by ETn LTRs, suggesting that different ERV groups could show a bias towards different mutational mechanisms. As in the case of LTR directed oncogene expression in humans, IAP LTR driven gene expression is highly reliant on the epigenetic state of the LTR, generating metastable epialleles [START_REF] Rakyan | Metastable epialleles in mammals[END_REF] and leading to differential expression of genes in genetically identical mice (reviewed in [START_REF] Maksakova | Retroviral elements and their hosts: Insertional mutagenesis in the mouse germ line[END_REF]. [START_REF] Whitelaw | Retrotransposons as epigenetic mediators of phenotypic variation in mammals[END_REF] offer the very intriguing hypothesis that the random insertion of imperfectly epigenetically controlled LTRs might in part explain the phenotypic variations in mammals. Since epigenetic regulations are imperfectly inherited during reproduction, even genetically identical individuals would present differential expression patterns due to differentially active LTRs. Post-insertion locus. In this example the inserted ERV contains LTRs presention a cryptic splice acceptor (sa) site, in addition to the canonical polyadenylation (polyA) and transcript initiation functions. The canonical retroviral splice donor (sd) and acceptor sites are also represented. Cellular transcription can be disrupted through several ways, three are presented here: aberrant splicing leads to addition of ERV sequences into the mRNA (green), premature termination leads to interruption of the transcript and polyadenylation (AAAAA) and abberant initiation leads to formation of incomplete transcripts. Adapted form [START_REF] Maksakova | Retroviral elements and their hosts: Insertional mutagenesis in the mouse germ line[END_REF].

Regulation of gene expression by

work on maize (see for example [START_REF] Mcclintock | The origin and behavior of mutable loci in maize[END_REF][START_REF] Mcclintock | Controlling Elements and the Gene[END_REF], and was later present in the concept of gene batteries put forward by [START_REF] Britten | Gene Regulation for Higher Cells: A Theory[END_REF]. Since then, the role of repeated sequences and especially ERVs in regulation of transcription has become increasingly clear (reviewed in [START_REF] Thompson | Long Terminal Repeats: From Parasitic Elements to Building Blocks of the Transcriptional Regulatory Repertoire[END_REF][START_REF] Chuong | Regulatory activities of transposable elements: from conflicts to benefits[END_REF]. Once more this role is mostly dependent on the LTR sequences and solo LTRs, but in contrast to the insertional mutagenesis events discussed above, the regulation events discussed here are part of cell physiology, the LTR sequence having been exapted. Exaptation (also called co-option or domestication) is a concept coined by [START_REF] Gould | Exaptation-a Missing Term in the Science of Form[END_REF] to describe the process by which "a character, previously shaped by natural selection for a particular function [...] is co-opted for a new use". In the present context, this term applies when the host cell uses features provided by the viral genome for its own ends. The LTRs have evolved to accomplish a very particular role in retroviral replication, but their ability to regulate transcription can also be used by the host cell for control over its own genes.

There have now been a number of reports of LTRs able to act not only as cell gene expression promoters, but even as tissue-specific promoters, responding to particular cellular conditions or the presence of specific cellular factors (reviewed in [START_REF] Thompson | Long Terminal Repeats: From Parasitic Elements to Building Blocks of the Transcriptional Regulatory Repertoire[END_REF]. [START_REF] Lynch | Ancient transposable elements transformed the uterine regulatory landscape and transcriptome during the evolution of mammalian pregnancy[END_REF] for example show that the endometrial expression of numerous genes involved in mammalian placentation has recently been shown to be initiated by repeated elements, LTRs among them. Comparisons between viviparous and non-viviparous species additionally indicated that many of these genes were recruited from other organs, probably through usage of the repeated sequences as promoters [START_REF] Lynch | Ancient transposable elements transformed the uterine regulatory landscape and transcriptome during the evolution of mammalian pregnancy[END_REF]. In mice, [START_REF] Peaston | Retrotransposons Regulate Host Genes in Mouse Oocytes and Preimplantation Embryos[END_REF] show that during the early pre-implantation stages, specifically from the oocyte to the 2-cell stage, 6-12% of genes are expressed under control of mouse ERVs , many of them belonging to the ERV-L group present in most placental mammals [START_REF] Cordonnier | Isolation of novel human endogenous retrovirus-like elements with foamy virus-related pol sequence[END_REF][START_REF] Bénit | ERV-L elements: a family of endogenous retrovirus-like elements active throughout the evolution of mammals[END_REF]. The involvement of ERVs in this stage of development was later strengthened by the discovery of a subset of mouse embryonic stem cells that behaved like cells of the 2-cell stage (being totipotent). Analysis of the transcriptome of these cells confirmed that many of the transcripts responsible for this transient totipotent state are under control of the same ERV elements as described previously [START_REF] Macfarlan | Embryonic stem cell potency fluctuates with endogenous retrovirus activity[END_REF]. Concerning later stages of placentation, it has been shown that the mouse ERV group RLTR13D5 provides numerous transcriptional activators in trophoblast cells, able to bind key trophoblastic regulatory factors [START_REF] Chuong | Endogenous retroviruses function as species-specific enhancer elements in the placenta[END_REF]. The ERV sequences involved in all these regulations are generally a recent acquisition by the host and often species specific (reviewed in [START_REF] Thompson | Long Terminal Repeats: From Parasitic Elements to Building Blocks of the Transcriptional Regulatory Repertoire[END_REF][START_REF] Chuong | Regulatory activities of transposable elements: from conflicts to benefits[END_REF]. A stunning illustration of this can be found when considering the endometrial expression of prolactin in mice and primates. In both cases an LTR drives prolactin expression, but the ERVs used in mice and primates are independent and have been exapted independently [START_REF] Emera | Convergent evolution of endometrial prolactin expression in primates, mice, and elephants through the independent recruitment of transposable elements[END_REF]. The best-studied example of ERV LTR involvement in early human development concerns the HERV-H group, which is abundantly (2% of all polyA RNA) and specifically expressed in embryonic stem cells [START_REF] Santoni | HERV-H RNA is abundant in human embryonic stem cells and a precise marker for pluripotency[END_REF]. Additionally the HERV-H LTR presents binding

2.2

sites for the pluripotency transcription factors NANOG and LBP9 [START_REF] Santoni | HERV-H RNA is abundant in human embryonic stem cells and a precise marker for pluripotency[END_REF][START_REF] Wang | Primate-specific endogenous retrovirus-driven transcription defines naive-like stem cells[END_REF]. HERV-H driven expression is dependent on the presence of these factors as well as OCT4 (another marker of pluripotency) and 10 % of the transcripts are long non-coding RNAs (lncRNA) [START_REF] Wang | Primate-specific endogenous retrovirus-driven transcription defines naive-like stem cells[END_REF]. Knock-down of some of these lncRNAs leads to rapid differentiation (reviewed in [START_REF] Chuong | Regulatory activities of transposable elements: from conflicts to benefits[END_REF]. While the exact function of lncRNAs is not know it has been suggested that they act as scaffolds recruiting OCT4 and the co-activator complexes Mediator and p300 to regulate expression of neighboring genes [START_REF] Lu | The retrovirus HERVH is a long noncoding RNA required for human embryonic stem cell identity[END_REF].

b. Impact of retroviral gene acquisition on host physiology

Pathologies linked to endogenous retroviral genes. Expression of retroviral genes can have deleterious consequences on both host cell and organism biology. Many retroviral genes have evolved specifically to modify host cell functions so as to create a more favorable environment for viral replication. This is especially true of regulatory and accessory genes (see 2.1.1.e.), though these are rare in ERVs (with the notable exception of HERV-K which presents a complex genome, [START_REF] Dewannieux | Identification of an infectious progenitor for the multiple-copy HERV-K human endogenous retroelements[END_REF], but also applies to canonical retroviral proteins. To keep HERV-K as an example, it has been shown that its Env was able to interfere with Tetherin restriction and even to possess oncogenic potential, with a strong impact on cell physiology [START_REF] Lemaitre | The HERV-K Human Endogenous Retrovirus Envelope Protein Antagonizes Tetherin Antiviral Activity[END_REF][START_REF] Lemaître | A human endogenous retrovirus-derived gene that can contribute to oncogenesis by activating the ERK pathway and inducing migration and invasion[END_REF]. The Env of the related JSRV retrovirus was also shown to be sufficient to induce tumorigenesis when expressed at a sufficiently high level [START_REF] Wootton | Sheep retrovirus structural protein induces lung tumours[END_REF]. Besides these admittedly rare cases in which a structural viral protein can carry oncogenic properties, rodent and avian retroviruses often carry oncogenes such as src, myc or ras (reviewed in [START_REF] Vogt | Retroviral oncogenes: a historical primer[END_REF]. While the potential impact of such genes after integration is obvious, endogenizations that present a strong oncogenic or otherwise deleterious impact have a low chance of becoming fixed in the population and as such rarely result in observable ERVs (Fig. 18). This aspect might also explain why there are only a few endogenized complex retroviruses.

There are many mostly correlative studies showing that expression of human ERVs is associated with a range of diseases. This is especially true for HERV-K which has for example been associated with melanoma, ovarian, breast, and prostate cancer [START_REF] Büscher | Expression of Human Endogenous Retrovirus K in Melanomas and Melanoma Cell Lines[END_REF][START_REF] Wang-Johanning | Expression of multiple human endogenous retrovirus surface envelope proteins in ovarian cancer[END_REF][START_REF] Esnault | A placenta-specific receptor for the fusogenic, endogenous retrovirus-derived, human syncytin-2[END_REF][START_REF] Agoni | Detection of Human Endogenous Retrovirus K (HERV-K) Transcripts in Human Prostate Cancer Cell Lines[END_REF], as well as amyotrophic lateral sclerosis and multiple sclerosis [START_REF] Li | Human endogenous retrovirus-K contributes to motor neuron disease[END_REF][START_REF] Bhetariya | Analysis of Human Endogenous Retrovirus Expression in Multiple Sclerosis Plaques[END_REF] and auto-immune diseases (reviewed in [START_REF] Trela | The role of molecular mimicry and other factors in the association of Human Endogenous Retroviruses and autoimmunity[END_REF]. Other studies however have shown that expression of HERV-K derived proteins also occurs in many healthy tissues [START_REF] Schmitt | HERV-K(HML-2) Rec and Np9 transcripts not restricted to disease but present in many normal human tissues[END_REF] and it remains to be seen to what extent (if any) ERV proteins expression plays a direct role in the different pathologies.

Endogenous retroviral genes as restriction factor.

A number of the still coding ERV genes have been maintained in a coding state following exaptation. Since exaptation of Env sequences for use as syncytins is critical to the present work, it will be treated in more detail in chapter 3 and other examples will be given here. In most of those cases, ERV genes have been exapted by the cell to protect it from infection by exogenous viruses. Two of the most famous examples are the mouse restriction factors Friend virus susceptibility (Fv) 1 and Fv4. Fv1 shows strong sequence similarity to spumaretroviral ERV-L gag [START_REF] Best | Positional cloning of the mouse retrovirus restriction gene Fvl[END_REF] and is able to restrict MLV infection in vivo by interfering with the viral PIC [START_REF] Jolicoeur | Effect of Fv-1 gene product on proviral DNA formation and integration in cells infected with murine leukemia viruses[END_REF]. Fv4 on the other hand is an MLV related env gene that has been shown to inhibits MLV infection [START_REF] Ikeda | Cellular expression of murine leukemia virus gp70-related antigen on thymocytes of uninfected mice correlates with Fv-4 gene-controlled resistance to friend leukemia virus infection[END_REF]. The mechanism employed by Fv4 to restrict MLV infection is twofold. First, Fv4 can interact with MLV Env specific receptors, blocking exogenous Env proteins from accessing the receptor and initiating fusion. This process, called receptor interference, is commonly observed and also allows exogenous retroviruses to avoid super-infection, i.e., entering an already infected cell (reviewed in [START_REF] Nethe | Retroviral superinfection resistance[END_REF]. Receptor interference is dependent only on Env-receptor binding and does not require fusion [START_REF] Granowitz | Analysis of Mutations in the Envelope Gene of Moloney Separation of Infectivity from Superinfection Murine Leukemia Virus : Resistance[END_REF]. Second, during MLV viral assembly in an infected cell, the Fv4 protein can be integrated in the virus instead of the exogenous Env. Since Fv4 presents a mutation that renders it non-fusiogenic, this leads to a virion with defective Env proteins at its surface, diminishing infectivity [START_REF] Taylor | Fv-4: identification of the defect in Env and the mechanism of resistance to ecotropic murine leukemia virus[END_REF].

Exaptation of viral genes as restriction factors using similar mechanisms has since been observed in other species. In sheep, an endogenous copy of JSRV Gag (enJS56A1) is able to interfere with viral assembly by integrating the assembly of exogenous Gag and blocking its intra-cellular addressing, leading to aggregation of defective viral particles in the cytoplasm [START_REF] Arnaud | Mechanisms of late restriction induced by an endogenous retrovirus[END_REF]. Other ERV copies of JSRV can express Env able to block exogenous JSRV entry by receptor interference [START_REF] Spencer | Receptor usage and fetal expression of ovine endogenous betaretroviruses: implications for coevolution of endogenous and exogenous retroviruses[END_REF]. In cats and humans two soluble factors (Refrex-1 and Suppressyn respectively) that result from the expression of truncated ERV env genes were recently discovered [START_REF] Ito | Refrex-1, a Soluble Restriction Factor against Feline Endogenous and Exogenous Retroviruses[END_REF][START_REF] Sugimoto | A novel human endogenous retroviral protein inhibits cell-cell fusion[END_REF]. They can specifically bind to cell surface receptors used during infection by exogenous retroviruses [START_REF] Ito | Refrex-1, a Soluble Restriction Factor against Feline Endogenous and Exogenous Retroviruses[END_REF][START_REF] Sugimoto | A novel human endogenous retroviral protein inhibits cell-cell fusion[END_REF]. While Refrex-1 has been shown to be able to inhibit receptor binding of a subset of exogenous FLV strains, there is no known extant human retrovirus for Suppressyn to counteract. Suppressyn blocks interaction of Env proteins with the solute carrier family 1 member 5 (SLC1A5) protein [START_REF] Sugimoto | A novel human endogenous retroviral protein inhibits cell-cell fusion[END_REF], which is used as a specific receptor by a strikingly high amount of retroviruses in diverse species (see 3.1.2.a., reviewed in [START_REF] Sinha | Retroviruses of the RDR superinfection interference group: ancient origins and broad host distribution of a promiscuous Env gene[END_REF], but which is also the necessary partner for Syncytin-1 activity in the human placenta (see 3.2.1 and Blond et al., 2000). It is possible that Suppressyn counteracted an older human retrovirus, or that it serves as a defense mechanism against an eventual cross-species transmission of another virus using SLC1A5 as receptor, as such events are relatively common (reviewed in [START_REF] Sinha | Retroviruses of the RDR superinfection interference group: ancient origins and broad host distribution of a promiscuous Env gene[END_REF].

In conclusion, it is important to remember that while retroviral infection and endogenization events can have a strong deleterious impact on the host, there are cases where domestication of viral elements provides hosts with new genetic material, able to play a role in normal host physiology. Retroviral insertions can be both a burden and a blessing, but in any case they represent a major evolutionary driving force.

2.2.3 Fate of ERVs and control by the host

Since production of viral particles or expression of parts of the viral genome is not always in the interest of the host, several ways of dealing with ERV integrations have developed during evolution. Often, but not always, they lead to inactivation of the proviral genome.

a. Epigenetic regulation of ERV expression

The fastest and most direct way for the host cell to regulate ERV expression is through epigenetic control. Cell enzymes are able to chemically modify DNA and the histones it is wound around so as to create zones of enhanced or repressed expression. At the DNA level, this regulation can take the form of CpG dinucleotide methylation to inhibit gene expression, and it has been shown that methylation of CpG in the HERV-K LTR for example can indeed block its transcriptional activity [START_REF] Lavie | CpG methylation directly regulates transcriptional activity of the human endogenous retrovirus family HERV-K (HML-2)[END_REF]. More generally, micro-array based studies reveal that CpG methylation is a silencing mechanism of all HERV in healthy tissues, though there are indications that this mechanism is age-dependent, younger HERV being more methylated than older ones [START_REF] Szpakowski | Loss of epigenetic silencing in tumors preferentially affects primate-specific retroelements[END_REF].

At a higher organizational level, epigenetic regulation of gene expression is achieved by chemically modifying histones, especially through acetylation and methylation, though the former does not seem to play a major role in HERV expression regulation (reviewed in [START_REF] Hurst | Epigenetic control of human endogenous retrovirus expression: Focus on regulation of long-terminal repeats (LTRs)[END_REF]. Methylation of histones can either have an activating or inhibiting effect on gene expression and the predominant associated modifications are H3K4me3 and H3K9me3 respectively. In silico analyses of IAP and HERV-K integration sites revealed an over-representation of inhibiting and an under-representation of activating histone marks, coherent with repression of ERV expression [START_REF] Campos-Sánchez | Integration and Fixation Preferences of Human and Mouse Endogenous Retroviruses Uncovered with Functional Data Analysis[END_REF]. The major cellular actors of histone based epigenetic repression of ERV expression are the Krüppel associated box zinc finger proteins (KRAB-ZFP). The proteins of this family are able to bind to specific DNA sequences with their zinc finger domain and to recruit histone modification complexes through their KRAB domain and interaction with transcription intermediary factor 1-beta (previously called tripartite motif-containing protein 28, TRIM28) (reviewed in [START_REF] Hurst | Epigenetic control of human endogenous retrovirus expression: Focus on regulation of long-terminal repeats (LTRs)[END_REF]. It has been shown that KRAB-ZFP proteins are encoded in genomic regions showing a propensity to recombination events capable of generating new random KRAB-ZFP with altered sequence specificity, and that the apparition and fixation of new copies is correlated with the acquisition of new ERV sequences during evolution [START_REF] Thomas | Coevolution of retroelements and tandem zinc finger genes[END_REF][START_REF] Lukic | The diversity of zinc-finger genes on human chromosome 19 provides an evolutionary mechanism for defense against inherited endogenous retroviruses[END_REF]. A more recent comprehensive study of human KRAB-ZFP found that two thirds of them bound sequences of ERVs and retrotransposons and confirmed the correlated emergence of new ERVs and corresponding new KRAB-ZFP proteins [START_REF] Imbeault | KRAB zinc-finger proteins contribute to the evolution of gene regulatory networks[END_REF]. However, the study also showed that a third of all KRAB-ZFP (among which most of the older and most conserved ones) bind to host sequences and that ERV/retrotransposon directed KRAB-ZFP are conserved long after the corresponding element lost its replicative potential, suggesting that KRAB-ZFP play additional roles in gene expression regulation [START_REF] Imbeault | KRAB zinc-finger proteins contribute to the evolution of gene regulatory networks[END_REF]. If the KRAB-ZFP/ERV co-evolution was purely an arms race, there would be no selective pressure to maintain proteins binding non-functional ERV elements.

It seems important to note here that i) these epigenetic regulation mechanisms do not repress all ERV expression, transcriptome studies show that in humans about a third of ERV sequences are transcribed [START_REF] Pérot | Microarray-Based Sketches of the HERV Transcriptome Landscape[END_REF], and ii) epigenetic regulation is a reversible process and in some situations ERV expression can be reactivated in vivo. Hypomethylation is a common phenomenon in cancers and accordingly enhanced ERV expression is observed in many cases (discussed above and reviewed in [START_REF] Hurst | Epigenetic control of human endogenous retrovirus expression: Focus on regulation of long-terminal repeats (LTRs)[END_REF]. Epigenetic regulation is also impacted by the early stages of reproduction, with for example global demethylation events. This might be part of the reason why ERV sequences often seem to play a role in early mammalian development as discussed above. In an intriguing example investigated by [START_REF] Barbot | Epigenetic regulation of an IAP retrotransposon in the aging mouse: progressive demethylation and de-silencing of the element by its repetitive induction[END_REF] describes the progressive demethylation of a mouse IAP sequence located in the vicinity of a gene with a circadian expression profile. As the mouse ages, the continual rythmic transcription of the cellular gene induces an age-dependent reactivation of the suppressed IAP sequence.

b. Accumulation of mutations and solo LTR formation

In most cases, ERV insertion has a neutral to deleterious effect on the host, leading to a neutral or negative selection of ERV sequences (Fig. 18). As such after integration the viral genome is subject to the accumulation of mutations, indels and recombination events which often disrupt the viral ORFs and render the element non-functional for autonomous expression (de Parseval et al., 2003;[START_REF] Villesen | Identification of endogenous retroviral reading frames in the human genome[END_REF]. In contrast to the epigenetic silencing discussed above, this process occurs mostly passively and quickly becomes irreversible through the sheer amount of accumulated mutations. A commonly observed deletion in ERV sequences is the loss of env, though this might be due to the higher amplification potential of these sequences rather than an increased probability of losing this ORF (see Fig. 18 and 2.2.1.b.).

By far the most common form of old ERV elements is the solo LTR, outnumbering more complete elements by a factor of about 10 (Lander et al., 2001). Solo LTR result from nonallelic homologous recombination (NAHR) events in which the high sequence identity of two LTRs (often the two identical LTRs bordering a single young provirus) leads to the excision of the genetic material contained between them (reviewed in [START_REF] Jern | Effects of Retroviruses on Host Genome Function[END_REF]. As such processes need 200-500 bp of sequence identity, LTRs are perfectly capable to mediate NAHR [START_REF] Stankiewicz | Genome architecture, rearrangements and genomic disorders[END_REF]. The excised portion of genome most commonly does not contain a centromere and will thus be lost. A solo LTR is the smallest possible form for an ERV, the rest of the viral genome has entirely disappeared from the host genome, but the LTR itself cannot be excised by the cell. Since point mutations and indels can accumulate in LTRs just as well as in the other parts of the viral genome, it stands to reason that NAHR events become less and less likely as the ERV ages and indeed, studies on HERV-K have shown that young insertions present 8 × 10 -5 recombinations per locus per generation, while in older insertions this number drops to 4 × 10 -7 recombinations per locus per generation [START_REF] Belshaw | Rate of Recombinational Deletion among Human Endogenous Retroviruses[END_REF]. Using an in silico simulation, the authors further determined that a single nucleotide substitution reduced the odds of NAHR tenfold, two substitutions reduced it a hundredfold [START_REF] Belshaw | Rate of Recombinational Deletion among Human Endogenous Retroviruses[END_REF]. This age-dependent pattern is also observed in other major HERV groups, except for HERV-H which presents an intriguingly low amount of solo LTR elements, suggesting that group-specific factors can influence these excision events [START_REF] Gemmell | Phylogenetic Analysis Reveals That ERVs "Die Young" but HERV-H Is Unusually Conserved[END_REF]. Since the LTR is able to mediate transcription initiation and transcript polyadenylation [START_REF] Pérot | Microarray-Based Sketches of the HERV Transcriptome Landscape[END_REF], solo LTR can still have a major impact on host physiology (see 2.2.2.a.) and the only way to completely control the impact of ERV insertion is epigenetic regulation.

c. Exaptation and purifying selection

In the relatively rare case of exaptation of retroviral sequences by the host, these sequences are generally under purifying selection, like would be expected for any other cellular gene. This is for example the case of the exapted syncytin genes (see 3, reviewed in [START_REF] Lavialle | Paleovirology of 'syncytins', retroviral env genes exapted for a role in placentation[END_REF] and of members of the HERV-H group [START_REF] Gemmell | Phylogenetic Analysis Reveals That ERVs "Die Young" but HERV-H Is Unusually Conserved[END_REF]. Under purifying selection, the ratio of synonymous to non-synonymous nucleotide mutations is strongly biased towards the former as it conserves amino acid sequence coding and identity. Additionally, deletion of sequences under purifying selection is generally counter-selected on the population level. Exaptation of retroviral sequences does not mean that they are free of epigenetic control however, just like many other cellular genes their expression has to be and is often regulated. Syncytins for example are almost exclusively expressed in the placenta (see chapter 3, reviewed in [START_REF] Lavialle | Paleovirology of 'syncytins', retroviral env genes exapted for a role in placentation[END_REF]Denner, 2016).

Syncytins

Retroviral envelopes

Studies of the human genome revealed that about 50% of still coding ERV ORFs are env genes [START_REF] Villesen | Identification of endogenous retroviral reading frames in the human genome[END_REF]. This might be due to the fact that retroviral Env present, among others, a function that is uncommon among cellular genes: fusogenicity. Among the handful of fusogenic proteins identified in eukaryotic cells, many show strong similarity to viral sequences (see [START_REF] Fédry | The Ancient Gamete Fusogen HAP2 Is a Eukaryotic Class II Fusion Protein[END_REF]. The following part describes the structure of orthoretroviral Env, with the exception of epsilonretroviral Env who present a number of differences and are not relevant to this work (see [START_REF] Rovnak | Establishment of productively infected walleye dermal sarcoma explant cells[END_REF], as well as 2.1.2.a.).

Functional domains a. Domains of the SU

Signal peptide. The N-terminus of SU is composed of a hydrophobic sequence of about 20-30 amino-acids forming the signal peptide (Fig. 21). Signal peptides are a commonly used addressing signal to the ER and the secretory pathway, and hydrophobicity plays an important role in their recognition by the signal recognition particle (SRP). Binding of this complex to the nascent Env protein leads to its co-translational insertion into the ER, after which the signal peptide is cleaved (reviewed in [START_REF] Nyathi | Co-translational targeting and translocation of proteins to the endoplasmic reticulum[END_REF]. While this sequence is therefore not part of the functional mature retroviral Env, it is nevertheless indispensable for its synthesis. Loss of the signal peptide leads to incorrect processing and post-translational modification of the Env polyprotein.

Receptor binding domain. Since the SU is the domain exposed to the exterior, it is responsible for recognition and binding of the cognate receptor domain. This implies the receptor binding domain (RBD) at the surface of SU. The exact nature of the residues implicated in this process is variable depending on the SU and receptor, and often unknown, but seems to often be contained in the N-terminal part of SU (Fig. 21, reviewed in [START_REF] Overbaugh | Receptors and Entry Cofactors for Retroviruses Include Single and Multiple Transmembrane-Spanning Proteins as well as Newly Described Glycophosphatidylinositol-Anchored and Secreted Proteins[END_REF]. Studies on Syncytin-1 SU binding to its receptor SLC1A5 for example demonstrated that its 124 N-terminal amino acids constituted the minimal receptor binding domain [START_REF] Cheynet | Identification of the hASCT2-binding domain of the Env ERVWE1/syncytin-1 fusogenic glycoprotein[END_REF]. A comparison with the SU domain of other retroviruses using SLC1A5 as receptor showed that only about a dozen residues were conserved in this 124 aa, grouped in three cysteine containing motifs (PCXP, CYX 5 C and CX 8-9 CW) and one longer SDGGGX 2 DX 2 R motif (Fig. 24, Cheynet et al., 2006).

Similarly, the minimal domains needed for receptor binding in MLV and HTLV-1 and -2 are contained in the N-terminal part of SU [START_REF] Battini | Receptor choice determinants in the envelope glycoproteins of amphotropic, xenotropic, and polytropic murine leukemia viruses[END_REF][START_REF] Battini | Receptor-binding domain of murine leukemia virus envelope glycoproteins[END_REF][START_REF] Kim | No Title[END_REF]. Subsequent studies showed that here too very short domains are directly implicated in receptor interaction by MLV [START_REF] Battini | Definition of a 14 amino-acid peptide essential for the interaction bet the murine leukemia virus amphotropic envelope glycoprotein and its receptor[END_REF] and HTLV-1 SU (reviewed in [START_REF] Jones | Molecular aspects of HTLV-1 entry: Functional domains of the HTLV-1 surface subunit (SU) and their relationships to the entry receptors[END_REF]. Additionally, replacing the RBD of MLV Env with that of HTLV-1 changes the viral tropism of MLV to that of HTLV-1, confirming the importance of this domain [START_REF] Kim | Definition of an amino-terminal domain of the human T-cell leukemia virus type 1 envelope surface unit that extends the fusogenic range of an ecotropic murine leukemia virus[END_REF]. Importantly, while the RBD is the main determinant of receptor binding, there are other regions in the SU that play an important role in this process (reviewed in [START_REF] Jones | Molecular aspects of HTLV-1 entry: Functional domains of the HTLV-1 surface subunit (SU) and their relationships to the entry receptors[END_REF] Proline-rich region. The proline-rich region (PRR) is found in gamma-type envelopes and separates the N-terminal and C-terminal parts of SU (Fig. 21). The PRR seems to form a flexible hinge that is implicated in stability changes of the SU/TM dimer after receptor binding, potentially being a part of the signal inducing TM conformational changes and fusion [START_REF] Lavillette | A proline-rich motif downstream of the receptor binding domain modulates conformation and fusogenicity of murine retroviral envelopes[END_REF]. The hinge-like structure of the PRR has also been confirmed by structure analyses which show that insertions within this region do not contribute to or majorly impact the structure of Env [START_REF] Riedel | Native structure of a retroviral envelope protein and its conformational change upon interaction with the target cell[END_REF].

CXXC.

A conserved CXXC motif (consensus CWLC) is necessary to regulate SU-TM interaction in non-alpharetroviral gamma-type Env (Fig. 21, [START_REF] Wallin | Isomerization of the intersubunit disulphide-bond in Env controls retrovirus fusion[END_REF]. The exact localization of the CXXC varies between different retroviruses (see for example de Parseval et al., 2003), but its presence is crucial to fusogenic potential of gamma-type Env. This domain was first discov-ered as being part of a disulfide bond between SU and TM (in association with the TM CX n CC motif, see below), strengthening the association of both subunits and stabilizing the pre-fusion TM conformation [START_REF] Pinter | Localization of the labile disulfide bond between SU and TM of the murine leukemia virus envelope protein complex to a highly conserved CWLC motif in SU that resembles the active-site sequence of thiol-disulfide exchange enzymes[END_REF][START_REF] Li | Intersubunit Disulfide Isomerization Controls Membrane Fusion of Human T-Cell Leukemia Virus Env[END_REF]. After interaction with the receptor, this SU-TM bond is converted into an intra-CXXC disulfide bond by a reactive thiol group carried in this motif, allowing separation of SU and TM and fusion (Fig. 23, [START_REF] Wallin | Isomerization of the intersubunit disulphide-bond in Env controls retrovirus fusion[END_REF][START_REF] Li | Intersubunit Disulfide Isomerization Controls Membrane Fusion of Human T-Cell Leukemia Virus Env[END_REF].

Furin-like cleavage site. By definition, the cleavage site between SU and TM forms the Cterminal end of SU (Fig. 21). Cleavage occurs in the Golgi network and is performed by cellular furin or furin-like proteases (reviewed in Henzy & Johnson, 2013). These proteases cleave precursor proteins after an RXKR or RXRR motif [START_REF] Hosaka | Arg-X-Lys/Arg-Arg motif as a signal for precursor cleavage catalyzed by furin within the constitutive secretory pathway[END_REF], and these sequences serve to distinguish the SU and TM domains of retroviral Env.

b. Domains of the TM Fusion peptide. Situated immediately after the furin-like cleavage site, the fusion peptide is the N-terminal part of TM in all retroviral envelopes, except for avian gamma-type in which it is situated about nine amino acids downstream (Fig. 21). This region is a 20-30 aa hydrophobic stretch that is crucial for fusion, being able to insert itself into the target membrane (Fig. 23, reviewed in Henzy & Johnson, 2013). Mutations in the fusion peptide are therefore extremely deleterious to viral survival. In addition to its shifted position, the fusion peptide of avian gamma-type Env is flanked by cysteines linked by a disulfide bond (Fig. 21, Gallaher, 1996).

Heptad repeats. The TM contains two heptad repeat regions (HR1 and HR2), one on each side of the immunosuppressive domain (when present) and the CX n C 1-2 domain (Fig. 21, reviewed in Henzy & Johnson, 2013). These heptad repeats are implicated in coiled-coil structure formation, important for the conformation of Env, but also the assembly of the Env trimer that constitutes the functional fusogenic arrangement (Fig. 21, Fass et al., 1996;[START_REF] Chan | Core structure of gp41 from the HIV envelope glycoprotein[END_REF][START_REF] Aydin | Crystal Structures of Beta-and Gammaretrovirus Fusion Proteins Reveal a Role for Electrostatic Stapling in Viral Entry[END_REF]. The HR1 domain forms a trimeric inter-TM coiled-coil, and its general length and structure is conserved among retroviruses [START_REF] Bénit | Identification, Phylogeny, and Evolution of Retroviral Elements Based on Their Envelope Genes[END_REF][START_REF] Aydin | Crystal Structures of Beta-and Gammaretrovirus Fusion Proteins Reveal a Role for Electrostatic Stapling in Viral Entry[END_REF]. The HR2 domain forms coils that are apposed to the inner HR1 coiled-coil (forming the so-called 6 helix bundle), in a slanted fashion and interacting with hydrophobic grooves on the outside of the latter [START_REF] Chan | Core structure of gp41 from the HIV envelope glycoprotein[END_REF][START_REF] Aydin | Crystal Structures of Beta-and Gammaretrovirus Fusion Proteins Reveal a Role for Electrostatic Stapling in Viral Entry[END_REF]. The structure of HR2 is more variable, in beta-type TM it forms a single long helix, while in gamma-type TM HR2 is much smaller and forms a short helical domain attached to an N-terminal tether region [START_REF] Aydin | Crystal Structures of Beta-and Gammaretrovirus Fusion Proteins Reveal a Role for Electrostatic Stapling in Viral Entry[END_REF].

Immunosuppressive domain. The immunosuppressive domain (ISD) is a conserved 20 aa region (Fig. 21) first identified in FLV TM and that of other gamma-type Env [START_REF] Cianciolo | Inhibition of lymphocyte proliferation by a synthetic peptide homologous to retroviral envelope proteins[END_REF]. Following studies identified immunosuppressive peptides in the corresponding regions of lentiviral TM [START_REF] Cianciolo | Human retrovirus-related synthetic peptides inhibit T lymphocyte proliferation[END_REF] and other beta-type TM such as MPMV and HERV-K [START_REF] Blaise | The envelope of Mason-Pfizer monkey virus has immunosuppressive properties[END_REF][START_REF] Morozov | The Transmembrane Protein of the Human Endogenous Retrovirus -K (HERV-K) Modulates Cytokine Release and Gene Expression[END_REF]. High conservation of the ISD sequence (the so-called CKS17 consensus sequence) is observed only among gamma-type TM (Fig. 22A, [START_REF] Cianciolo | Inhibition of lymphocyte proliferation by a synthetic peptide homologous to retroviral envelope proteins[END_REF][START_REF] Bénit | Identification, Phylogeny, and Evolution of Retroviral Elements Based on Their Envelope Genes[END_REF], and as such the term ISD is sometimes reserved to these envelopes (reviewed in Henzy & Johnson, 2013). In vivo tumor rejection assays in mice (Fig. 22B) seem to confirm the in vitro immunosuppressive properties for a number of envelopes, such as MLV [START_REF] Mangeney | Tumor cells expressing a retroviral envelope escape immune rejection in vivo[END_REF], MPMV [START_REF] Blaise | The envelope of Mason-Pfizer monkey virus has immunosuppressive properties[END_REF], Syncytin-2 and -B [START_REF] Mangeney | Placental syncytins: Genetic disjunction between the fusogenic and immunosuppressive activity of retroviral envelope proteins[END_REF] and FLV [START_REF] Schlecht-Louf | A Targeted Mutation within the Feline Leukemia Virus (FeLV) Envelope Protein Immunosuppressive Domain To Improve a Canarypox Virus-Vectored FeLV Vaccine[END_REF] for example. The presence of an ISD is also coherent with the fact that many exogenous retroviral infections lead to immunodeficiencies (reviewed in [START_REF] Denner | The transmembrane proteins contribute to immunodeficiencies induced by HIV-1 and other retroviruses[END_REF].

The effects of the ISD on the host immune system are numerous including inhibition of NK cells and cytotoxic T lymphocytes, down-regulation of pro-inflammatory and up-regulation of immuno-suppressive cytokines for example, but the exact mechanisms by which the ISD induces these changes are unknown (reviewed in [START_REF] Denner | The transmembrane proteins contribute to immunodeficiencies induced by HIV-1 and other retroviruses[END_REF].

CX n CC or CX n C motif. These two motifs, collectively referred to herein as CX n C 1-2 , are present immediately after the ISD in all retroviral envelopes and form one of the major distinguishing factors between beta-and gamma-type TM (Fig. 22, reviewed in Henzy & Johnson, 2013). For gamma-types the motif is CX 6 CC, with the two first cysteines forming an intra-TM disulfide bond which creates a loop between the two HR regions so that HR1 and HR2 are apposed [START_REF] Aydin | Crystal Structures of Beta-and Gammaretrovirus Fusion Proteins Reveal a Role for Electrostatic Stapling in Viral Entry[END_REF]. The third cysteine forms a disulfide bond with the CXXC motif in the SU, binding both subunits covalently until SU/receptor interaction upon which this bond is isomerized into the intra-CXXC bond (Fig. 23, Pinter et al., 1997). In beta-type envelopes, this motif takes the form of CX 4-7 C, presenting the two cysteines necessary to form the disulfide bond of the TM loop, but none to bind the SU. As a result in beta-type Env, the SU is not bound covalently to TM (Henzy & Coffin, 2013).

Transmembrane domain and proximal regions.

As is to be expected, the TM subunit contains a transmembrane domain, a hydrophobic stretch of about 40 aa that forms a helical domain spanning the membrane (Fig. 21). Deletion or cleavage of this domain turns the remaining N-terminal part of Env into a soluble factor, released from the cell as observed for Refrex-1 [START_REF] Ito | Refrex-1, a Soluble Restriction Factor against Feline Endogenous and Exogenous Retroviruses[END_REF], suppressyn [START_REF] Sugimoto | A novel human endogenous retroviral protein inhibits cell-cell fusion[END_REF], ERV3 [START_REF] Cohen | The nucleotide sequence of the env gene from the human provirus ERV3 and isolation and characterization of an ERV3-specific cDNA[END_REF] and human endogenous MER34 ORF (HEMO, Heidmann et al., 2017) for example.

The two domains immediately N-and C-terminally of the transmembrane are referred to as the membrane proximal external region (MPER) and the cytoplasmic tail respectively. The length of both regions is variable depending on the virus and can play an important role in regulating virion incorporation, membrane fusion and other Env properties (reviewed in Henzy & Johnson, 2013). The MPER is 20-30 aa longer in beta-than in gamma-type TM [START_REF] Bénit | Identification, Phylogeny, and Evolution of Retroviral Elements Based on Their Envelope Genes[END_REF], and it has been shown that in HIV-1 this stretch plays a role in Env incorporation and during fusion [START_REF] Vishwanathan | Importance of the Membrane-Perturbing Properties of the Membrane-Proximal External Region of Human Immunodeficiency Virus Type 1 gp41 to Viral Fusion[END_REF]. Studies of the cytoplasmic tail of MLV Env have shown that this 32 aa long sequence is cleaved to release the 16 aa R-peptide during maturation and this cleavage is essential to render the protein fusogenic [START_REF] Januszeski | Functional analysis of the cytoplasmic tail of Moloney murine leukemia virus envelope protein[END_REF]. The tails of JSRV and HERV-K TM have been shown to play a role in their oncogenic activity, probably through interaction with cellular signaling factors [START_REF] Hull | Mutational analysis of the cytoplasmic tail of jaagsiekte sheep retrovirus envelope protein[END_REF][START_REF] Lemaître | A human endogenous retrovirus-derived gene that can contribute to oncogenesis by activating the ERK pathway and inducing migration and invasion[END_REF]. Finally the cytoplasmic tail seems to be an important factor in Env incorporation into lentiviral particles [START_REF] Christodoulopoulos | Sequences in the Cytoplasmic Tail of the Gibbon Ape Leukemia Virus Envelope Protein That Prevent Its Incorporation into Lentivirus Vectors Sequences in the Cytoplasmic Tail of the Gibbon Ape Leukemia Virus Envelope Protein That Prevent Its Incorporation i[END_REF][START_REF] Freed | HIV-1 assembly, release and maturation[END_REF].

CKS-17 ALV RSV BaEV FLV GaLV MLV MPMV PERV REV BLV HTLV-1 Syncytin-1 Syncytin-2 Syncytin-A Syncytin-B Syncytin-Ory1 Syncytin-Car1 Syncytin-Rum1 Syncytin-Mar1 Syncytin-Ten1 Syncytin-Opo1 EnvV2 HEMO pan-Mars-Env2 Env-Cav1
N R R G L D L L F L K E G G L LQ N R A A I D F L L L A Q G H G C Q D V E -G M C C LQ N R A A I D F L L L A H G H G C E D V A -G M C C LQ N R R G L D L L T A E Q G G I C L A L Q -E K C C LQ N R R G L D I L F L Q E G G L C A A L K -E E C C LQ N R R G L D L L F L K E G G L C A A L K -E E C C LQ N R R G L D L L F L K E G G L C A A L K -E E C C LQ N R R G L D L L T A E Q G G I C L A L Q -E K C C LQ N R R G L D L L F L K E G G L C V A L K -E E C C LQ N R R G L D L L T A E Q G G I C L A L Q -E K C C AQ N R R G L D W L Y I R L G F Q C P T I N -E P C C AQ N R R G L D L L F W E Q G G L C K A L Q -E Q C C LQ N R R A L D L L T A E R G G T C L F L G -E E C C LQ N R R G L D M L T A A Q G G I C L A L D -E K C C LQ N R R A L D L I V A E R G G T C L F L Q -E E C C LQ N R R A L D L I T A E K G G T C L F L Q -E E C C LQ N R R G L D L L T A E R G G I C L A L Q -E K C C LQ N R M A L D I L T A A Q G G T C A I V K -T E C C LQ N R M A L D I L T A A Q G G T C A I I K -V E C C YQ N R L A L D Y L L A E E G G V C G K F N S S D C C LD N R I A L D F L L A E Q G G V C A I A N -T S C C IQ N R R S L D L L T L H Q R G L C A M H G -M E C C MD N R L A L D Y L L A E Q G G V C A V I N -K S C C SR K D H V L D I P T T Q R Q T A C G T V G -K Q C C VQ S R M A L D M K Q A Q Q G G V C A -Y N -T M C C YQ N R L A L D Y L L A K E G G V C G K F N L T S C C

Env induced membrane fusion

Retroviral Env proteins are class I membrane fusion proteins, the best-studied of which is the influenza hemagglutinin (HA) fusion protein. Interestingly the class I fusion proteins show very little sequence identity, but are structurally highly similar. HA is also synthesized as a precursor before being cleaved into the SU equivalent HA1 and the TM equivalent HA2, which also presents an N-terminal fusion peptide, an intra-molecular disulfide bond and two heptad repeat regions (reviewed in [START_REF] Podbilewicz | Virus and cell fusion mechanisms[END_REF][START_REF] Harrison | Viral membrane fusion[END_REF].

a. Implication of the cognate receptor in fusion

Necessity of destabilizing the metastable SU/TM. The uncleaved Env folds during synthesis to assume its most stable possible conformation. Separation of SU and TM after cleavage however, yields a metastable dimer as the new free energy minimum conformation of the TM subunit is its post-fusion state, in which the two HR domains are interacting to form the six helix bundle (reviewed in [START_REF] Harrison | Viral membrane fusion[END_REF]. The interaction of SU and TM is necessary to maintain TM in the less stable pre-fusion state and to prevent inclusion of the fusion peptide into the membrane TM is anchored to. Conversely, SU needs to dissociate from TM to allow the conformational changes necessary for productive fusion (reviewed in [START_REF] Harrison | Viral membrane fusion[END_REF]. Studies on HA have shown that cleavage of the precursor does not change its conformation in a major way, demonstrating the stabilizing effect of subunit interaction, the only major change being burial of the fusion peptide in a sheltered cavity formed by HA trimerization [START_REF] Chen | Structure of the hemagglutinin precursor cleavage site, a determinant of influenza pathogenicity and the origin of the labile conformation[END_REF].

Nature of the receptor and additional triggers. To release TM from its metastable form, SU has to interact with one or more cellular partners depending on the retrovirus. Commonly a single interaction is assumed to be sufficient as a trigger for fusion. Retroviruses presenting a gamma-type TM usually interact with cellular solute carrier (SLC) proteins (Fig. 24A), a family of functionally related multiple pass transmembrane proteins that are however highly divergent on the sequence and structure levels, with the exception of most ALV strains (reviewed in [START_REF] Greenwood | Transmission, Evolution, and Endogenization: Lessons Learned from Recent Retroviral Invasions[END_REF]. The latter use single-pass or GPI anchored molecules, as do most beta-type TM retroviruses (Fig. 24A, reviewed in [START_REF] Greenwood | Transmission, Evolution, and Endogenization: Lessons Learned from Recent Retroviral Invasions[END_REF].

In some cases, priming of the envelope for fusion is a bit more complex and needs several triggers. In both HIV-1 and HTLV-1, for example, specific interactions with several proteins are necessary. HIV-1 first binds to CD4 [START_REF] Klatzmann | T-lymphocyte T4 molecule behaves as the receptor for human retrovirus LAV[END_REF][START_REF] Dalgleish | The CD4 (T4) antigen is an essential component of the receptor for the AIDS retrovirus[END_REF], inducing a first shift in conformation and freeing an SU domain that then interacts with the co-receptor CCR5 [START_REF] Deng | Identification of a major co-receptor for primary isolates of HIV-1[END_REF][START_REF] Dragic | HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5[END_REF][START_REF] Alkhatib | CC CKR5: A RANTES, MIP-1alpha , MIP-1beta Receptor as a Fusion Cofactor for Macrophage-Tropic HIV-1[END_REF][START_REF] Choe | The β-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates[END_REF][START_REF] Doranz | A dual-tropic primary HIV-1 isolate that uses fusin and the β-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors[END_REF] or CXCR4 [START_REF] Feng | HIV-1 Entry Cofactor: Functional cDNA Cloning of a Seven-Transmembrane, G Protein-Coupled Receptor[END_REF], inducing the second and final conformation change necessary as trigger for fusion. In HTLV-1, the virus first attaches to heparan sulfate proteoglycans present at the surface of the cell, then interacts with a first receptor (Neuropilin-1, NRP-1; [START_REF] Ghez | Neuropilin-1 Is Involved in Human T-Cell Lymphotropic Virus Type 1 Entry[END_REF], probably going through a first conformational change. Subsequent interaction with SLC2A1 [START_REF] Manel | The Ubiquitous Glucose Transporter GLUT-1 Is a Receptor for HTLV[END_REF], also called GLUT-1, provokes further conformation changes and completes the triggering process (reviewed in [START_REF] Jones | Molecular aspects of HTLV-1 entry: Functional domains of the HTLV-1 surface subunit (SU) and their relationships to the entry receptors[END_REF]. While in most cases retroviral envelope fusion is pH-independant (in direct opposition to model class I fusogen HA, [START_REF] Podbilewicz | Virus and cell fusion mechanisms[END_REF], in some cases an acidification of the medium is a necessary secondary trigger for fusion. In ALV for example, binding to the cognate receptor induces a first conformational shift, but fusion only occurs after endosomal acidification [START_REF] Diaz-Griffero | Endocytosis is a critical step in entry of subgroup B avian leukosis viruses[END_REF].

Receptor specificity: example of the SLC1A5/RDR interference group. The SLC1A5 interference group (originally called RDR interference group) contains a number of gamma-type envelopes that use the SLC1A5 (RDR, ASCT2) multiple-pass transmembrane protein as a receptor, thus being able to block each other through receptor interference (reviewed in [START_REF] Sinha | Retroviruses of the RDR superinfection interference group: ancient origins and broad host distribution of a promiscuous Env gene[END_REF]. There are over two dozen exogenous and endogenous Env that have been sug- gested to be able to use this receptor for fusion, including Syncytin-1, Syncytin-Ory1, MMTV, baboon endogenous virus (BaEV), MPMV and the feline RD114 virus that gave the receptor its original name: RD114 and D-type receptor, RDR (reviewed in [START_REF] Sinha | Retroviruses of the RDR superinfection interference group: ancient origins and broad host distribution of a promiscuous Env gene[END_REF]. As mentioned above, the members of this interference group show poor sequence similarity except for a few conserved cysteine containing domains, especially an SDGGGX 2 DX 2 R motif (Fig. 24B, [START_REF] Cheynet | Identification of the hASCT2-binding domain of the Env ERVWE1/syncytin-1 fusogenic glycoprotein[END_REF]. Use of SLC1A5 as receptor confers a very broad species tropism to the virus (as illustrated in Miyaho et al., 2015, for example), implying that it may be involved in numerous cross-species transmission events after viral genome recombinations (reviewed in [START_REF] Sinha | Retroviruses of the RDR superinfection interference group: ancient origins and broad host distribution of a promiscuous Env gene[END_REF]. Recombination events in this envelope's history are clearly apparent with the examples of MMTV and Syncytin-Ory1, in which a gamma-type SLC1A5 using Env is associated with a betaretroviral RT sequence.

receptor multiple pass virus

SLC20A1 SLC19A2 SLC20A2 SLC7A1 SLC5A3 SLC1A5 SLC1A4 SLC49A1,SLC49A2 SLC53A1/XPR1 SLC5A1 SL19A1 SLC9A1 SLC2A1 MFSD2A CXCR4 (co) CCR5 (co) KoRV-A, GALV, FeLV-B, FeLV-T, WMV, 10A1-MLV KoRV-A, FeLV-A A-MLV, 10A1-MLV, GALV, FeLV-B E-MLV, BLV M813, HEMV RD-114, BaEV, REV, SRV, MPMV, Syncytin-1, Syncytin-Ory1 BaEV, Syncytin-1 FeLV-C X-MLV, P-MLV PERV-A GLN, MLV ALV-J HTLV Syncytin-2 FIV, HIV HIV single pass TFRC TVA TVB TVC CD4 CD134 NRP-1 (co) MMTV ALV-A ALV-B, -D, -E ALV-C HIV FIV HTLV GPI-anchored HYAL2 Ly6E EFNA1-5 JSRV Syncytin-A IAPE A B M A L P 1 3 T L T A P P P C -R C M T S 3 7 M P R N C Y H S A T L C M HA N TH Y W T G K M 1 M G F T 2 6 Q K R Y G R P C -D C S G G 5 4 V H S S C Y T S Y Q Q C R SG N KT Y Y T A T L 2 1 M N F N 3 0 Q Q K H G K P C -D C A G G 5 7 V H A T C Y N H Y Q Q C T IG N KT Y L T A T M 2 4 M T V K 3 0 H Q Q H G K P C -D C A G G 5 4 V H S S C Y T T Y Q E C F FG N KT Y Y T A I L 2 4 M N F N 3 0 Q Q K H G K P C -D C A G G 5 7 V H A S C Y N H Y Q Q C N IG N KT Y L T A T I 2 4 M N F E 3 0 Q N K H G K P C -E C K G G 5 7 V H S T C Y S S Y Q Q C T IG N KT Y F T A T I 2 4 M L C I 2 5 T A L F G A P C -D C K G G 6 8 M H S S C Y S S F S Q C T QG N NT Y F T A I L 2 2 M D C L 3 5 Q Q L W G L P C -D C S G G 7 0 M H S T C Y E K T Q E C T LL G KT Y F T A I L 1 6 M A L S 3 1 Q Q H H --P C -T C S G G 6 5 V H S S C Y T Q Y Q T C N KG N QR L L F A V I 1 8 M H S F 2 9 K T L Y G T P C -E C R G G 6 5 M H V H C Y S E V Q S C T RN N KT Y Y T A I L 2 5 M I S T 2 3 H A L F G A P C -D C K G G 6 4 L H S L C Y T S T Q Q C T GK S GT Y L T S R Q 2 4 M D C L 3 5 Q Q L W G L P C -D C S G G 6 8 M H S T C Y E K A Q E C T LM G KT Y F T A I L 1 6 M F S Q 2 7 D A L F G P P C -D C Q G G 7 5 H S A C C Y Y K V Y T C S KG G QT Y F T A R I 2 8 M K L P 2 6 Q K Q H G K P C -E C S G G 5 5 M H S S C Y T E Y R Q C R AN N KT Y Y T A T L 2 1 Syncytin-1 BaEV SRV-1 SRV-2 MPMV SRV-4 SMRV SNV Syncytin-Ory1 Dasy-Env1.1 TvERV(D) REV-A DrERV RD114 Syncytin-1 BaEV SRV-1 SRV-2 MPMV SRV-4 SMRV SNV Syncytin-Ory1 Dasy-Env1.1 TvERV(D) REV-A DrERV RD114 N P S C P -G G L G V T V C W T Y F T Q T G M S D G G G V Q D Q A R E K H V K EV I 1 8 0 R N K R Q S P C -N G I K G Q S I C W S T T A P I H V S D G G G P L D T T R I K S V Q RK L 1 8 9 R P K R I A G C P E N K K G Q V V C W N S Q P S V H M S D G G G P Q D K V R E I I V N KK F 1 9 7 R P K R S A G C -T G T V G Q H I C W N P K A P V H I S D G G G P Q D K A R E I A V Q KR L 1 8 8 R A R R T A G C P N G K K G Q V V C W N S R P S V H I S D G G G P Q D K A R D I I V N KK F 1 9 6 K A K R S A G C P K D E L G K T A C W S A T P P V H V S D G G G P Q D K V R E I L V Q KK F 1 9 2 R T K R Q A G C -D G T V G K S V C W N Q Q A P I H V S D G G G P Q D A V R E L Y V Q KQ I 1 8 5 R Q K R Q A S C -T G T V G K P V C W D P V A P V Y V S D G G G P T D M I R E E S V R ER L 1 9 1 R H K R S P S C P --T K G Q V A C W Y P I A P V G V S D G G G P Q D T L T K L K T Q KV L 1 8 6 R Q K R Q A G C -Y G T I G D T V C W N K I P P A H I S D G G G P Q D Q A R Q I I V Q ER M 1 8 4 R S K R Q A S C D K I N I G K N V C W S L H A P I H V S D G G G P T D Q I R E M E V K ER V 1 8 9 R P K R Q A S C -T G I I G K P V C W D P A A P V Y V S D G G G P T D M I R E E S V R ER L 1 9 1 R H K R P D K C P G -E I G K P A C W K L D S P L G -S D G G G P Q D K R R T E R V R IQ I 1 7 9 R H K R Q S P C -R G S I N Q P V C W S A T A P I H I S D G G G P L D T K R V W T V Q KR L 1 9 0 R P K R
Rodent cells form an exception to this broad species tropism, being highly resistant to infection using most SLC1A5 group Env. A comparative study of human and mouse SLC1A5 revealed the presence of a hypervariable 21 aa sequence in an extracellular loop, and replacing the corresponding sequence with that of the other species was able to completely restore/abrogate fusion [START_REF] Marin | N-Linked Glycosylation and Sequence Changes in a Critical Negative Control Region of the ASCT1 and ASCT2 Neutral Amino Acid Transporters Determine Their Retroviral Receptor Functions[END_REF]. These results suggest that the sequence variability in a short stretch of SLC1A5 (which also includes 10 aa insertions) is sufficient to completely block Env/receptor interaction. The observation that some members of the SLC1A5 interference group (such as Syncytin-1 and BaEV) could still infect rodent cells, led to the identification of SLC1A4 (RDR2, ASCT1) as an alternative receptor for these envelopes [START_REF] Marin | Sodium-Dependent Neutral Amino Acid Transporter Type 1 Is an Auxiliary Receptor for Baboon Endogenous Retrovirus[END_REF]. It became apparent that in the case of SLC1A4, N-linked glycosylations were responsable for its resistance to most members of the interference group, extending the notion of receptor specificity beyond just the amino acid sequence. This can be further exemplified by the hamster SLC1A4 which was unusable even for Syncytin-1 and BaEV, which was linked by [START_REF] Marin | N-Linked Glycosylation and Sequence Changes in a Critical Negative Control Region of the ASCT1 and ASCT2 Neutral Amino Acid Transporters Determine Their Retroviral Receptor Functions[END_REF] to the presence of a third N-glycosylation site that is absent in all other rodents.

b. Model of viral fusion protein class I induced membrane fusion

Metastable SU/TM. The metastable SU/TM can be likened to a spring-loaded state in which the SU keeps the TM in an unstable form. Env forms hollow homo-trimers at the surface of the membrane with the fusion peptide tucked into the cavity [START_REF] Riedel | Native structure of a retroviral envelope protein and its conformational change upon interaction with the target cell[END_REF]. Following SU/receptor interaction, the conformation of the SU changes and it dissociates at least in part from TM, therefore no longer stabilizing the TM pre-fusion conformation (Fig. 23). In the case of gamma-type Env, this release implies rupture of the disulfide bond linking SU and TM. It has been shown in both MLV and HTLV-1 that this is due to activation of a thiol group carried by the cysteine of the SU CXXC motif that is not implicated in the SU-TM bond [START_REF] Wallin | Isomerization of the intersubunit disulphide-bond in Env controls retrovirus fusion[END_REF][START_REF] Li | Intersubunit Disulfide Isomerization Controls Membrane Fusion of Human T-Cell Leukemia Virus Env[END_REF]. Activation of this thiol leads to isomerization of the bond, i.e., shifting it from SU-TM to an intra-SU bond between the two cysteines of the CXXC motif [START_REF] Wallin | Isomerization of the intersubunit disulphide-bond in Env controls retrovirus fusion[END_REF][START_REF] Li | Intersubunit Disulfide Isomerization Controls Membrane Fusion of Human T-Cell Leukemia Virus Env[END_REF].

3.2

Extended intermediate. Release of the now unstable TM subunit leads to its extension, during which the fusion peptides of each unit of the trimer are inserted in the opposite cell membrane, due to their hydrophobic properties (Fig. 23). While there is no direct evidence for the existence of this structure, it is extremely probable (reviewed in [START_REF] Harrison | Viral membrane fusion[END_REF]. In a recent study on MLV, [START_REF] Riedel | Native structure of a retroviral envelope protein and its conformational change upon interaction with the target cell[END_REF] show that the length bridged by TM between the viral and cellular membrane is about 120 Å, significantly longer than the 85 Å height of pre-fusion Env.

Six-helix bundle. The intermediate extended structure in which TM is inserted in both membranes still does not represent the minimum energy-free state of the system, to reach this conformation the TM slowly fold in onto themselves, the region around the CX n C 1-2 domain serving as hinge (Fig. 23, reviewed in [START_REF] Harrison | Viral membrane fusion[END_REF]. This folding brings the fusion peptide closer to the transmembrane region and draws the viral and cellular membranes closer, until they form a hemifusion stalk and finally a fusion pore (reviewed in [START_REF] Podbilewicz | Virus and cell fusion mechanisms[END_REF][START_REF] Harrison | Viral membrane fusion[END_REF]. Achievement of the fusion pore marks the end of TM refolding, the fusion peptide and transmembrane domains are next to each other and HR1 and HR2 form the stable six helix bundle, the free-energy minimum state of TM (Fig. 23, reviewed in [START_REF] Harrison | Viral membrane fusion[END_REF]. Interestingly, this conformational change represents an inversion of TM organization, as in its pre-fusion state the C-terminal part was in the center of the trimer, while in the post-fusion state the N-terminal HR1 form the inner coiled-coil. Such a major rearrangement implies a probable dissociation and re-association of the trimer, but no transitory monomeric form has been observed so far (reviewed in Podbilewicz, 2014).

Syncytins in mammals

Bridging the gap between the two previous chapters, syncytins are a series of ERV envelope genes that have been exapted in different mammalian taxa for a role in placentation (Fig. 25. A syncytin is a captured retroviral envelope that is i) expressed specifically (or at the least preferentially) in the placenta, ii) conserved over the course of evolution, and iii) fusogenic. In this first part I will present a comprehensive list of described syncytins and their particularities and relationship to fused placental cells. As often in biology, the human and mouse counterparts have been studied more extensively and as such will be described in more details.

Human syncytins: Syncytin-1 and Syncytin-2

In humans, two syncytins have been described and named Syncytin-1 and -2. They are exapted env genes from an HERV-W and an HERV-FRD copy, respectively [START_REF] Blond | An Envelope Glycoprotein of the Human Endogenous Retrovirus HERV-W Is Expressed in the Human Placenta and Fuses Cells Expressing the Type D Mammalian Retrovirus Receptor[END_REF][START_REF] Mi | Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis[END_REF]Blaise et al., 2003).

a. Syncytin-1

Discovery. The screening of a placental cDNA library by [START_REF] Blond | Molecular characterization and placental expression of HERV-W, a new human endogenous retrovirus family[END_REF] led to the first description of the HERV-W group and the single conserved env ORF with placental expression belonging to it. The next year, both [START_REF] Blond | An Envelope Glycoprotein of the Human Endogenous Retrovirus HERV-W Is Expressed in the Human Placenta and Fuses Cells Expressing the Type D Mammalian Retrovirus Receptor[END_REF] and [START_REF] Mi | Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis[END_REF] confirm that this Env is indeed expressed in the human placenta and show that expression occurs in the ST. Additionally, they show that transfection of the Env induces formation of large syncytia, demonstrating fusogenic activity. [START_REF] Blond | An Envelope Glycoprotein of the Human Endogenous Retrovirus HERV-W Is Expressed in the Human Placenta and Fuses Cells Expressing the Type D Mammalian Retrovirus Receptor[END_REF] also identify SLC1A5 as the major cognate receptor of Syncytin-1, with SLC1A4 as alternative receptor [START_REF] Lavillette | The Envelope Glycoprotein of Human Endogenous Retrovirus Type W Uses a Divergent Family of Amino Acid Transporters / Cell Surface Receptors[END_REF]. On the basis of these results, [START_REF] Mi | Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis[END_REF] call this protein Syncytin, in reference to its probable role in establishing syncytia in vivo. It will be renamed to Syncytin-1 upon the discovery of Syncytin-2 by Blaise et al. (2003) a few years later. The expression profile of Syncytin-1 was later expanded to include all subpopulations of villous (ST and CT) and extravillous trophoblast cells [START_REF] Malassiné | Expression of HERV-W Env glycoprotein (syncytin) in the extravillous trophoblast of first trimester human placenta[END_REF][START_REF] Muir | Human endogenous retrovirus-W envelope (syncytin) is expressed in both villous and extravillous trophoblast populations[END_REF].

Subsequent studies revealed that the locus and sequence of syncytin-1 as well as the LTR driving its expression are conserved not only among humans, but also other Hominoidea (apes), displaying purifying selection [START_REF] Mallet | The endogenous retroviral locus ERVWE1 is a bona fide gene involved in hominoid placental physiology[END_REF][START_REF] Bonnaud | Evidence of selection on the domesticated ERVWE1 env retroviral element involved in placentation[END_REF]. Interestingly, when compared to a consensus sequence of non-conserved HERV-W Env, Syncytin-1 displays a 4 aa deletion in the cytoplasmic tail which is crucial for its fusogenic activity and also observed in the other investigated Hominoidea species, suggesting it represents an early adaptation to its new role [START_REF] Bonnaud | Evidence of selection on the domesticated ERVWE1 env retroviral element involved in placentation[END_REF]. The insertion of the Syncytin-1 encoding provirus occurred about 40 Mya (Fig. 25), before separation of Hominoidae from old world monkeys, but the gene was lost in the latter (Bonnaud et al., 2005, see 3.3.1.a. for discussion as to why Syncytin-1 was lost in old world monkeys).

Syncytin-1 and cell-cell fusion. This conservation for over 25 My in Hominoidea strongly suggested that Syncytin-1 plays an important physiological role in these species. Due to its nature as an ERV envelope and its expression in a fused cell layer of the placenta, the first proposed role for Syncytin-1 was the establishment of the ST in the placenta [START_REF] Blond | An Envelope Glycoprotein of the Human Endogenous Retrovirus HERV-W Is Expressed in the Human Placenta and Fuses Cells Expressing the Type D Mammalian Retrovirus Receptor[END_REF][START_REF] Mi | Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis[END_REF]. Accordingly, suppression of Syncytin-1 expression in primary cytotrophoblasts ex vivo was shown to inhibit cell-cell fusion [START_REF] Frendo | Direct Involvement of HERV-W Env Glycoprotein in Human Trophoblast Cell Fusion and Differentiation[END_REF]. Investigation of the expression profile of the cognate receptor SLC1A5 in the placenta revealed that it was specifically expressed in the CT layer, but somewhat surprisingly not in the ST [START_REF] Hayward | Distribution of human endogenous retrovirus type W receptor in normal human villous placenta[END_REF]. The absence of expression in the fused layer might be due to a down-regulation of receptor expression postfusion, as is observed in trophoblast derived cell lines [START_REF] Hayward | Distribution of human endogenous retrovirus type W receptor in normal human villous placenta[END_REF]. [START_REF] Hayward | Distribution of human endogenous retrovirus type W receptor in normal human villous placenta[END_REF] also show that the expression of the receptor is more or less constant during pregnancy, raising the question as to why and how the CT layer persists as individualized cells up to term instead of fusing with the ST. A series of later studies suggested that, in vivo, the CT cells needs to become fusion competent, implicating a number of intracellular changes (reviewed in [START_REF] Gerbaud | Review: An overview of molecular events occurring in human trophoblast fusion[END_REF]. It seems that the fusion competency of CT can be regulated both positively and negatively by ST secretions and that commitment to fusion implies an increase in cell-cell adhesion, probably bringing the CT closer to the ST (reviewed in [START_REF] Gerbaud | Review: An overview of molecular events occurring in human trophoblast fusion[END_REF].

Other roles of Syncytin-1 in human placentas. Syncytin-1 knock-down in primary CT cells inhibited differentiation of the latter, in addition to fusion [START_REF] Frendo | Direct Involvement of HERV-W Env Glycoprotein in Human Trophoblast Cell Fusion and Differentiation[END_REF], and its expression in other unfused trophoblastic tissues [START_REF] Muir | Human endogenous retrovirus-W envelope (syncytin) is expressed in both villous and extravillous trophoblast populations[END_REF], especially the EVT [START_REF] Malassiné | Expression of HERV-W Env glycoprotein (syncytin) in the extravillous trophoblast of first trimester human placenta[END_REF], hints at other potential roles than just cell-cell fusion. Several studies have for example confirmed a role for Syncytin-1 in cell differentiation as well as proliferation and anti-apoptotic processes (reviewed in [START_REF] Bolze | Chapter Five -Contribution of Syncytins and Other Endogenous Retroviral Envelopes to Human Placenta Pathologies[END_REF]. Syncytin-1 presents a conserved ISD (Fig. 22A, de Parseval et al., 2003), though a later study showed that it does not display immunosuppression in tumor rejection assays in mice [START_REF] Mangeney | Placental syncytins: Genetic disjunction between the fusogenic and immunosuppressive activity of retroviral envelope proteins[END_REF]. Still, it has been shown that Syncytin-1 expression on trophoblast derived exosomes was able to modulate immune responses, and that surprisingly this phenomenon is mediated at least in part by a region of SU [START_REF] Holder | Immune cell activation by trophoblast-derived microvesicles is mediated by syncytin 1[END_REF][START_REF] Tolosa | The endogenous retroviral envelope protein syncytin-1 inhibits LPS/PHAstimulated cytokine responses in human blood and is sorted into placental exosomes[END_REF]. The implication of Syncytin-1 in other processes than just cell-cell fusion might also be implicated in the abnormal expression observed in a number of placental pathologies, such as pre-eclampsia (reviewed in [START_REF] Bolze | Chapter Five -Contribution of Syncytins and Other Endogenous Retroviral Envelopes to Human Placenta Pathologies[END_REF], see also 3.3.3.c.).

Regulation of Syncytin-1 expression.

Due to its diversity of functions during human placentation, Syncytin-1 expression needs to be tightly regulated through both epigenetic modifications and transcription factors (reviewed in [START_REF] Huang | Epigenetic and non-epigenetic regulation of syncytin-1 expression in human placenta and cancer tissues[END_REF][START_REF] Bolze | Chapter Five -Contribution of Syncytins and Other Endogenous Retroviral Envelopes to Human Placenta Pathologies[END_REF]. As for many other ERV sequences, expression of Syncytin-1 is highly dependent on the CpG methylation state of the 5' promoter, strongly methylated in nearly all non placental tissues [START_REF] Matoušková | CpG methylation suppresses transcriptional activity of human syncytin-1 in non-placental tissues[END_REF][START_REF] Gimenez | Comparative methylation of ERVWE1/syncytin-1 and other human endogenous retrovirus LTRs in placenta tissues[END_REF]. Interestingly, the Syncytin-1 promoter is unmethylated during the beginning of pregnancy, but methylated towards term, in accordance with an observed decrease of fusion and increase of trophoblast apoptosis at the end of gestation, and suggesting an important temporal regulation of expression [START_REF] Gimenez | Comparative methylation of ERVWE1/syncytin-1 and other human endogenous retrovirus LTRs in placenta tissues[END_REF]. Immediately upstream of the Syncytin-1 associated 5' LTR is another ERV-derived regulatory sequence which contributes to transcriptory regulation and presents binding sites for "chorion-specific transcription factor GCMa" (GCMa, from its original name glial cells missing homolog a, reviewed in [START_REF] Bolze | Chapter Five -Contribution of Syncytins and Other Endogenous Retroviral Envelopes to Human Placenta Pathologies[END_REF]. Inhibition of GCMa expression ex vivo has been shown to decrease syncytialization and EVT differentiation and to increase cell proliferation [START_REF] Baczyk | Glial cell missing-1 transcription factor is required for the differentiation of the human trophoblast[END_REF]. In the 5' HERV-W LTR itself is a site able to bind steroid hormones to activate Syncytin-1 expression [START_REF] Strick | Proliferation and cell-cell fusion of endometrial carcinoma are induced by the human endogenous retroviral Syncytin-1 and regulated by TGF-β[END_REF]. This pathway however also leads to the expression of TGFβ whose effects seem to augment cell proliferation and reduce syncytialization [START_REF] Strick | Proliferation and cell-cell fusion of endometrial carcinoma are induced by the human endogenous retroviral Syncytin-1 and regulated by TGF-β[END_REF]. A TGFβ mediated reduction of GCMa activity, and thus Syncytin-1 expression, has been proposed as an explanation [START_REF] Xu | Aberrant TGFβ signaling contributes to altered trophoblast differentiation in preeclampsia[END_REF].

b. Syncytin-2

Discovery. Following identification of Syncytin-1, a genome-wide screening by Blaise et al. (2003) for new fusogenic human ERV Env revealed the existence of Syncytin-2. The same study shows that Syncytin-2 transfection is able to induce syncytialization ex vivo, is expressed specifically in the placenta and that it is highly conserved and fusogenic in all primates at the same locus, suggesting it inserted over 40 Mya (Fig. 25) and has been conserved since under purifying selection (Blaise et al., 2003). Using the GeneBridge4 human/Chinese hamster radiation hybrid panel [START_REF] Gyapay | A radiation hybrid map of the human genome[END_REF], [START_REF] Esnault | A placenta-specific receptor for the fusogenic, endogenous retrovirusderived, human syncytin-2[END_REF] were able to identify the multiple-pass major facilitator superfamily domain containing 2 A (MFSD2A) carbohydrate transporter as the cognate receptor necessary for Syncytin-2 mediated fusion.

Syncytin-2 and in-fusion. In accordance with a role of Syncytin-2 in CT fusion and ST establishment in the human placenta, siRNA mediated knockdown of both Syncytin-2 or its receptor significantly reduced the fusion of primary trophoblastic cells and trophoblast-derived cells lines [START_REF] Vargas | Syncytin-2 Plays an Important Role in the Fusion of Human Trophoblast Cells[END_REF][START_REF] Toufaily | MFSD2a, the Syncytin-2 receptor, is important for trophoblast fusion[END_REF]. Interestingly, the effect of Syncytin-2 knockdown on cell-cell fusion is much more important than that observed for Syncytin-1 knockdown, suggesting that the former might be the principal mediator of cell-cell fusion in vivo [START_REF] Vargas | Syncytin-2 Plays an Important Role in the Fusion of Human Trophoblast Cells[END_REF]. The expression profile of both Syncytin-2 and MFSD2A strongly differ from those of Syncytin-1 and SLC1A5. While Syncytin-1 expression occurs in a more or less constant fashion in all trophoblast cells, Syncytin-2 is expressed transiently at the ST facing membrane of some CT only, suggesting a much tighter control of its spatio-temporal expression [START_REF] Malassiné | Expression of the fusogenic HERV-FRD Env glycoprotein (syncytin 2) in human placenta is restricted to villous cytotrophoblastic cells[END_REF]. Conversely, while SLC1A5 was expressed in CT but not ST cells, MFSD2A is expressed exclusively in the ST [START_REF] Esnault | A placenta-specific receptor for the fusogenic, endogenous retrovirusderived, human syncytin-2[END_REF]. With regards to these expression profiles, [START_REF] Esnault | A placenta-specific receptor for the fusogenic, endogenous retrovirusderived, human syncytin-2[END_REF] propose that Syncytin-2 mediated fusion occurs through "in-fusion" in which CT can only fuse into the ST since it is the only layer expressing the cognate receptor. This model also explains why the CT cannot form a second fused layer, as the absence of MFSD2A at their surface prevents Syncytin-2 mediated CT-CT fusion.

Other roles of Syncytin-2 in human placentas. Syncytin-2 presents a conserved gamma-type ISD (Fig. 22, de Parseval et al., 2003) and is indeed immunosuppressive in in vivo mouse tumor rejection assays [START_REF] Mangeney | Placental syncytins: Genetic disjunction between the fusogenic and immunosuppressive activity of retroviral envelope proteins[END_REF]. Its very localized expression profile might indicate that Syncytin-2 has only limited opportunities to be presented to the maternal immune system, but is has been shown that it is also present in trophoblast derived exosomes, alongside Syncytin-1 [START_REF] Vargas | Syncytin proteins incorporated in placenta exosomes are important for cell uptake and show variation in abundance in serum exosomes from patients with preeclampsia[END_REF]. Similarly to Syncytin-1, Syncytin-2 expression is abnormal in a number of placental pathologies such as pre-eclampsia (reviewed in [START_REF] Bolze | Chapter Five -Contribution of Syncytins and Other Endogenous Retroviral Envelopes to Human Placenta Pathologies[END_REF].

Regulation of Syncytin-2 expression. While epigenetic controls seem to play a role in restricting expression of Syncytin-2 to the placenta, there is no temporal variation in CpG methylation of the placental LTR as was observed for Syncytin-1, it remains unmethylated from beginning to term [START_REF] Gimenez | Comparative methylation of ERVWE1/syncytin-1 and other human endogenous retrovirus LTRs in placenta tissues[END_REF][START_REF] Liang | GCM1 Regulation of the Expression of Syncytin 2 and Its Cognate Receptor MFSD2A in Human Placenta1[END_REF]. On the transcription factor level, [START_REF] Liang | GCM1 Regulation of the Expression of Syncytin 2 and Its Cognate Receptor MFSD2A in Human Placenta1[END_REF] revealed that intriguingly expression of both Syncytin-2 and MFSD2A is regulated by GCMa, the same transcription factor regulating expression of Syncytin-1. Ectopic expression of GCMa can induce ectopic expression of Syncytin-2 and its receptor provoking syncytialization ex vivo [START_REF] Liang | GCM1 Regulation of the Expression of Syncytin 2 and Its Cognate Receptor MFSD2A in Human Placenta1[END_REF]. Additionally, it seems that GCMa plays an important role in the demethylation of the Syncytin-2 promoter [START_REF] Liang | GCM1 Regulation of the Expression of Syncytin 2 and Its Cognate Receptor MFSD2A in Human Placenta1[END_REF]. It is interesting to consider that while they are all inducible by GCMa, Syncytin-1 and -2 and MFSD2A present very distinct expression patterns, suggesting that other factors play an important role in regulating their spatiotemporal expression. Nevertheless, it emerges that this transcription factor plays a major role in human placenta ST establishment, being able to induce two syncytin genes and the cognate receptor of one of them. Recently JunD has been proposed to be one of the additional factors regulating Syncytin-3 expression [START_REF] Toufaily | A CRE/AP-1-like motif is essential for induced syncytin-2 expression and fusion in human trophoblast-like model[END_REF].

Mouse syncytins: Syncytin-A and Syncytin-B a. Discovery

Following identification of human syncytins, both rodent syncytin genes were discovered in the same study by [START_REF] Dupressoir | Syncytin-A and syncytin-B, two fusogenic placenta-specific murine envelope genes of retroviral origin conserved in Muridae[END_REF] through an in silico screen of the mouse genome. Both genes are gamma-type ERV envelopes that are specifically expressed in the placenta and able to mediate cell-cell fusion ex vivo [START_REF] Dupressoir | Syncytin-A and syncytin-B, two fusogenic placenta-specific murine envelope genes of retroviral origin conserved in Muridae[END_REF]. Interestingly, both envelope genes are unrelated to the previously described human syncytins, deriving from distinct endogenization events by different retroviruses [START_REF] Dupressoir | Syncytin-A and syncytin-B, two fusogenic placenta-specific murine envelope genes of retroviral origin conserved in Muridae[END_REF]. While the original study only reported conservation of Syncytin-A and -B in Muridae (mouse, rat) and Cricetidae (hamster, vole) with an estimated integration time of 20 Mya, a subsequent study by [START_REF] Vernochet | The captured retroviral envelope syncytin-A and syncytin-B genes are conserved in the Spalacidae together with hemotrichorial placentation[END_REF] revealed that is was also conserved in Spalacidae (blind mole rats), pushing back the age of endogenization to about 40 Mya (Fig. 25), during the radiation of Muroidea. While both Syncytin-A and -B present a conserved ISD sequence (Fig. 22, Dupressoir et al., 2005), only Syncytin-B is immunosuppressive in tumor rejection assays, a remarkable parallel to the situation in humans where Syncytin-2 but not -1 displays immunosuppressive properties in this assay [START_REF] Mangeney | Placental syncytins: Genetic disjunction between the fusogenic and immunosuppressive activity of retroviral envelope proteins[END_REF]. Recently the receptor of Syncytin-A was shown to be the lymphocyte antigen 6E (Ly6E) protein [START_REF] Bacquin | A Cell Fusion-Based Screening Method Identifies Glycosylphosphatidylinositol-Anchored Protein Ly6e as the Receptor for Mouse Endogenous Retroviral Envelope Syncytin-A[END_REF], this was further confirmed by showing the importance of Ly6E for placenta formation in vivo [START_REF] Langford | Deletion of the Syncytin A receptor Ly6e impairs syncytiotrophoblast fusion and placental morphogenesis causing embryonic lethality in mice[END_REF]. Interestingly, Ly6E is a GPI-anchored and not a multiple pass protein, an unusual receptor for an otherwise classical gamma-type envelope. The cognate receptor of Syncytin-B has not been described so far.

b. Expression in 2 segregated layers

While the definitive human placenta is hemomonochorial with a fused cell layer overlying the discontinuous remains of an unfused layer, Muroidae all present a hemotrichorial placenta

3.2

presenting, in most cases, two internal fused cell layers and an external continuous but loose and fenestrated non-fused layer (see 1.2.2.d. and Vernochet et al., 2014). The presence of two fused layers and two distinct syncytins with different cognate receptors raised the question of whether each syncytin was responsible for the establishment of one ST layer. This hypothesis was confirmed by in situ hybridization [START_REF] Simmons | Early patterning of the chorion leads to the trilaminar trophoblast cell structure in the placental labyrinth[END_REF] and through two separate electron microscopy studies showing that knockout of syncytin-A disrupted establishment of ST-I and knockout of syncytin-B did the same for ST-II [START_REF] Dupressoir | Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene[END_REF](Dupressoir et al., , 2011)). Similarly, the knockout of Ly6E prevents syncytialization of ST-I but not ST-II [START_REF] Langford | Deletion of the Syncytin A receptor Ly6e impairs syncytiotrophoblast fusion and placental morphogenesis causing embryonic lethality in mice[END_REF]. It is therefore likely that the independent capture and exaptation of two distinct ERV Env with a distinct receptor preference was instrumental in the establishment of the very particular hemotrichorial placental phenotype observed in mouse and other Muroidea.

Interestingly, GCMa has been shown to be necessary in mice for correct establishment of placental structures [START_REF] Schreiber | Placental failure in mice lacking the mammalian homolog of glial cells missing, GCMa[END_REF], and the loci expressing Syncytin-A and -B present several putative GCMa binding sites [START_REF] Dupressoir | Syncytin-A and syncytin-B, two fusogenic placenta-specific murine envelope genes of retroviral origin conserved in Muridae[END_REF]. A link between GCMa and expression of Syncytin-B is also suggested by the observation of their co-expression in the same placental cell layer, with GCMa expression preceding that of Syncytin-B [START_REF] Simmons | Early patterning of the chorion leads to the trilaminar trophoblast cell structure in the placental labyrinth[END_REF]. Another study reports that Syncytin-A expression might also be under control of GCMa [START_REF] Schubert | Identification of integrin-α4, Rb1, and syncytin A as murine placental target genes of the transcription factor GCMa/Gcm[END_REF] and while this would be in accordance with the fact that GCMa deprived mice display embryonic lethality at the same date as mice knocked out for both syncytin-A and -B (but earlier than in single knockouts, Fig. 26, Schreiber et al., 2000;Dupressoir et al., 2011), no GCMa expression in the ST-I progenitor cells has been reported by [START_REF] Simmons | Early patterning of the chorion leads to the trilaminar trophoblast cell structure in the placental labyrinth[END_REF]. In any case, the importance of GCMa for syncytin expression and placental morphology in mice is a striking parallel to the case of human syncytins and a noteworthy example of evolutive convergence.

c. Impact of Syncytin knockout on placental physiology

The knockout experiments discussed above are of immense interest to the study of syncytins, as they are the most conclusive proof of their placental role available today. In other species, such experiments are either inconceivable (humans) or entirely less feasible for technical reasons (other mammals), but in mice it is possible to specifically knockout the syncytin genes and observe the direct effect on placental morphology. Interestingly, knockout of syncytin-A is lethal, all embryos dying at about mid-gestation, while in the case of syncytin-B viable embryos are born, albeit in smaller numbers and with moderate growth retardation [START_REF] Dupressoir | Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene[END_REF](Dupressoir et al., , 2011)). The lessened impact of syncytin-B inactivation might be at least partly explained through the observed induction of gap junction beta-6 protein expression, a protein that is not present in the mouse placenta under normal conditions (Dupressoir et al., 2011). Expression of this protein could lead to the establishment of communicating gap junctions, both between cells of the unfused ST-II or with the fused ST-I layer, thereby restoring a syncytium-like state in which there is a certain cytoplasmic continuity across the layer (Dupressoir et al., 2011).

3.2

A different impact depending on the inactivated syncytin is also observed at the morphological level. While knockout of each syncytin disrupts the syncytialization of the corresponding layer, they have more far-reaching impacts on other placental tissues [START_REF] Dupressoir | Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene[END_REF](Dupressoir et al., , 2011)). Syncytin-A knockout leads to an over-proliferation of the individualized ST-I cells, significantly reducing the volume of maternal blood spaces and probably seriously impacting blood flow and blood supply to the placenta [START_REF] Dupressoir | Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene[END_REF]. Syncytin-B knockout on the other hand leads to an over-expansion of maternal blood spaces, once again impacting the volume and flow speed of placental blood supply, but in this case there is no trophoblast over-proliferation (Dupressoir et al., 2011). A double knockout for both syncytin genes displays complete embryonic lethality a few days earlier than the syncytin-A knockout, suggesting a cumulative effect of both inactivations (Dupressoir et al., 2011).

Other mammalian syncytins

In the years since the discovery of human and mice syncytins, the investigation of the implication of ERV Env in other mammalian placentas led to rapid identification of new syncytins in many mammalian clades. All presently described syncytins will be briefly presented in this section, classified by the species in which it was first described. Of note, they all correspond to distinct endogenization events that are specific to the clade in question and implicate a variety of viral species (reviewed in [START_REF] Lavialle | Paleovirology of 'syncytins', retroviral env genes exapted for a role in placentation[END_REF]Denner, 2016). Unless mentioned otherwise, all these syncytins are specifically expressed in cells of the maternofetal interface and are fusogenic ex vivo.

a. Rabbit: Syncytin-Ory1

Syncytin-Ory1 was identified in the rabbit (Oryctolagus cuniculus) by Heidmann et al. (2009). It seems to be conserved among Leporidae (rabbits and hares) but not Ochotonidae (pikas), which put the time of insertion around 12 Mya at the time of publication (Heidmann et al., 2009), a date that has been pushed back to about 20 Mya (Fig. 25) according to the TimeTree database [START_REF] Kumar | TimeTree: A Resource for Timelines, Timetrees, and Divergence Times[END_REF]. Syncytin-Ory1 is a gamma-type Env of the SLC1A5 interference group and therefore uses the same receptor as Syncytin-1. Interestingly, the envelope is close to that of MPMV (reviewed in [START_REF] Sinha | Retroviruses of the RDR superinfection interference group: ancient origins and broad host distribution of a promiscuous Env gene[END_REF], and the syncytin-Ory1 associated RT is also close to that of MPMV as well as other betaretroviral RTs [START_REF] Malicorne | Genome-Wide Screening of Retroviral Envelope Genes in the Nine-Banded Armadillo (Dasypus novemcinctus, Xenarthra) Reveals an Unfixed Chimeric Endogenous Betaretrovirus Using the ASCT2 Receptor[END_REF]. This suggests that the ancestral retrovirus was a recombinant betaretrovirus that acquired a gamma-type SLC1A5 interference group env, a somewhat remarkable happening as the immense majority of syncytins seem to originate from gammaretroviruses. In this case the use of SLC1A5 as receptor is again linked to hemochorial placentation with a single fused cell layer (see 1.2.2.d.).

b. Cat and dog: Syncytin-Car1

Carnivorans (cats, dogs) all present a common endotheliochorial general placental organization (see 1.2.2.c.; except hyenas which present a hemochorial placenta [START_REF] Morton | Placentation in the spotted hyena (Crocuta crocuta Erxleben)[END_REF][START_REF] Wynn | Placentation in the Spotted Hyena (Crocuta crocuta), with Particular References to the Circulation[END_REF][START_REF] Oduor Okelo | The chorioallantoic placenta of the spotted hyena (Crocuta crocuta Erxleben): An electron microscopic study[END_REF] and were therefore postulated to all present the same syncytin gene. [START_REF] Cornelis | Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora[END_REF] were able to identify such a gene and named it syncytin-Car1. Syncytin-Car1 is a gamma-type Env expressed by an ERV of gammaretroviral origin and is orthologous and conserved under purifying selection in the 26 tested carnivoran species, suggesting an insertion date of about 75 Mya according to TimeTree [START_REF] Kumar | TimeTree: A Resource for Timelines, Timetrees, and Divergence Times[END_REF] and making Syncyin-Car1 the oldest known syncytin (Fig. 25 Cornelis et al., 2012).

c. Old world monkey: EnvV2

The curious case of EnvV2 is mentioned here for completeness, but will be discussed in more details in 3.3.1.a. This ERV envelope gene was endogenized in the common ancestor of simians, about 43 Mya (Fig. 25, TimeTree, [START_REF] Kumar | TimeTree: A Resource for Timelines, Timetrees, and Divergence Times[END_REF], but conserved all characteristics of a syncytin only in Old World monkeys [START_REF] Esnault | Differential Evolutionary Fate of an Ancestral Primate Endogenous Retrovirus Envelope Gene, the EnvV Syncytin, Captured for a Function in Placentation[END_REF]. It seems that in other clades, EnvV2 was replaced by Syncytin-1 over the course of evolution and is only present as a decaying syncytin that still displays some purifying selection [START_REF] Esnault | Differential Evolutionary Fate of an Ancestral Primate Endogenous Retrovirus Envelope Gene, the EnvV Syncytin, Captured for a Function in Placentation[END_REF].

d. Cattle and sheep: Syncytin-Rum1 and Fematrin-1

Ruminants display the very particular and rare style of synepitheliochorial placentation which displays only limited syncytialization of the materno-fetal interface (see 1.2.2.b.). [START_REF] Cornelis | Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants[END_REF] describe placental expression of two ERV Env in cows: Syncytin-Rum1 and the previously identified BERV-K1/Bos-Env4. The latter was also investigated a few months later by [START_REF] Nakaya | Fematrin-1 Is Involved in Fetomaternal Cell-to-Cell Fusion in Bovinae Placenta and Has Contributed to Diversity of Ruminant Placentation[END_REF], who renamed it somewhat confusingly fetomaternal trinucleate cell inducer 1 (Fematrin-1).

Syncytin-Rum1 is a gamma-type env displaying limited similarity with Syncytin-Car1, potentially resulting from the infection of a related but distinct gammaretrovirus [START_REF] Cornelis | Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants[END_REF]. Syncytin-Rum1 is present at an orthologous position in 16 tested Pecora (all ruminants except chevrotain and mouse-deer) species, suggesting integration followed by purifying selection about 45 Mya (Fig. 25, Cornelis et al., 2013). Interestingly, the expression levels of Syncytin-Rum1 are significantly higher in sheep than in cow, correlating with the higher amount of syncytialization observed in the sheep placenta (see 1.2.2.b.) and potentially linking the expression level of syncytins with the structure of the placenta [START_REF] Cornelis | Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants[END_REF].

Fematrin-1 is a beta-type Env that is, contrarily to Syncytin-Rum1, conserved only in Bovinae (cattle) and not in Caprinae (sheep) [START_REF] Cornelis | Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants[END_REF][START_REF] Nakaya | Fematrin-1 Is Involved in Fetomaternal Cell-to-Cell Fusion in Bovinae Placenta and Has Contributed to Diversity of Ruminant Placentation[END_REF] leading to an estimated insertion time of 20 Mya (Fig. 25) according to TimeTree [START_REF] Kumar | TimeTree: A Resource for Timelines, Timetrees, and Divergence Times[END_REF]. The fusogenic activity ex vivo of Fematrin-1 is about 2.5 fold higher than that observed with Syncytin-Rum1 at pH 7.0, the latter being more fusogenic at a lower pH of 5.0 [START_REF] Cornelis | Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants[END_REF] 3.2 [START_REF] Nakaya | Fematrin-1 Is Involved in Fetomaternal Cell-to-Cell Fusion in Bovinae Placenta and Has Contributed to Diversity of Ruminant Placentation[END_REF], the physiological relevance of which has not been established. Interestingly, a subsequent study showed that the fusogenic potential of Fematrin-1 in bovine trophoblast cell lines is also increased at low pH [START_REF] Koshi | Induction of ovine trophoblast cell fusion by fematrin-1 in vitro[END_REF]. Taking into account the relatively recent and Bovinae-specific acquisition of Fematrin-1, [START_REF] Nakaya | Fematrin-1 Is Involved in Fetomaternal Cell-to-Cell Fusion in Bovinae Placenta and Has Contributed to Diversity of Ruminant Placentation[END_REF] suggest that Syncytin-Rum1 might represent an ancestral syncytin that is currently being replaced by Fematrin-1 in Bovinae and endogenous JSRV Env in Caprinae (see also 3.3.1).

e. Squirrel and marmot: Syncytin-Mar1

While it had been previously shown that some rodents clades presented Syncytin-A and -B, the Sciuridae do not present these two genes. Taking advantage of the recent sequencing of the thirteen-lined ground squirrel (Ictidomys tridecemlineatus) genome, [START_REF] Redelsperger | Capture of syncytin-Mar1, a Fusogenic Endogenous Retroviral Envelope Gene Involved in Placentation in the Rodentia Squirrel-Related Clade[END_REF] were able to identify Syncytin-Mar1. This syncytin has been conserved in a coding state in all members of the Marmotini tribe (ground squirrels), but is present as a non-coding sequence in other Sciuridae [START_REF] Redelsperger | Capture of syncytin-Mar1, a Fusogenic Endogenous Retroviral Envelope Gene Involved in Placentation in the Rodentia Squirrel-Related Clade[END_REF], suggesting an endogenization about 35 Mya and an exaptation only in the Marmotini which radiated approximately 27 Mya (Fig. 25, TimeTree, [START_REF] Kumar | TimeTree: A Resource for Timelines, Timetrees, and Divergence Times[END_REF]. This integration date would also mean that Syncytin-A and -B and Syncytin-Mar1 never co-existed in a common ancestor of Marmotini and Muroidea [START_REF] Redelsperger | Capture of syncytin-Mar1, a Fusogenic Endogenous Retroviral Envelope Gene Involved in Placentation in the Rodentia Squirrel-Related Clade[END_REF].

f. Tenrec: Syncytin-Ten1

While at this point a number of syncytins had been described in Euarchontoglires (Syncytin-1, -2, -A, -B, -Ory1, -Mar1) and Laurasiatheria (Syncytin-Car1, -Rum1), no members of the Xenartha or Afrotheria had been shown to present these kind of genes. To investigate the latter group, [START_REF] Cornelis | Retroviral envelope syncytin capture in an ancestrally diverged mammalian clade for placentation in the primitive Afrotherian tenrecs[END_REF] screened the genome of the lesser hedgehog tenrec (Echinops telfairi) and identified Syncytin-Ten1. It is conserved at an orthologous position and under purifying selection in all Malagasy tenrec and probably present in the non-Malagasy subfamily [START_REF] Cornelis | Retroviral envelope syncytin capture in an ancestrally diverged mammalian clade for placentation in the primitive Afrotherian tenrecs[END_REF], suggesting an insertion date of 40 to 50 Mya (Fig. 25, TimeTree, [START_REF] Kumar | TimeTree: A Resource for Timelines, Timetrees, and Divergence Times[END_REF].

g. Short-tailed opossum: Syncytin-Opo1

Marsupials present a very particular and short-lived placenta when compared to that of eutherian mammals. To try to investigate the potential role of syncytins in a marsupial placenta, [START_REF] Cornelis | Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials[END_REF] searched the gray short-tailed opossum (Monodelphis domestica) genome in silico for the presence of coding ERV env and identified syncytin-Opo1. The expression profile of this syncytin is less placenta-specific that for the ones described previously, with non-negligible expression occurring in other tissues such as the spleen [START_REF] Cornelis | Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials[END_REF]. Conservation of the envelope is limited to a subset of the Monodelphis genus, as it is present in a non-coding form in Monodelphis theresa and emiliae and absent in the sister group of Monodelphis [START_REF] Cornelis | Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials[END_REF]. This makes this syncytin a recent acquisition, about 27 Mya (TimeTree, [START_REF] Kumar | TimeTree: A Resource for Timelines, Timetrees, and Divergence Times[END_REF] with a conservation for about 10 Mya only (Fig. 25, Cornelis et al., 2015). The syncytialization status of the materno-fetal interface in Monodelphis theresa and emiliae is unknown [START_REF] Cornelis | Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials[END_REF].

Syncytin-like Env

In addition to the syncytins described above, a number of conserved envelopes lacking one or several of the canonical syncytin properties have been discovered. Nevertheless, these envelopes are often conserved with signs of purifying selection that hint at a physiological role, very probably distinct from the classical fusogenic role of syncytins.

a. Conserved ERV Env with placental expression in human

ERV3. ERV3 (also called HERV-R), was the third ERV discovered in the human genome [START_REF] O'connell | ERV3, A full-length human endogenous provirus: Chromosomal localization and evolutionary relationships[END_REF], though it became quickly apparent that the env ORF was truncated before the transmembrane anchoring domain, theoretically yielding a secreted Env-like protein [START_REF] Cohen | The nucleotide sequence of the env gene from the human provirus ERV3 and isolation and characterization of an ERV3-specific cDNA[END_REF]. Since ERV3 Env is lacking its membrane attachment, it cannot be fusogenic. Expression of this truncated envelope in the placenta was shown on both the RNA [START_REF] Kato | Tissue-specific expression of human provirus ERV3 mRNA in human placenta: two of the three ERV3 mRNAs contain human cellular sequences[END_REF] and the protein level [START_REF] Venables | Abudnance of an endogenous retroviral envelope protein trophoblasts suggests a biological function[END_REF], and investigation of its expression in a range of human tissues revealed that it is preferentially expressed in the placenta and adrenal gland (de Parseval et al., 2003). Due to its apparent conservation not only in humans but in all Catarrhini (except in gorillas), which puts its time of endogenization at about 40 Mya (Fig. 25), it was proposed to play an important physiological role (reviewed in Bustamante [START_REF] Bustamante Rivera | Endogenous retrovirus 3-History, physiology, and pathology[END_REF]. Using a trophoblast derived cell line, [START_REF] Lin | Expression of endogenous retrovirus ERV-3 induces differentiation in BeWo, a choriocarcinoma model of human placental trophoblast[END_REF] show that overexpression of ERV3 env induces cell differentiation, as revealed by a decrease in proliferation, morphological changes and synthesis of human chorionic gonadotropin (hCG) mRNA. In a follow-up study, the same authors show that the pro-differentiation effect of ERV3 involves an increase in intracellular cAMP levels and the activity of protein kinase A (PKA) [START_REF] Lin | The cellular mechanism by which the human endogenous retrovirus ERV-3 env gene affects proliferation and differentiation in a human placental trophoblast model, BeWo[END_REF]. Intriguingly, an increase in cAMP and PKA activity was later shown to stimulate GCMa transcriptional activity [START_REF] Knerr | Stimulation of GCMa and syncytin via cAMP mediated PKA signaling in human trophoblastic cells under normoxic and hypoxic conditions[END_REF][START_REF] Chang | Stimulation of GCMa Transcriptional Activity by Cyclic AMP/Protein Kinase A Signaling Is Attributed to CBP-Mediated Acetylation of GCMa[END_REF], which as mentioned contributes to the expression of both human syncytins and MFSD2A. ERV3 Env is a gamma-type Env containing an ISD (Fig. 22) that was shown to be functional in mice rejection assays [START_REF] Mangeney | Placental syncytins: Genetic disjunction between the fusogenic and immunosuppressive activity of retroviral envelope proteins[END_REF]. The main argument against a fundamental role of ERV3 Env in placentation comes from an early study by de Parseval & Heidmann (1998) which revealed the presence of homozygous early truncated alleles in about 1% of the caucasian population. While this does not mean that ERV3 Env has no function in physiology, it proves that it is not essential, unlike Syncytin-A for example.

HEMO. HEMO is a newly described conserved simian ERV env, that was somehow overlooked for a long time [START_REF] Heidmann | HEMO, an ancestral endogenous retroviral envelope protein shed in the blood of pregnant women and expressed in pluripotent stem cells and tumors[END_REF]. It is specifically expressed in the placenta and conserved in all simians (Fig. 25), though it entered the mammalian genome before separation of Laurasiatheria and Euarchontoglires [START_REF] Heidmann | HEMO, an ancestral endogenous retroviral envelope protein shed in the blood of pregnant women and expressed in pluripotent stem cells and tumors[END_REF], about 96 Mya (TimeTree, [START_REF] Kumar | TimeTree: A Resource for Timelines, Timetrees, and Divergence Times[END_REF]. HEMO is expressed in CT and EVT and does not disclose any fusogenic activity ex vivo, which is perhaps not surprising considering that the human envelope has lost its furin cleavage site, preventing correct maturation [START_REF] Heidmann | HEMO, an ancestral endogenous retroviral envelope protein shed in the blood of pregnant women and expressed in pluripotent stem cells and tumors[END_REF]. Intriguingly HEMO has however acquired a new cleavage site shortly upstream of the transmembrane sequence [START_REF] Heidmann | HEMO, an ancestral endogenous retroviral envelope protein shed in the blood of pregnant women and expressed in pluripotent stem cells and tumors[END_REF]. Cleavage seems to occur under the action of metalloproteinases and leads to the release of a shed, still linked, SU/most of TM protein from the cell membrane, accordingly HEMO can be detected in the blood of pregnant women [START_REF] Heidmann | HEMO, an ancestral endogenous retroviral envelope protein shed in the blood of pregnant women and expressed in pluripotent stem cells and tumors[END_REF]. This separation from the membrane anchoring domain is another reason HEMO might not be fusogenic. HEMO Env is also strongly expressed in stem cells, in a way reminiscent of HERV-H, and is related to the marsupial conserved Env pan-Mars-Env2 [START_REF] Heidmann | HEMO, an ancestral endogenous retroviral envelope protein shed in the blood of pregnant women and expressed in pluripotent stem cells and tumors[END_REF]. It remains to be seen what role, if any, HEMO plays in primate physiology, but its high amount of sequence conservation and peculiar properties suggest a new, non-syncytin role.

b. Non-human syncytin-like genes enJSRV. The case of endogenous copies of the still active JSRV virus (enJSRV) is complicated by their relatively young age. There have been several descriptions of enJSRV sequences that were exapted and positively selected during sheep domestication, such as the previously mentioned enJS56A1 locus gag which acts as a late restriction factor [START_REF] Arnaud | Mechanisms of late restriction induced by an endogenous retrovirus[END_REF]. While it is possible that enJSRV plays an important placental role by simply protecting the embryo from viral infections, other studies hint at a more complex role. Indeed, enJSRV env sequences from several loci are expressed in specific tissues in the placenta, in particular the BNC and the syncytial plaques [START_REF] Dunlap | Sheep Endogenous Betaretroviruses (enJSRVs) and the Hyaluronidase 2 (HYAL2) Receptor in the Ovine Uterus and Conceptus[END_REF]. Additionally, inhibition of enJSRV Env in utero leads to reduction in trophoblast cell proliferation, preventing implantation and leading to embryo death [START_REF] Dunlap | Endogenous retroviruses regulate periimplantation placental growth and differentiation[END_REF]. The implication of enJSRV Env in cell proliferation and differentiation is intriguing as it has been shown that this protein possesses strong oncogenic properties [START_REF] Wootton | Sheep retrovirus structural protein induces lung tumours[END_REF]. Implication of enJSRV Env in cell-cell fusion events is less clear, as no copy able to induce cell-cell fusion ex vivo has been identified so far (reviewed in Nakaya & Miyazawa, 2015).

Env-Cav1. The search for non-Muroidea rodent syncytins led [START_REF] Vernochet | A syncytin-like endogenous retrovirus envelope gene of the guinea pig specifically expressed in the placenta junctional zone and conserved in Caviomorpha[END_REF] to identify Env-Cav1 in the genome of guinea pigs (Cavia procellus). This Env seems conserved in all Caviomorpha under purifying selection, suggesting an endogenization about 30 Mya (Fig. 25), and is expressed in the syncytial streamers, a fused and invasive placental tissue [START_REF] Vernochet | A syncytin-like endogenous retrovirus envelope gene of the guinea pig specifically expressed in the placenta junctional zone and conserved in Caviomorpha[END_REF]. Ex vivo fusogenicity assays disclosed no fusogenic potential of Env-Cav1, suggesting that its placental role is not that of a classical syncytin [START_REF] Vernochet | A syncytin-like endogenous retrovirus envelope gene of the guinea pig specifically expressed in the placenta junctional zone and conserved in Caviomorpha[END_REF]. The exact role of this envelope remains to be determined, but [START_REF] Vernochet | A syncytin-like endogenous retrovirus envelope gene of the guinea pig specifically expressed in the placenta junctional zone and conserved in Caviomorpha[END_REF] suggest that it might be implicated in placental invasion phenomena, drawing a parallel with the presence of Syncytin-1 on EVT cells. pan-Mars-Env2. The same study that described Syncytin-Opo1 also revealed the existence of pan-Mars-Env2 [START_REF] Cornelis | Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials[END_REF]. This Env is present at the orthologous site in all marsupials and has remained coding under purifying selection since its endogenization at least 80 Mya (Fig. 25, Cornelis et al., 2015). Interestingly, pan-Mars-Env2 is truncated by a stop codon just before the transmembrane sequence, similarly to what is observed for ERV3 [START_REF] Cornelis | Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials[END_REF]. While this means that pan-Mars-Env2 is unable to be fusogenic, its strict conservation still heavily implies that it plays a major role in marsupial placentation [START_REF] Cornelis | Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials[END_REF]. Discovery of HEMO a few years later revealed that the two Env sequences are related with a number of conserved regions in the extracellular domain [START_REF] Heidmann | HEMO, an ancestral endogenous retroviral envelope protein shed in the blood of pregnant women and expressed in pluripotent stem cells and tumors[END_REF]. While marsupials and simians are too distantly related to perform reliable synteny analyses, it is probable that the two sequences at least result from endogenization of the same retrovirus, if not from the same endogenization [START_REF] Heidmann | HEMO, an ancestral endogenous retroviral envelope protein shed in the blood of pregnant women and expressed in pluripotent stem cells and tumors[END_REF]. It is intriguing that both HEMO and pan-Mars-Env2 are released into the extra-cellular medium, but using two different ways: metalloproteinase shedding for the former and stop codon induced absence of a membrane anchoring domain for the latter [START_REF] Cornelis | Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials[END_REF][START_REF] Heidmann | HEMO, an ancestral endogenous retroviral envelope protein shed in the blood of pregnant women and expressed in pluripotent stem cells and tumors[END_REF].

Syncytins and evolution

Ancestral syncytin and syncytin replacement

The involvement of syncytins in mammalian placentation poses a few conceptual problems, chief among them the relatively recent acquisition of some of these genes in their respective clade. The mammalian placenta emerged in the last common ancestor of marsupials and eutherians, about 175 Mya (Fig. 25 dos Reis et al., 2012), while the oldest syncytin, Syncytin-Car1 is less than half as old at 75 My [START_REF] Cornelis | Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora[END_REF]. The oldest conserved syncytin-like gene pan-Mars-Env2 is about 80 My old [START_REF] Cornelis | Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials[END_REF], and the oldest still conserved ERV envelope across all animal clades, percomORF, described to date is only 110 My old [START_REF] Henzy | An Intact Retroviral Gene Conserved in Spiny-Rayed Fishes for over 100 My[END_REF]. While mammalian placentas can be entirely functional without cell-cell fusion (as is the case in epitheliochorial placentas, see 1.2.2.a.), syncytin and syncytin-like genes also seem to play an important role in other aspects of placentation, such as cell differentiation. It is unlikely that these phenomena arose only in the last 10-80 My after endogenization of syncytins and even more unlikely that this emergence occurred in all the above mentioned lineages independently. A working hypothesis to explain this apparent discrepancy is the acquisition of an ancestral syncytin at the time of emergence of the mammalian placenta, followed by its replacement with newly acquired ERV Env over the course of evolution (Fig. 25, reviewed in [START_REF] Lavialle | Paleovirology of 'syncytins', retroviral env genes exapted for a role in placentation[END_REF]. [START_REF] Kumar | TimeTree: A Resource for Timelines, Timetrees, and Divergence Times[END_REF].The presence of HERV-FRD Env (FRD), EnvV2 (V) and HERV-W Env (W) as fully functional syncytins is indicated on the tree by colored lines and letters. A fading line and gray letter means loss of at least one syncytin property (fusogenicity for EnvV2, all three for HERV-W Env) in this clade. In the last common ancestor of apes and Old World monkey, all three envelopes were present and probably displayed all syncytin characteristics at the same time. Following split of these clades, either EnvV2 was conserved and HERV-W Env lost (lower arrow), or EnvV2 lost its fusogenic properties and HERV-W Env replaced it as syncytin (upper arrow, baton pass event). Present day syncytins are indicated on the right, using the same colors as in the tree. Adapted from [START_REF] Esnault | Differential Evolutionary Fate of an Ancestral Primate Endogenous Retrovirus Envelope Gene, the EnvV Syncytin, Captured for a Function in Placentation[END_REF].

a. Example of EnvV2, a decaying syncytin in humans

An example of syncytin replacement and decay can be found when examining the fate of EnvV2 following the primate/Old World monkey split about 45 Mya (Fig. 27, Esnault et al., 2013). EnvV2 is the full-length ORF of a very old ERV-V that integrated the genome of the last common ancestor of simians and is present orthologously and under apparent purifying selection in New World monkeys, Old World monkeys and apes (Fig. 25 and 27, Esnault et al., 2013). Specific placental expression and expression in the ST layer is conserved between humans and the Old World monkey macaque, but fusogenic potential was conserved only in the Old World monkey clade [START_REF] Esnault | Differential Evolutionary Fate of an Ancestral Primate Endogenous Retrovirus Envelope Gene, the EnvV Syncytin, Captured for a Function in Placentation[END_REF]. While there are some species with fusogenic EnvV2 in apes and New World monkeys, they are the exception and not the rule [START_REF] Esnault | Differential Evolutionary Fate of an Ancestral Primate Endogenous Retrovirus Envelope Gene, the EnvV Syncytin, Captured for a Function in Placentation[END_REF]. These results suggest that EnvV2 had all the properties of a syncytin before the separation of apes and Old World monkeys, being fusogenic, expressed in the placenta and conserved in the clade, however it maintained these three properties only in Old World monkeys and not in its ape sister clade (Fig. 27, Esnault et al., 2013). Another study using isolated Rhesus monkey trophoblasts confirmed a probable implication of EnvV2 in Old World monkey ST formation [START_REF] Kumar | Differential Effects of Sodium Butyrate and Lithium Chloride on Rhesus Monkey Trophoblast Differentiation[END_REF]. Interestingly, an inverse pattern can be observed for Syncytin-1 which was endogenized by the last common ancestor of these two clades. Syncytin-1 has remained fusogenic in apes, but has lost this property in Old World monkeys in which the ORF has not been conserved at all [START_REF] Bonnaud | Natural history of the ERVWE1 endogenous retroviral locus[END_REF][START_REF] Esnault | Differential Evolutionary Fate of an Ancestral Primate Endogenous Retrovirus Envelope Gene, the EnvV Syncytin, Captured for a Function in Placentation[END_REF]. From these results, [START_REF] Esnault | Differential Evolutionary Fate of an Ancestral Primate Endogenous Retrovirus Envelope Gene, the EnvV Syncytin, Captured for a Function in Placentation[END_REF] propose a model in which the two 45 My syncytin-2 and envV2 genes were the functional syncytins in the common simian ancestor. Following endogenization of syncytin-1 about 25 Mya, it replaced envV2 as placental fusiogen in apes but not Old World monkeys, in which EnvV2 maintained its fusogenic activity and Syncytin-1 was lost (Fig. 27, Esnault et al., 2013).

Interestingly, even though it lost its fusogenic properties, EnvV2 still displays signs of purifying selection in all simians [START_REF] Esnault | Differential Evolutionary Fate of an Ancestral Primate Endogenous Retrovirus Envelope Gene, the EnvV Syncytin, Captured for a Function in Placentation[END_REF]. This might either be due to syncytin decay taking a long time, or to the fact that EnvV2 still plays another role in non-Old World monkey placentas, such as immunosuppression, as it contains a functional ISD [START_REF] Mangeney | Placental syncytins: Genetic disjunction between the fusogenic and immunosuppressive activity of retroviral envelope proteins[END_REF].

b. Baton pass hypothesis

The case of EnvV2/Syncytin-1 illustrates a model proposed initially several years earlier by Thierry Heidmann [START_REF] Saib | Endogenous retroviruses: Thierry Heidmann wins the 2009 Retrovirology prize[END_REF] and which was more formally established afterwards by his team (Dupressoir & Heidmann, 2011;[START_REF] Dupressoir | From ancestral infectious retroviruses to bona fide cellular genes: role of the captured syncytins in placentation[END_REF][START_REF] Lavialle | Paleovirology of 'syncytins', retroviral env genes exapted for a role in placentation[END_REF] and the team of [START_REF] Nakamura | Retroviral Endogenization and Its Role in the Genital Tract during Mammalian Evolution[END_REF] who coined the term "baton pass hypothesis" to refer to it. The model formally proposes that emergence of mammalian placentation followed the acquisition of an ancestral ERV Env that provided the organism with new functionalities, the most important of which might have been immunosuppression via the presence of the ISD, since cell-cell fusion is not necessary for mammalian placentation, but tolerance of fetal tissue by the maternal immune system is (reviewed in [START_REF] Lavialle | Paleovirology of 'syncytins', retroviral env genes exapted for a role in placentation[END_REF]. Over the course of evolution, the random acquisition of new Env genes due to endogenization and exaptation events has then led to the appearance of new genes with slightly different properties that might present an evolutionary advantage for the host. These new genes could functionally replace the older syncytins, leading to the conservation of the former but the loss of the latter. In a fashion, the older syncytin passes the baton of placental functionality to the newly acquire gene and, since it is no longer essential for placentation and reproduction, is lost over the course of evolution (reviewed in [START_REF] Lavialle | Paleovirology of 'syncytins', retroviral env genes exapted for a role in placentation[END_REF]. This model would explain why genes strictly necessary for placentation such as Syncytin-A and -B could have been exapted by pure chance over 100 My after the emergence of the mammalian placenta, it also provides a more parsimonious explanation to the seemingly independent use of ERV Env in the placentation of a large number of mammalian species. Finally, this model might contribute to the explanation as to why the mammalian placenta is by far the most diversely structured organ. The ancestral syncytin, which should be shared orthologously by all eutherian and marsupial mammals, must have been exapted over 175 Mya. Sadly such an ancient origin associated with a probable absence of purifying selection following its replacement, makes it probable that this funding gene has been lost to the ages, and indeed no such gene has been described to date.

Syncytin diversity and placental diversity

It is important to realise that, as mentioned above, every syncytin described so far has resulted from an independent endogenization event (as illustrated by their non-orthologous status), and this is also the case for most syncytin-like genes except maybe HEMO and pan-Mars-Env2 [START_REF] Heidmann | HEMO, an ancestral endogenous retroviral envelope protein shed in the blood of pregnant women and expressed in pluripotent stem cells and tumors[END_REF]. Phylogenetic analyses on syncytin TM subunits show that while, except for Fematrin-1, they all cluster (expectedly) with exogenous gamma-type Env, even more specifically the gamma-type Env of gammaretroviruses, they are nevertheless spread across this family of envelopes, clearly originating from distinct exogenous retroviruses (Fig. 28B). The same can be said for an analysis based on the RT sequences associated with syncytins (when they are available), which almost exclusively represent gammaretroviral type RTs, except for the Syncytin-Ory1 and Fematrin-1 associated RT which is clearly betaretroviral (Fig. 28). This suggests that different syncytins possess related but slightly different innate characteristics with regards to fusion, immunosuppression and other potential roles of retroviral envelopes, originating from different retroviral species or strains. Accordingly, shared receptors seem to be the exception rather than the norm amongst syncytins.

The diversity of syncytins is further enhanced by their time of acquisition (varying by almost an order of magnitude between 10-80 Mya [START_REF] Cornelis | Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora[END_REF][START_REF] Cornelis | Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials[END_REF] and the manner of their exaptation (Fig. 29). Some syncytins such as syncytin-Car1 are associated with a recognizable provirus containing two LTRs, an identifiable TSD and PBS and are expressed following the canonical retroviral pathway of LTR transcript initiation, single splicing and LTR transcript termination [START_REF] Cornelis | Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora[END_REF]. Other syncytins are associated with highly degraded proviruses and are expressed using alternative cellular promoters, or as in the case of syncytin-Rum1 for example a transcript initation site located in the degenerate gag-pol region [START_REF] Cornelis | Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants[END_REF]. The recently described HEMO Env presents an incredibly disrupted provirus that contains deletions and inversions of the the ancestral retroviral sequence and its expression is driven by a cellular CpG island and involves several subsequent and alternative splicing events [START_REF] Heidmann | HEMO, an ancestral endogenous retroviral envelope protein shed in the blood of pregnant women and expressed in pluripotent stem cells and tumors[END_REF]. The vast diversity of transcription mechanisms and contexts that surround different syncytins and syncytin-like genes doubtlessly can have an impact on the ease and specificity of their transcriptional regulation as well as on their expression level. The different expression levels of Syncytin-Rum1 in sheep and cow placentas has for example been proposed to be a causative factor of the varying extent of syncytialization observed in these placentas [START_REF] Cornelis | Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants[END_REF]. Further variation between species is introduced by the potential exaptation of different syncytin genes present and functional concurrently, as was probably the case with EnvV2 and Syncytin-1.

Keeping this diversity in mind, it is intriguing to remember the vast diversity of placental structures observed in mammals (Fig. 3 and4, reviewed in [START_REF] Enders | What Can Comparative Studies of Placental Structure Tell Us?-A Review[END_REF]Wooding & Burton, 2008, chap. 1). Placentas vary from the rather simple epitheliochorial pig placenta to the much more complex hemochorial placentas of humans and mice, even though there does

0.5 K o R V BL V W D S V S y n -O r y 1 JS R V W E H V -1 S y n -R u m 1 FL V RD11 4 S y n -2 E I A V HIV -1 B F V S F V Ga LV M P M V M M T V F F V H IV -2 S M R V W E H V -2 S y n -1 P T L V -2 R S V M L V P T L V -1 AMV E F V REV A L V S y n -C a r 1 C A E V S yn -O po 1 β β α δ ε γ λ λ spuma A B 0.8 S y n c y t i n - 1 R E V A L V Syncytin-Mar1 S y n c y t in - B M L V M P M V K o R V R D 1 1 4 E I A V C A E V Syncy tin-A E n v -C a v 1 S y n c y t in -O r y 1 H I V -2 P T L V -2 GaLV p a n -M a rs -E n v 2 Sy nc ytin -Te n1 A M V SMR V H IV -1 E n v V 2 B L V F L V Sy nc yti n-C ar1 M M T V F e m a t r i n - 1 Sy nc yt in -O po 1 P T L V -1 R S V S y n c y ti n - R u m 1 E R V -3 S y n c y ti n -2 J S R V HE MO β-type CX n C -ISD γ-type CX 6 CC +ISD TM RT

Figure 28 -Phylogeny of syncytins among retroviral genera based on RT or TM analysis. (A)

Unrooted RT-based PhyML tree using selected retroviral species as well as all syncytins for which it RT is available, with RT-based genera indicated on the tree. The tree was obtained with the PhyML program and the RtREf+Γ model selected by the Smart Model Selection program (Lefort et al., 2017). (B) Unrooted TM-based tree using selected gamma-and beta-type TM as well as all syncytins and selected syncytinlikes.RT-based genera, other than gammaretrovirus, are indicated on the tree. Non-specified genera are assumed to be gammaretroviral in view of RT and TM repartitions. TM type is indicated in gray alongside the associated CX n C 1-2 motif and the presence or absence of the CKS-17 ISD (see also Fig. 22). The tree was obtained with the PhyML program and the LG+Γ+I+F model, selected by the Smart Model Selection program (Lefort et al., 2017). Syncytin-like Env are highlighted in green. For both trees, syncytins are highlighted in blue, branch length is proportional to the average number of substitutions per site (see scale below each tree), and RT/TM recombinant retroviruses are highlighted in red. For abbreviations see Fig. 11 and HEMO: human endogenous mer34 ORF. In some cases like Syncytin-Car1, expression is simply driven by its original LTR promoter and the canonical retroviral splice is present. In other cases like that Syncytin-Rum1, the original promoter LTR has been lost during evolution and transcription is initiated by a cryptic promoter present in the otherwise degenerated gag-pol region. Only the splice acceptor site is canonical. Finally, in the case of the syncytin-like HEMO, transcription is initiated by a cellular CpG island promoter and presents multiple splicing events that can yield alternative transcripts containing different parts of the cellular genome 5' of the HEMO Env ORF. Faded out boxes represent non-functional ERV sequences, color coding is the same as Fig. 9. The gradient covering the colors of gag, pro and pol indicates that the three cannot be told apart. Green lines represent transcripts and dotted line splicing events.

not seem to be a particular advantage to one form or the other. The involvement of stochastic gene acquisitions through endogenizations might explain the breadth of structures and strategies observed across mammals by introducing the necessary randomness to placental evolution. It has been suggested that following the parsimony principle establishes the hemochorial placenta as an ancestral state while epitheliochorial placentation would be a derived condition (reviewed in Roberts et al., 2016). This suggests a counter-intuitive path for evolution in which the originally separated mother and egg immediately transitioned to a highly invasive phenotype in which the maternal immune system is in direct contact with fetal tissues, before returning to a more distant partnership in which fetal and maternal tissues are merely apposed, closer to an egg/mother relationship. These analyses often do not take into account the potential impact of random gene acquisitions on placental structures and the gradual replacements occurring in the baton pass model.

Other roles of syncytins

While the placental functions of syncytins are the best described role and their placenta-specific or preferential expression pattern strongly suggests a major role in placental physiology, some of them have been described as being involved in other processes.

a. Cell-cell fusion in muscles

Formation and regeneration of animal muscles is heavily reliant on controlled cell-cell fusion processes in which myoblasts fuse into myotubes. While there exist a number of non-viral fusiogens described as implicated in this process, the demonstration of syncytin mediated cell fusion in placentas led to the investigation of a potential role of these virus-derived fusiogens.

The first results in this direction were obtained by [START_REF] Bjerregaard | The Endogenous Envelope Protein Syncytin Is Involved in Myoblast Fusion[END_REF] who show that SLC1A5 is expressed in growing muscles and that induction of myoblast fusion triggered Syncytin-1 expression. Additionally, Syncytin-1 is specifically expressed at the cell membrane and inhibition of its expression leads to a significant decrease of myoblast-myotube fusion [START_REF] Bjerregaard | The Endogenous Envelope Protein Syncytin Is Involved in Myoblast Fusion[END_REF][START_REF] Bjerregaard | Syncytin-1 in differentiating human myoblasts: relationship to caveolin-3 and myogenin[END_REF]. The expression of Syncytin-1 was colocalized with that of other known effectors of muscle fusion and often observed in filopoda that established contact between myoblasts and myotubes [START_REF] Bjerregaard | Syncytin-1 in differentiating human myoblasts: relationship to caveolin-3 and myogenin[END_REF]. An implication of Syncytin-1 in muscle fusion events was later confirmed by [START_REF] Frese | Long-Term Endurance Exercise in Humans Stimulates Cell Fusion of Myoblasts along with Fusogenic Endogenous Retroviral Genes In Vivo[END_REF], who also show that muscle biopsies taken from athletes after their competitive season show strongly increased cell-cell fusion as well as Syncytin-1 expression when compared to pre-season samples.

Redelsperger et al. ( 2016) investigated the contribution of syncytins to mouse muscle fusion and showed that in syncytin-B knockout male mice the muscle mass is reduced by about 20%. Intriguingly, this effect was not observed in female mice, suggesting it is a sex specific effect and that Syncytin-B might contribute to sex-differences in this species [START_REF] Redelsperger | Genetic Evidence That Captured Retroviral Envelope syncytins Contribute to Myoblast Fusion and Muscle Sexual Dimorphism in Mice[END_REF]. Similarly, in male but not female mice, the knockout of syncytin-B leads to slower muscular regeneration after injury. The effect of syncytin-A knockout was not studied as it is lethal for all embryos [START_REF] Dupressoir | Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene[END_REF].

In the same study, [START_REF] Redelsperger | Genetic Evidence That Captured Retroviral Envelope syncytins Contribute to Myoblast Fusion and Muscle Sexual Dimorphism in Mice[END_REF] also assays fusion of primary myoblasts ex vivo, using cells originating from human, mouse, dog and sheep and inhibiting the corresponding syncytin each time. In each case there is a 20-40 percentage point reduction of the fusion index, suggesting that involvement of syncytins in muscle fusion is a widespread phenomenon [START_REF] Redelsperger | Genetic Evidence That Captured Retroviral Envelope syncytins Contribute to Myoblast Fusion and Muscle Sexual Dimorphism in Mice[END_REF]. For human myoblasts, both Syncytin-1 and -2 were inhibited via siRNA and the decrease of the fusion index is similar in both cases, about 20 percentage points [START_REF] Redelsperger | Genetic Evidence That Captured Retroviral Envelope syncytins Contribute to Myoblast Fusion and Muscle Sexual Dimorphism in Mice[END_REF].

Taken together these results suggest that syncytins play a significant role in muscular cell-cell fusion in a number of mammalian species, but that they are not the only actors involved by far, unlike in the placenta where their knockout can completely abolish fusion [START_REF] Dupressoir | Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene[END_REF](Dupressoir et al., , 2011)).

b. Osteoclast fusion

Contrary to popular belief, bone is a rather dynamic structure that is constantly resorbed and rebuilt by osteoclasts and osteoblasts respectively. The resorption of bone is inefficient in the absence of fusion of osteoclast precursors into a multinucleated osteoclast. [START_REF] Søe | Involvement of human endogenous retroviral syncytin-1 in human osteoclast fusion[END_REF] showed that both Syncytin-1 and SLC1A5 were expressed in differentiating and fusing osteoclasts, and that inhibition of Syncytin-1 fusion resulted in a significant decrease of the number of nuclei per osteoclast, but did not completely abrogate fusion. This suggests that Syncytin is involved in osteoclast fusion but is not the only actor, and that it is mainly involved in later fusion events that augment the number of nuclei present in an already formed syncytium [START_REF] Søe | Involvement of human endogenous retroviral syncytin-1 in human osteoclast fusion[END_REF]. This observation was confirmed by a later time-lapse study which revealed that CD47 was responsible for promoting fusion of mononuclear pre-osteoclasts, while Syncytin-1 mediated fusion of two multinucleated osteoclasts [START_REF] Møller | Osteoclast Fusion: Time-Lapse Reveals Involvement of CD47 and Syncytin-1 at Different Stages of Nuclearity[END_REF]. Interestingly, Syncytin-1 expression at the cell surface colocalized with f-actin expression, another component necessary for osteoclast fusion, and was especially visible in filopodia involved in cell-cell contact sensing and osteoclast movement [START_REF] Søe | Involvement of human endogenous retroviral syncytin-1 in human osteoclast fusion[END_REF]. Another recent ex vivo study by [START_REF] Ethiraj | Microgravity modulation of syncytin-A expression enhance osteoclast formation[END_REF] extends the potential involvement of syncytins to murine osteoclasts in which stimulation of Syncytin-A expression leads to increased osteoclast formation and bone resorption.

c. Syncytins and pathologies

As is the case for non-syncytin ERV env genes (see 2.2.2), abnormal syncytin expression has been linked to a number of pathologies in humans (reviewed in [START_REF] Bastida-Ruiz | The Dark Side of Cell Fusion[END_REF][START_REF] Bolze | Chapter Five -Contribution of Syncytins and Other Endogenous Retroviral Envelopes to Human Placenta Pathologies[END_REF], while their implication in non-human pathologies has not been extensively studied. In most cases the abnormal expression observed in a pathological context seems to be a consequence rather than a cause (reviewed in [START_REF] Bolze | Chapter Five -Contribution of Syncytins and Other Endogenous Retroviral Envelopes to Human Placenta Pathologies[END_REF]. In the placental pathology preeclampsia (in which maternal artery remodelling is insufficient, reviewed in [START_REF] Brosens | The "Great Obstetrical Syndromes" are associated with disorders of deep placentation[END_REF] for example, both Syncytin-1 and -2 expression is significantly down-regulated (correlating with severity of the disease) and cell-cell fusion is impaired [START_REF] Langbein | Impaired cytotrophoblast cell-cell fusion is associated with reduced Syncytin and increased apoptosis in patients with placental dysfunction[END_REF][START_REF] Vargas | Syncytin-2 Plays an Important Role in the Fusion of Human Trophoblast Cells[END_REF]. The reason for this down-regulation might be the hypoxic condition of the placenta (due to insufficient blood supply) which has been shown to induce degradation of GCMa, thereby being able to affect syncytin expression (reviewed in [START_REF] Bolze | Chapter Five -Contribution of Syncytins and Other Endogenous Retroviral Envelopes to Human Placenta Pathologies[END_REF].

The only example of a direct contribution to a disease can be found in some cancers. As with other ERV sequences, ectopic upregulations of syncytin expression is observed in a number of cancers (reviewed in [START_REF] Bastida-Ruiz | The Dark Side of Cell Fusion[END_REF]. It has been shown that in some cancers, tumor cells are implicated in cell fusion events with both tumor and non-tumor cells, thereby creating hybrid cancerous cells with novel properties and increasing the overall heterogeneity of the cancer (reviewed in [START_REF] Bastida-Ruiz | The Dark Side of Cell Fusion[END_REF]. As a consequence the cancer can become more difficult to treat and at the same time more invasive and dangerous (reviewed in [START_REF] Bastida-Ruiz | The Dark Side of Cell Fusion[END_REF]. A study by [START_REF] Bjerregaard | Syncytin is involved in breast cancer-endothelial cell fusions[END_REF] shows that about a third of breast cancer derived cell lines express Syncytin-1, and these cells are able to fuse with endothelial cell lines, presenting a potential pathway for a contribution of Syncytin-1 to tumor virulence, though its relevance in vivo has not been confirmed so far.

Objectives

In view of the results described in the previous section, it has been proposed that syncytin implication in placental evolution is two-fold: i) an ancestral syncytin capture even was responsible for the emergence of the mammalian placenta and ii) subsequent stochastic acquisition of new syncytins and syncytin-like genes led to structural changes resulting in the high diversity of modern day placentas. In the present study, we tried to strengthen the link between syncytin exaptation and both the establishment and subsequent structural transitions of placentas. To do so we approached the two aspects separately and investigated two different placental species, each illustrating one of them.

First, we studied placentation in the matrotrophic lizard genus Mabuya. Complex placentas have been described for several members of these genus and they display a highly organized structural arrangement with regions specialized for maternofetal exchanges that are, at least structurally, homologous with those observed in some mammalian placentas (Fig. 8 and see 1.3.2.c.). Of note, in the Mabuya placentome the maternofetal interface presents a syncytial tissue layer, similar to those observed in mammals. In the latter it has been shown that syncytins are indispensible for the establishment of the syncytial layers. The emergence of this placenta is a recent event since the Mabuya genus separated from its closest relatives (the Heremites genus) about 25 Mya (Karin et al., 2016), while the mammalian placenta is thought to have emerged about 175 Mya (dos Reis et al., 2012). This recent emergence led us to investigate if appearance of the Mabuya placenta is linked to the acquisition of a founding syncytin, as is hypothesized for mammals in which the ancient emergence of matrotrophy prohibits identification of a founding exaptation. Identification of a Mabuya syncytin would not only be the first identification of a non-mammalian syncytin gene, but also the first identification of a potential founding syncytin, which contributed directly to a shift to matrotrophic placentation.

Second, we investigated the curious case of Hyaenidae placentation. Contrary to every other carnivoran, hyenas present a hemochorial and not endotheliochorial placenta. The hemochorial hyena placenta is much more invasive that that of its carnivoran relatives and it has been repeatedly suggested that syncytins and syncytin-like genes might play a role in placental invasion processes. The major structural endo/hemochorial transition occurred specifically in the Hyaenidae family and has been studied in detail in one of its four extant species: Crocuta crocuta (the spotted hyena). As with the emergence of the Mabuya placenta, this structural transition took place recently in evolution, as Hyaenidae split from their sister family Eupleridae about 30 Mya [START_REF] Nyakatura | Updating the evolutionary history of Carnivora (Mammalia): a new species-level supertree complete with divergence time estimates[END_REF]. Since a direct link between variable syncytin expression

Implication of ERV env genes in non-mammalian Mabuya placentation 1.1 Summary

Following the identification of syncytins in most major mammalian clades, we investigated their involvement in non-mammalian placentation. As discussed in 1.3, placentation is not restricted to mammals and is present in a number of lizards, including the Mabuya genus which presents one of the most complex non-mammalian placentas described to date. Due to its structural similarity to mammalian placentas, and in particular the presence of a fused cell layer at the maternofetal interface, we searched for a fusogenic exapted env gene with placental expression, as is observed for mammalian syncytins.

Since no Mabuya genome has been published to date and the closest sequenced species is the distantly related green anole (Anolis carolinensis, last common ancestor about 200 Mya), we performed high-throughput RNA sequencing of a Mabuya sp. IV placental transcriptome. Comparison of this transcriptome to an equivalent mammalian placental transcriptome revealed that many implicated pathways are shared, reinforcing the notion that placentation is based on similar processes in these two distantly related clades. An in silico search of the Mabuya transcriptome led to the identification of four coding env ORFs, named Mab-Env1 through 4. These new env can be sorted into two groups: Mab-Env1 and 2 presenting strong similarities, as do Mab-Env3 and 4. All 4 sequences present the major domains of retroviral env and cluster alongside gamma-type envelopes on a TM based phylogenetic tree, however none of them seem to be classical gammaretroviral envelopes. Mab-Env3/4 present characteristics of the avian gamma-type, namely a shifted fusion peptide that is flanked by two cysteines, and cluster with RSV on the TM tree. Mab-Env1/2 show a much more uncommon organization, associating a gamma-type ISD with a beta-type CX 7 C motif. Their position on the phylogenetic tree puts them among other gamma-type TMs, suggesting that their main phenotype is gamma-like. Further characterization of Mab-Env1 led to the identification of the associated pol gene and allowed construction of an RT based phylogeny, in which it takes an intermediary place, between gammaretroviruses and all other retroviral genera.

RT-qPCR based analysis of Mab-Env expression in a panel of tissues show that none displays placenta-specific or preferential expression, but that Mab-Env1 is nevertheless significantly expressed in the placenta while the others show a lower level of expression. We therefore 1.1

. . Summary focused on Mab-Env1 in this study. To examine the expression profile of Mab-Env1 in the different layers of the Mabuya placentome (the region presenting a fused cell layer) and the adjacent paraplacentome, we performed in situ hybridization as well as immunohistochemistry (using anti-Mab-Env1 antibodies we obtained by immunizing mice). In the placentome, Mab-Env1 is expressed on both the fetal and maternal sides of the interface, with a stronger expression in the fused maternal layer. In the paraplacentome, strong expression is observed at the apical end of the fetal layer forming the border with maternal tissues This expression profile is in accordance with a physiological role of Mab-Env1 at the maternofetal interface.

To study conservation of Mab-Env1 in the Mabuya genus we attempted to amplify the entire ORF as well as a 400 bp internal fragment in 10 other related Scincidae genera. Amplification of a full length coding ORF was observed in all Mabuya species tested and only within that genus, suggesting that Mab-Env1 was conserved only in Mabuya. The internal fragment however was also amplified from genomic DNA of four closely related genera, suggesting that insertion of a Mab-Env1 containing provirus occurred, but that the sequence was not conserved.

Having established that Mab-Env1 is expressed in the placenta and conserved in the genus, we examined its fusogenic properties, the third characteristic of syncytin genes. Ex vivo fusion assays showed that Mab-Env-pseudotyped MLV particles were highly infectious (up to 10 7 ffu/mL) in most tested cell lines, except for rodents which display lower titers. In cell-cell fusion assays, Mab-Env1 is strongly fusogenic in acid pH conditions, as was observed for Syncytin-Rum1.

The tropism conferred by Mab-Env1 allowed us to search for its cognate receptor using the GenBridge4 human/hamster irradiation hybrid panel, which was previously used to identify the Syncytin-2/MFSD2A pair. The infection profile of Mab-Env1 pseudotyped-particles using the hybrid panel led to the identification of myelin protein zero-like 1 (MPZL1, a single transmembrane signal transducer; previously called protein zero related, PZR) as the cognate receptor. Cloning of Mabuya MPZL1 from placental cDNA confirmed that it is indeed able to render restrictive rodent cells infectable by Mab-Env1 pseudotyped particles.

Expression of MPZL1 was assayed both by RT-qPCR and in situ hybridization. Similarly to Mab-Env1, MPZL1 does not show placenta-specific or preferential expression, being highly expressed in a number of tissues, but does display significant expression in the placenta. In situ hybridization shows that MPZL1 is strongly expressed in the maternal layers of the maternofetal interface, both the fused layer in the placentome and the unfused layer in the paraplacentome. Fetal tissues are less stained indicating lower expression. This expression profile supports interaction of MPZL1 and Mab-Env1 in vivo, as they are expressed in the same tissue layer (placentome) or in two adjacent layers (paraplacentome).

In mammals, previous studies have implicated MPZL1 in cell migration and invasion processes in both oncogenic and physiological contexts. To study if interaction with Mab-Env1 can induce MPZL1 signal transduction, we transduced untransformed MCF10A cells with a Mab-Env1 expressing vector and showed that expression of Mab-Env1 is sufficient to trigger phosphorylation of MPZL1, an important step in its signal transduction and reflecting its . Implication of ERV env genes in non-mammalian Mabuya placentation 1.1 activation.

In conclusion, Mab-Env1 is expressed in the placenta, conserved during evolution and fusogenic, it therefore presents the three characteristic of mammalian syncytin genes and was renamed syncytin-Mab1. Syncytin-Mab1 is clearly distinct from all other previously identified syncytins (its unusual ISD/CX 7 C association sets it apart from all other gamma-type Env), and was exapted in a very distantly related clade. This exaptation of an ERV Env for a potential function in placental physiology is a remarkable example of convergent evolution and might be indicative of the involvement of Env in many more non-mammalian placentas. Additionally, the identification of the Syncytin-Mab1 cognate receptor hints at an involvement of Syncytin-Mab1 in placental invasiveness, a role long proposed for mammalian syncytins and syncytin-likes.

An endogenous retroviral envelope syncytin and its cognate receptor identified in the viviparous placental Mabuya lizard Syncytins are envelope genes from endogenous retroviruses that have been captured during evolution for a function in placentation. They have been found in all placental mammals in which they have been searched, including marsupials. Placental structures are not restricted to mammals but also emerged in some other vertebrates, most frequently in lizards, such as the viviparous Mabuya Scincidae. Here, we performed high-throughput RNA sequencing of a Mabuya placenta transcriptome and screened for the presence of retroviral env genes with a full-length ORF. We identified one such gene, which we named "syncytin-Mab1," that has all the characteristics expected for a syncytin gene. It encodes a membrane-bound envelope protein with fusogenic activity ex vivo, is expressed at the placental level as revealed by in situ hybridization and immunohistochemistry, and is conserved in all Mabuya species tested, spanning over 25 My of evolution. Its cognate receptor, required for its fusogenic activity, was searched for by a screening assay using the GeneBridge4 human/Chinese hamster radiation hybrid panel and found to be the MPZL1 gene, previously identified in mammals as a signal-transducing transmembrane protein involved in cell migration. Together, these results show that syncytin capture is not restricted to placental mammals, but can also take place in the rare nonmammalian vertebrates in which a viviparous placentotrophic mode of reproduction emerged. It suggests that similar molecular tools have been used for the convergent evolution of placentation in independently evolved and highly distant vertebrates.

endogenous retrovirus | envelope protein | placenta | syncytin | receptor S yncytins are "captured" genes of retroviral origin that corre- spond to the envelope gene of ancestrally endogenized retroviruses. These genes encode fusogenic proteins that are involved in the formation, by cell-cell fusion, of the syncytiotrophoblast at the placental materno-fetal interface in eutherian mammals (reviewed in refs. 1 and2). Furthermore, genetically modified mice in which the two syncytin genes, syncytin-A and syncytin-B, were knocked out showed deficiencies in placental development, with altered structure of the materno-fetal interface resulting in inhibition of growth or death of the embryo at midgestation [START_REF] Dupressoir | Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene[END_REF][START_REF] Dupressoir | A pair of co-opted retroviral envelope syncytin genes is required for formation of the two-layered murine placental syncytiotrophoblast[END_REF]. Syncytins have been found in all placental mammals in which they have been searched, with independently captured syncytins occurring across all major clades of placental mammals, including Euarchontoglires (primates, rodents, and lagomorphs), Laurasiatherians (ruminants and carnivores), Afrotherians (tenrec), and even Marsupials (opossum) (Fig. 1) [START_REF] Mi | Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis[END_REF][START_REF] Blond | An envelope glycoprotein of the human endogenous retrovirus HERV-W is expressed in the human placenta and fuses cells expressing the type D mammalian retrovirus receptor[END_REF][START_REF] Blaise | Genomewide screening for fusogenic human endogenous retrovirus envelopes identifies syncytin 2, a gene conserved on primate evolution[END_REF](8)[START_REF] Heidmann | Identification of an endogenous retroviral envelope gene with fusogenic activity and placenta-specific expression in the rabbit: A new "syncytin" in a third order of mammals[END_REF][START_REF] Cornelis | Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora[END_REF][START_REF] Cornelis | Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants[END_REF](12)(13)(14)(15). This has led to the proposal that these genes, which are absolutely required for placentation as shown by the knockout mice experiments, are most likely involved in the emergence and evolution of placental mammals from egg-laying animals [START_REF] Lavialle | Paleovirology of 'syncytins', retroviral env genes exapted for a role in placentation[END_REF]. Analysis of the conservation of these genes further indicates that they have been subjected to purifying selection in the course of evolution, as expected for any bona fide cellular gene.

Remarkably, placental structures are not restricted to mammalian species. Placentation emerged independently and in a stochastic manner in several groups of vertebrates, with the noticeable exception of birds (reviewed in refs. 16 and 17). In particular, complex placentas have been described in some South American or African species of Scincidae lizards (18-21; reviewed in refs. 16, 22, and 23). In one Scincidae genus, Mabuya, placental structures form specialized regions very similar to those found in mammals, e.g., the placentome where maternal and invasive fetal tissues are highly folded and interdigitated. Ultrastructural analysis of this region further revealed the presence of a syncytial structure at the materno-fetal interface, as observed in numerous mammalian species. However, in contrast to most mammals, the syncytial layer of the Mabuya placenta is formed by maternal epithelial cells rather than by fetal trophoblast cells, which remain individualized Significance Retroviral envelope gene capture and exaptation for a placental function has been demonstrated in mammals. Remarkably, placental structures have also emerged on rare occasions in nonmammalian vertebrates, resulting in related modes of reproduction. The Mabuya lizard, which emerged 25 Mya, possesses a placenta closely related to that of mammals. Here, we identified a specific retroviral envelope gene capture that shows all the characteristic features of a bona fide mammalian syncytin, being conserved in Mabuya evolution, expressed in the placenta, and fusogenic. Together with the present identification of its cognate receptor, these results show that syncytin capture is not restricted to mammals and is likely to be a major driving force for placenta emergence. cells throughout gestation (19,20,[START_REF] Jerez | Morphogenesis of extraembryonic membranes and placentation in Mabuya mabouya (Squamata, Scincidae)[END_REF][START_REF] Ramírez-Pinilla | Allantoplacental ultrastructure of an Andean population of Mabuya (Squamata, Scincidae)[END_REF][START_REF] Leal | Morphological variation in the allantoplacenta within the genus Mabuya (Squamata: Scincidae)[END_REF](27). Yet these differences can be considered minor in view of the tremendous qualitative transition that must have taken place between egg-laying and placental lizards that have a mode of reproduction so closely related to that of placental mammals. Following our previous proposal that such transitions are likely due to the stochastic acquisition of new genes via the endogenization of retroviruses and the exaptation of their envelope gene, a process that has taken place on several occasions in mammalian evolution, we investigated whether, as in mammals, endogenous retroviral env genes could have been specifically captured and exapted for a role in placenta formation in the Mabuya genus and might even be involved in the refined structure of their materno-fetal interface. Such events would indeed represent a remarkable example of convergent evolution in two major classes of vertebrates.

Currently, no genome within the Mabuya genus has been sequenced, and the most closely related species whose genome is available is the green anole (Anolis carolinensis), a distant small oviparous lizard. As a result, we could not screen Mabuya genomes to identify candidate syncytin genes as was done previously for mammalian species. After having captured pregnant females from a Colombian Mabuya population [referred to as "Mabuya sp. IV" ( 28)], we therefore performed high-throughput RNA sequencing (RNA-seq) of their placenta transcriptome and searched for the presence of expressed, coding retroviral env genes. Remarkably, we identified such a gene, syncytin-Mab1, which proved to be a syncytin and was characterized for its fusogenic activity, its conservation during Mabuya evolution, and its expression at the materno-fetal interface. Using a genetic approach, we identified its cognate receptor as the MPZL1 transmembrane protein, previously characterized as a membrane-bound signal transducer implicated in cell-migration processes [START_REF] Zhao | Cell surface glycoprotein PZR is a major mediator of concanavalin A-induced cell signaling[END_REF][START_REF] Zannettino | Novel mesenchymal and haematopoietic cell isoforms of the SHP-2 docking receptor, PZR: Identification, molecular cloning and effects on cell migration[END_REF][START_REF] Roubelakis | The murine ortholog of the SHP-2 binding molecule, PZR accelerates cell migration on fibronectin and is expressed in early embryo formation[END_REF][START_REF] Jia | Amplification of MPZL1/PZR promotes tumor cell migration through Src-mediated phosphorylation of cortactin in hepatocellular carcinoma[END_REF]. This identification of a captured syncytin and its receptor in a distant viviparous lizard with a placentotrophic mode of reproduction shows that syncytin capture is not restricted to mammals and is likely to be a major driving force for placenta emergence and evolution.

Results

High-Throughput Sequencing and in Silico Search for Retroviral Env Genes Within the Mabuya Placental Transcriptome. Placental RNA was extracted from pregnant Andean Mabuya sp. IV females captured in the wild (Methods). The transcriptome of a placenta at embryonic development stage 35 (as defined in ref. 33), presenting a well-established syncytium, was determined by the French National Sequencing Center (Genoscope, Evry, France). Placental transcripts were assembled using de novo assembly methods in absence of a reference genome (Methods). Transcriptome reconstruction identified 72,763 transcripts ranging from 123 bp to 24.4 kb [N50 (the length N for which 50% of all bases in the sequences are in a sequence of length L < N) = 2,869 bp; median size = 1.1 kb], 26,690 of which (36.7%) could be positively matched to a known protein gene from the Refseq Vertebrate database. A set of 21,253 transcripts shows significant expression (more than two tags per million) ( 34), 16,398 of which were positively annotated (77.2%) and correspond to 13,189 unique genes, among which 10,079 were nonhousekeeping genes. This subset of transcripts contains a range of genes annotated as "placental" genes with key biological functions during placenta development, such as core placental transcription factors, placental hormones, iron transporters, gap junction proteins, interleukins and interleukin receptors, metalloproteinases, and angiogenesis factors (SI Transcriptome Analysis and Datasets S2-S4).

To identify putative env-derived syncytin genes in this transcriptome, we screened these assemblies using the method we previously devised to screen mammalian genomes for such genes. In short, a BLAST search for env ORFs longer than 400 aa (from the Met start codon to the stop codon) was performed using a set of env sequences including all previously identified syncytins (Methods). It yielded four Mabuya transcripts that we named "Mab-Env1" to "Mab-Env4." Analysis of the overall structure of the four identified env genes (Fig. 2 A andB) strongly suggests that they indeed correspond to retroviral Env proteins, with all or most of their characteristic features conserved, including the presence of a predicted signal peptide sequence at the N terminus, a putative furin/PACE cleavage site delineating a surface (SU) and a transmembrane (TM) subunit, and a hydrophobic domain >20 aa long located downstream of the highly conserved C-X 5-7 -C motif of retroviral envelopes. This hydrophobic domain is involved in anchoring the Env protein within the plasma membrane, a feature required for its fusogenic activity. Finally, all genes present a canonical immunosuppressive domain (ISD). The presence of the ISD classifies the four envelopes as gamma-type retroviral envelopes (reviewed in ref. 35); however only Mab-Env3 and Mab-Env4 present the gamma-type-specific C-X 6 -CC motif that contains the third cysteine necessary for the covalent link between SU and TM observed in gamma-type envelopes, with the other two cysteines forming an intra-TM loop. Mab-Env1 and Mab-Env2 present a C-X 7 -C motif, a characteristic of beta-type retroviral envelopes in which the SU and TM are not linked by a disulfide bond and only the intra-TM bond is present (reviewed in ref. [START_REF] Henzy | Pushing the endogenous envelope[END_REF].

Incorporation of Mab-Env1 to Mab-Env4 into a phylogenetic tree based on the alignment of TM subunits is shown in Fig. 2C and demonstrates that they are distinct from previously identified env genes and that all four of them cluster with gamma-type envelopes. Interestingly, Mab-Env1 and Mab-Env2 are grouped together and display 82% amino acid identity with strong conservation of most of the functional domains (Fig. 2D), suggesting that they correspond to two genomic copies of the same retroviral family. The main differences between Mab-Env1 and Mab-Env2 are a 10-aa deletion in the N-terminal domain of the TM subunit of Mab-Env2 (the domain presumably harboring the fusion peptide critical for Env fusion activity) and a short truncation of the (15,[START_REF] Vidal | The phylogeny of squamate reptiles (lizards, snakes, and amphisbaenians) inferred from nine nuclear protein-coding genes[END_REF][START_REF] Kumazawa | Mitochondrial genomes from major lizard families suggest their phylogenetic relationships and ancient radiations[END_REF]], as indicated in the scale below the tree.

cytoplasmic tail of Mab-Env1 due to the presence of a premature stop codon. Although Mab-Env3 and Mab-Env4 are also grouped together, they only display 56% amino acid identity, suggesting that they belong to distinct, but related, retroviral families. Surprisingly, Mab-Env3 and Mab-Env4 cluster with the envelope gene of the Rous sarcoma virus (RSV), an alpharetrovirus presenting an avian gamma-type envelope. Closer inspection further reveals that their fusion peptide is located about nine amino acids after the putative furin site, a characteristic of avian gamma-type envelopes (reviewed in ref. [START_REF] Henzy | Pushing the endogenous envelope[END_REF]. As their name indicates, this subset of gamma-type envelopes was thought to be specific to bird retroviruses.

Mab-Env1 Is Highly Expressed in Placental Tissues. We performed an RT-qPCR analysis of transcript levels for each candidate Mab-Env gene in different tissues (Fig. 3) using primers that were designed to be specific for each env sequence (Table S1). Mabuya placental RNA was analyzed at two developmental stages: stage 30, when the uterine cells start to fuse, forming the syncytial layer, and stage 37, when the syncytium is well formed. High expression levels of Mab-Env1 (similar to that of the ribosomal protein RPL19) could be observed in the placenta at both stages.

The expression of Mab-Env2, Mab-Env3, and Mab-Env4 in the placenta was 6-to 85-fold lower than that of Mab-Env1. Of note, none of these genes was specifically expressed in the placenta.

Mab-Env1, in particular, shows high levels of expression in several other organs, including organs of the maternal genital tract (ovary and oviduct), a feature not commonly observed for mammalian syncytins. Due to the high expression levels of Mab-Env1 in the placenta and the low expression levels of the three other env genes, we focused on Mab-Env1 in the present study.

Characterization of the Mab-Env1-Associated Pol Gene. To obtain the sequence of the provirus harboring the Mab-Env1 gene, we rescreened the placental transcriptome in an iterative fashion using BLAST and found additional overlapping transcripts, each sharing a 3′ region homologous with the 5′ region of the previous transcript, allowing us to reconstruct an internal proviral sequence by concatenating the transcripts (Fig. 4A). We confirmed the existence of this sequence through PCR on genomic DNA with primers designed at both extremities of the assembled RNA-seq sequence (Table S1). We were able to amplify and sequence 5.8 kb of internal proviral sequence containing the end of gag and the entirety of pol and Mab-Env1. The pol ORF is interrupted at the 3′ end by several stop codons and frameshifts, while the Mab-Env1 ORF is intact, as expected (Fig. 4A). Of note, an acceptor splice site can be predicted with a high score in the proviral sequence, about 400 bp upstream of the env start codon (using www.cbs.dtu.dk/ services/NetGene2), but the corresponding retroviral spliced env transcript could not be retrieved from the RNA-seq assemblies.

y E R V H T L V 1 RE V C A E V M P M V Syn -1 E n v -C a v 1 B L V Syn -Op o1 S y n -T e n 1 G a L V BaE V HERV -K P E R V T g E R V F Sy n-Ru m 1 S y n -C a r 1 M a b -E n v 3 S y n -A F IV M a b -E n v 2 M o M L V H I V 1 S y n -M a r1 Syn-2 JS RV M M T V C 0 -3 3 0 -3 3 0 -3 3
Identification of the conserved reverse transcriptase (RT) domain of the pol gene allowed us to compare it with previously described RT domains to determine the family of the Mab-Env1associated virus. A PhyML maximum likelihood tree using RTs from both exo-and endogenous retroviruses (Fig. 4B) places the Mab-Env1-associated RT closest to the gamma family of RTs. However, it remains external to all gammaretroviral RTs of the dataset, indicating that it might be a distant member of this family or a member of another, closely related family. In association with the noncanonical combination of an ISD and a C-X 7 -C motif in Mab-Env1 (see above and Fig. 2C), this finding suggests that this lizard retrovirus differs from previously described retroviral families.

Conservation Within the Mabuya Genus. To study Mab-Env1 conservation within the Mabuya genus, we tentatively PCR-amplified the entire Mab-Env1 ORF from the genomic DNA of several species of Scincidae. Fragments of the expected length were obtained for all Mabuya species shown in Fig. 5. Sequencing of the purified PCR products revealed intact reading frames belonging to the Mab-Env1 family (Fig. 5). No amplification was obtained in any non-Mabuya Scincidae, even though amplification of BDNF, a conserved control gene, indicated that the extracted genomic DNA was of good quality. We then used a less stringent approach by designing degenerate primers in conserved internal Mab-Env1 regions based on alignments of all available Mabuya sequences. This approach led to the amplification of a 400-bp fragment, ranging from the end of the SU to the start of the ISD, in all Mabuya tested, as expected, but also in the Trachylepis, Chioninia, Eumecia, and Lubuya genera (sequences are shown in Fig. S2). This unexpected result indicates that endogenization of the Mab-Env1-associated virus probably occurred before the separation of these genera, about 30 Mya (Fig. 5) [START_REF] Pereira | Arrival and diversification of mabuyine skinks (Squamata: Scincidae) in the Neotropics based on a fossil-calibrated timetree[END_REF], but that the full-length envelope was conserved only in the Mabuya genus. Of note, we have been unable to amplify even the 400-bp fragment from Heremites vitattus genomic DNA, which is not compatible with the entry date proposed above (Fig. 5).

Heremites is a newly created genus [START_REF] Karin | Resolving the higher-order phylogenetic relationships of the circumtropical Mabuya group (Squamata: Scincidae): An out-of-Asia diversification[END_REF], and its position on the Scincidae phylogenetic tree is only weakly supported so far, which might explain this apparent anomaly, although sequence divergence could also account for this negative result.

In Situ Analyses of Mab-Env1 Transcription and Protein Expression on Placental Sections. As described previously in refs. 19, 20, and 24-26, the Mabuya placenta is a complex structure composed of specialized regions (Fig. 6A) including areolae and absorptive plaques involved in histiotrophic nutrition and, at the embryonic pole, the placentome and paraplacentome. In the placentome region, maternal and fetal epithelia fold and interdigitate during gestation, and ramifications are highly vascularized. At the materno-fetal interface, the fetal side is mainly composed of giant columnar binucleated chorionic cells. On the maternal side, the uterine epithelium is replaced by large syncytial structures, most probably resulting from the cellular fusion of uterine cells. At the cellular interface, maternal and fetal cells form microvilli, further increasing the exchange surface of the placentome (19,20,[START_REF] Jerez | Morphogenesis of extraembryonic membranes and placentation in Mabuya mabouya (Squamata, Scincidae)[END_REF][START_REF] Ramírez-Pinilla | Allantoplacental ultrastructure of an Andean population of Mabuya (Squamata, Scincidae)[END_REF][START_REF] Leal | Morphological variation in the allantoplacenta within the genus Mabuya (Squamata: Scincidae)[END_REF]. Periodically the microvilli are interrupted by the presence of

P y E R V S y n -C a r1 C A E V G A L V Ba EV P E R V Mab-E nv1 F IV F L V H IV 1 RSV Sy n-Op o1 H T L V 1 S y n -1
support values <25% ≥25% ≥50% ≥75% ≥95% smaller invasive fetal cells located between the fetal and maternal epithelia, allowing a more intimate contact between fetal tissues and the maternal circulatory system [START_REF] Vieira | Invasive cells in the placentome of Andean populations of Mabuya: An endotheliochorial contribution to the placenta?[END_REF]. The paraplacentome surrounds the placentome and is limited on one side by the abrupt transition from a uterine syncytium to a uterine epithelium. The organization of fetal tissues in the paraplacentome does not differ significantly from that observed in the placentome, although tissues are not folded. The materno-fetal interface is still marked by interdigitated microvilli, although they are smaller than in the placentome (19,20,[START_REF] Jerez | Morphogenesis of extraembryonic membranes and placentation in Mabuya mabouya (Squamata, Scincidae)[END_REF][START_REF] Ramírez-Pinilla | Allantoplacental ultrastructure of an Andean population of Mabuya (Squamata, Scincidae)[END_REF][START_REF] Leal | Morphological variation in the allantoplacenta within the genus Mabuya (Squamata: Scincidae)[END_REF].

RT

To assess the potential physiological relevance of Mab-Env1 expression in the placenta, both in situ hybridization and immunohistochemistry experiments were performed on paraffin sections of stage 37 Mabuya placentas (Fig. 6B). Specific digoxigeninlabeled antisense riboprobes were synthesized for the detection of Mab-Env1 transcripts, and the corresponding sense riboprobes were used as negative controls. Anti-Mab-Env1 sera were obtained by immunizing mice with Mab-Env1 SU, and preimmune sera were used as controls (SI Methods). Both methods show that the expression of Mab-Env1 seems to be widespread, but the staining (at both the RNA and protein level) is more intense in the cell layers forming the interface between mother and fetus. In the placentome, the staining indicates stronger expression in the maternal syncytium (ms) than in the fetal chorionic cells (fc). In the paraplacentome, strong staining is observed at the apical side of the fetal chorionic cell layer, including the microvillous interface between mother and fetus. These expression profiles would support a physiological role for Mab-Env1 at the maternofetal interface (as further examined below).

Mab-Env1 Is Fusogenic in ex Vivo Assays. The functionality of Mab-Env1 as an ancestrally derived retroviral envelope protein was assayed as previously for other syncytins. First, we tested whether this envelope protein added in trans could render a recombinant retrovirus deprived of its native env gene able to infect target cells (Fig. 7A). To do so, the env gene was cloned and introduced into a CMV promoter-containing expression vector that was used to generate pseudotyped murine leukemia virus (MLV) particles (SI Methods). As illustrated in Fig. 7 B and C, Mab-Env1-pseudotyped MLV particles generated in human 293T cells could infect a panel of target cells of different origins, including human (293T, TE671, SHSY-5Y, HeLa), bovine (MDBK), carnivoran (G355.5, CrFK, MDCK), and rodent (WOP, 208F, A23) cells, in some cases at levels similar to those observed using the amphotropic MLV (A-MLV) Env protein (although all rodent cells tested exhibited a significantly lower titer). Since Mab-Env1 is a protein of reptilian origin, this suggests that its cognate receptor is a highly conserved protein among vertebrates. Rodent cells might be less permissive to infection due to subtle divergences in the evolution of the receptor in this rapidly evolving clade. Mab-Env2 was found to be negative for all target cells tested under the same experimental conditions. Although we cannot exclude the possibility that Mab-Env2 has lost its ability to interact with the mammalian ortholog of its cognate receptor or that Mab-Env1 and Mab-Env2 have different receptors, these are unlikely. Indeed, as described above, Mab-Env1 and Mab-Env2 are very similar, but Mab-Env2 presents a deletion within the putative fusion peptide (Fig. 2D), suggesting that Mab-Env2 has lost its fusogenic properties.

Cell-cell fusion experiments were performed by transfecting cells with the env expressing plasmids and assaying the formation of syncytia 48 h post transfection in the presence or absence of an acidic shock (Fig. 7D). Mab-Env1 was able to induce fusion only when the cells were submitted to a low-pH shock, and under these conditions it induced the formation of large, multinuclear syncytia (Fig. 7E) in all cell lines tested except rodent cells, as expected from the assay described in Fig. 7B. Mab-Env2 tested negative under all conditions, further confirming the results of the pseudotyping assay.

Of note, both Mab-Env3 and Mab-Env4 were negative in the two assays above.

Search for the Mab-Env1 Receptor. Since Mab-Env1 is able to efficiently mediate the infection of human cells but not hamster A23 cells, we took advantage of the human-hamster hybrid irradiation panel (Genbridge4;ref. 39) in which fragments of the human genome had been introduced into a panel of 93 hamster A23 clones (Fig. S3A) and which we had used successfully to identify the receptor of human Syncytin-2 [START_REF] Esnault | A placenta-specific receptor for the fusogenic, endogenous retrovirus-derived, human syncytin-2[END_REF]. We screened the Genebridge4 clones for infection using the pseudotyping assay described above. The infection results for the 93 ordered clones were [00000100000011100000000010000000100000010100000-000100000000001000000001011000000200000000001], where 1 denotes infection (>100 focus-forming units/mL), 0 denotes the absence of infection, and 2 indicates that the clone was unavailable for analysis (Fig. S3B). This pattern of infection was compared with a matrix of specific genomic markers by computing a matching score using the RH program [START_REF] Boehnke | Statistical methods for multipoint radiation hybrid mapping[END_REF]. The eight best-scoring markers were located within the same 2-Mb region of the human genome, on chromosome 1 (Fig. S3C). Among the genes located within this genomic region, 11 encode putative transmembrane proteins. The capacity of each of these genes to mediate the infection of A23 cells by Mab-Env1-pseudotyped particles, and thus to function as a receptor for Mab-Env1, was assayed using expression vectors for the corresponding cloned human cDNAs (Thermo Fisher) and revealed that only the myelin protein zero-like 1 (MPZL1) protein, a previously characterized single-pass transmembrane receptor [START_REF] Zhao | Cell surface glycoprotein PZR is a major mediator of concanavalin A-induced cell signaling[END_REF][START_REF] Zhao | Purification and cloning of PZR, a binding protein and putative physiological substrate of tyrosine phosphatase SHP-2[END_REF], was able to increase infection of A23 cells (see below). To verify that Mab-Env1 can also interact with Mabuya MPZL1, we searched by BLAST for the corresponding cDNA sequence in the Mabuya placental transcriptome and designed primers to amplify MPZL1 from placental cDNA. The Mabuya MPZL1 ORF that could be cloned shares 65% amino acid identity with human MPZL1 (Fig. 8A), with the glycosylation sites, the disulfide bond predicted for human MPZL1, and the intracellular C-terminal domain that contains two immunoreceptor tyrosine-based inhibitory motifs (ITIMs) (Fig. 8 A and B) [START_REF] Zhao | Purification and cloning of PZR, a binding protein and putative physiological substrate of tyrosine phosphatase SHP-2[END_REF] being conserved, whereas the extracellular N-terminal domains of the two proteins are much more variable. As illustrated in Fig. 8C, when infected with Mab-Env1-pseudotyped MLV particles, A23 cells transfected with either human or Mabuya MPZL1 exhibit titers 100 times Fig. 5. Mab-Env1 is conserved only within the Mabuya genus. (Left) Species tree of Scincidae with A. carolinensis as an outgroup. Nodes with black circles are dated in Mya and placed proportionally to their age [START_REF] Pinto-Sánchez | Molecular phylogenetics and biogeography of the Neotropical skink genus Mabuya Fitzinger (Squamata: Scincidae) with emphasis on Colombian populations[END_REF][START_REF] Pereira | Arrival and diversification of mabuyine skinks (Squamata: Scincidae) in the Neotropics based on a fossil-calibrated timetree[END_REF][START_REF] Vidal | The phylogeny of squamate reptiles (lizards, snakes, and amphisbaenians) inferred from nine nuclear protein-coding genes[END_REF]. Entry of Mab-Env1 is indicated by the white-tipped arrow; clades in which the envelope has been conserved are indicated by the black arrow. An asterisk indicates that a published genome is available (A. carolinensis). (Right) A plus sign indicates amplification of a Mab-Env1 internal 400-bp fragment (int) or the complete env ORF by genomic DNA PCR. Amplification of the complete ORF is restricted to the Mabuya genus, with no amplification in other Scincidae. higher than control cells, indicating that Mabuya MPZL1 can indeed act as a receptor for Mab-Env1.

Expression and Potential Activation of MPZL1 in Mabuya Placenta. To determine if MPZL1 can interact with Mab-Env1 in the placenta, we first compared its expression profile with that of Mab-Env1.

Using the same cDNA panel as in Fig. 3, we performed RT-qPCR and found that MPZL1 is indeed expressed in the placenta but also in other tissues (Fig. 8D). We then performed in situ hybridization on Mabuya placenta sections, as in Fig. 6B, using MPZL1-specific antisense probes and sense probes as negative controls. As illustrated in Fig. 8E, a specific antisense-only signal is observed at the materno-fetal interface. The maternal syncytium in the placentome and the maternal epithelium of the paraplacentome in particular show strong staining, while fetal chorionic cells are only moderately stained in both regions. This pattern is close to that observed for Mab-Env1 (Fig. 6B), especially for the placentome where both genes are more expressed at the level of the maternal syncytium, whereas in the paraplacentome the strongest expression of each gene takes place in distinct, although interdigitated, tissues of the materno-fetal interface. Therefore Mab-Env1 and MPZL1 could interact physically in vivo in both the placentome and paraplacentome, but in the former this interaction could contribute directly to the establishment of the maternal syncytium, which shows strong staining for both genes.

Previous studies have shown that MPZL1 is a signal transducer, the activation of which requires phosphorylation of the two tyrosine residues (Y241 and Y263) contained in its cytoplasmic ITIM motifs (Fig. 8 A andB) [START_REF] Zhao | Cell surface glycoprotein PZR is a major mediator of concanavalin A-induced cell signaling[END_REF]. To further demonstrate the interaction of Mab-Env1 with MPZL1, we investigated whether Mab-Env1 can trigger MPZL1 phosphorylation. To do so, we transduced untransformed human breast MCF10A cells with a lentiviral vector expressing Mab-Env1 or a negative control (empty vector or a vector expressing the mammalian syncytin-Car1). As a positive control, we treated the cells with concanavalin A (Con A), which is known to induce homodimerization and phosphorylation of MPZL1 [START_REF] Zhao | Cell surface glycoprotein PZR is a major mediator of concanavalin A-induced cell signaling[END_REF]. Cell lysates were then analyzed by Western blot using an antibody directed specifically against the phosphorylated form as well as an antibody detecting both forms of MPZL1. In three independent transduction experiments we observed that Mab-Env1 very significantly reduces the total amount of MPZL1 without decreasing (rather slightly increasing) its overall phosphorylation level, compared with the control syncytin-Car1 or empty vector (Fig. 9). This indicates that Mab-Env1 interacts with MPZL1, triggering both its degradation (reminiscent of receptor interference) and, as expected, a strong correlative increase of its phosphorylation. Maternal and fetal tissues are highly interdigitated in the placentome region. In addition to the placentome, the Mabuya placenta develops specialized regions for materno-fetal exchanges such as areolae and absorptive plaques. The brown/orange and gray/yellow areas represent the fetal and maternal tissues, respectively. (Right) Detailed scheme of the materno-fetal interface in the placentome and paraplacentome region. In the placentome, maternal and fetal tissues are highly interdigitated, with numerous microvilli between fetal and uterine cells. The fetal epithelium is formed by giant binucleated chorionic cells. The uterine epithelium is replaced by a large syncytial structure formed by the fusion of the uterine cells. In the paraplacentome the syncytium is abruptly replaced by uterine epithelium. Cells still present microvilli, but the tissues are no longer interdigitated. (B, Left) Hematoxylin eosin saffron (HES)-stained sections of placenta with the maternal uterine syncytium (ms), the maternal epithelium (me), and the fetal chorionic epithelium ( fc

Discussion

Analysis of the presently established Mabuya lizard placental transcriptome revealed the presence of an endogenous retroviral env gene, Mab-Env1. It is highly expressed in placental tissues, especially at the level of the materno-fetal interface, both in the maternal syncytium layer in the placentome and in the fetal epithelium in the paraplacentome. Mab-Env1 is a fusogenic protein that is able to functionally replace a present-day retroviral env gene within a recombinant infectious retrovirus. It also can mediate cell-cell fusion, although only under conditions of low pH as observed for the ruminant syncytin-Rum1 [START_REF] Cornelis | Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants[END_REF]. The cognate receptor of Mab-Env1 was also identified and found to be the previously described MPZL1 transmembrane signal transducer [START_REF] Zhao | Cell surface glycoprotein PZR is a major mediator of concanavalin A-induced cell signaling[END_REF]. Its expression is not restricted to the placenta, but in situ hybridization demonstrates expression at the materno-fetal interface, which colocalizes with that of Mab-Env1 in the placentome syncytial layer. Finally, we have shown that Mab-Env1 is conserved in the Mabuya species tested, covering ∼25 My of evolution of this specific placental clade [START_REF] Pereira | Arrival and diversification of mabuyine skinks (Squamata: Scincidae) in the Neotropics based on a fossil-calibrated timetree[END_REF]. Together, these canonical characteristics-i.e., fusion activity, expression in the placenta, and conservation during evolution-allow Mab-Env1 to be now named "syncytin-Mab1" and to be added to the series of previously characterized syncytins that we and others have identified so far in both Eutherian and Marsupial placental mammals [START_REF] Mi | Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis[END_REF][START_REF] Blond | An envelope glycoprotein of the human endogenous retrovirus HERV-W is expressed in the human placenta and fuses cells expressing the type D mammalian retrovirus receptor[END_REF][START_REF] Blaise | Genomewide screening for fusogenic human endogenous retrovirus envelopes identifies syncytin 2, a gene conserved on primate evolution[END_REF](8)[START_REF] Heidmann | Identification of an endogenous retroviral envelope gene with fusogenic activity and placenta-specific expression in the rabbit: A new "syncytin" in a third order of mammals[END_REF][START_REF] Cornelis | Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora[END_REF][START_REF] Cornelis | Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants[END_REF](12)(13)(14)(15).

The captured Mab-Env1 and associated pol genes have some unusual properties that could be linked to the remoteness of the species in which they were discovered. Although the Env gene falls within the family of gamma-type envelopes according to both the phylogenetic tree and the presence of an ISD, as shown in Fig. 2, it possesses a C-X 7 -C motif in the TM subunit that is not classically found in such envelopes. Gamma-type envelopes normally possess three cysteines in this motif (C-X 6 -CC), allowing the formation of a covalent SU-TM link, whereas a two-cysteine motif is observed in betaretroviruses and lentiviruses, in which the SU and TM are not covalently associated (reviewed in ref. [START_REF] Henzy | Pushing the endogenous envelope[END_REF]. The associated noncoding pol gene displays a characteristic RT domain that clusters more closely to gammaretroviral RTs but forms a sister clade to all remaining gammaretroviral sequences in our dataset (Fig. 4). This suggests that it is either a remotely related gammaretrovirus or that it belongs to another, potentially unknown, family. The latter might also explain the unusual characteristics of the syncytin-Mab1 gene discussed above. It is also noteworthy that Mab-Env3 and Mab-Env4 present characteristics of avian gammatype envelopes, further reinforcing the idea that the remoteness of the investigated species should reveal novel categories of endogenous retroviruses.

Other interesting results of the present investigation concern the nature of the identified receptor for this retroviral envelope and the properties of this previously characterized gene that could be relevant to Mabuya placenta physiology. The MPZL1 receptor is a single-pass transmembrane glycoprotein belonging to the immunoglobulin superfamily, with an intracellular moiety containing two ITIMs. Previous studies have shown that MPZL1 can promote the migration of mesenchymal-derived mouse embryonic fibroblasts [START_REF] Zannettino | Novel mesenchymal and haematopoietic cell isoforms of the SHP-2 docking receptor, PZR: Identification, molecular cloning and effects on cell migration[END_REF][START_REF] Roubelakis | The murine ortholog of the SHP-2 binding molecule, PZR accelerates cell migration on fibronectin and is expressed in early embryo formation[END_REF] or hepatocellular carcinoma cells [START_REF] Jia | Amplification of MPZL1/PZR promotes tumor cell migration through Src-mediated phosphorylation of cortactin in hepatocellular carcinoma[END_REF] and can be involved in adhesion-dependent signaling [START_REF] Eminaga | Noonan syndrome-associated SHP-2/Ptpn11 mutants enhance SIRPalpha and PZR tyrosyl phosphorylation and promote adhesion-mediated ERK activation[END_REF][START_REF] Kusano | Phosphorylation and localization of proteinzero related (PZR) in cultured endothelial cells[END_REF]. In this respect, it will be interesting to determine whether the presently demonstrated syncytin-Mab1-induced phosphorylation of MPZL1 could also trigger receptor-mediated signaling in vivo that could participate in some function essential for Mabuya placentation in addition to the canonical membrane fusion activity observed for syncytins. For instance, activation of MPZL1 could enhance placental cell migration/invasion into the maternal tissue or even inhibit some specific immune functions via its ITIM domains.

One remarkable aspect of vertebrate physiology concerns the highly diverse modes of reproduction, with clades such as birds having a strictly oviparous mode of reproduction and other clades containing both oviparous and some viviparous species. Even among viviparous species, a range of reproductive strategies exists. In some species, the egg and embryo develop with very limited maternal supply, being essentially dependent on yolk supply (lecithotrophy), while at the other extreme some species show strong dependence on maternal exchanges and almost no reliance on yolk (matrotrophy) (reviewed in refs. 16 and 45). The placental organization of mammals represents the latter extreme, with the mother supplying almost all nutrients necessary for fetal growth. Clearly, the mammalian placenta is likely to be a monophyletic trait that emerged about 150 Mya and has since evolved into several variations on a common theme. In all cases in which it has been searched, there is evidence that retroviral capture has played a role in the diversity of placental structures, and possibly even in the emergence of the mammalian placenta via the capture of a primitive retroviral envelope during the radiation of this family (reviewed in refs. 1 and 2). Of note, there is increasing evidence that some nonmammalian vertebrates, among them Mabuya Scincidae and other lizards such as the African Lubuya ivensii (21,[START_REF] Blackburn | Invasive implantation and intimate placental associations in a placentotrophic African lizard, Trachylepis ivensi (Scincidae)[END_REF], possess a mode of viviparity with placental structures that closely resembles that found in mammals. (D) Schematic representation of the cell-cell fusion assay. 293T cells were cotransfected with a plasmid expressing a nuclear β-galactosidase and a plasmid expressing either Mab-Env1 or a control plasmid. After 48 h a 5-min acidic shock was performed (or not) using Dulbecco's phosphate-buffered saline (DPBS)•HCl at either pH 4 or 7. Cells were X-gal stained 6 h afterward. (E) Mab-Env1 is able to induce cell-cell fusion after an acidic shock. After exposition to acid medium, cells transfected with the Mab-Env1-expressing plasmid show a degree of fusion equivalent to that of cells transfected with a plasmid expressing the Env protein of mouse mammary tumor virus (MMTV), which was used as a positive control. These species have developed placentas that possess several of the characteristic features of the mammalian placenta and enable very similar reproductive strategies, including high maternal dependence for embryo growth, very tiny eggs almost devoid of yolk supply, prolonged pregnancy, and a large increase in conceptus dry mass during gestation (18,22,[START_REF] Flemming | Extraordinary case of matrotrophy in the African skink Eumecia anchietae[END_REF]. Accordingly, and taking the present results into account, we propose as a working hypothesis an extended version of a model previously proposed for mammals, in which emergence of this reproductive strategy is associated with the stochastic acquisition of a retroviral envelope gene that provides the corresponding species with syncytin functions. This model can be substantiated as follows. First, it is now established that placenta-like structures have emerged among egg-laying vertebrates throughout vertebrate evolution, ranging from 400 Mya in fish to 150 Mya in mammals and 25 Mya in the Mabuya lizards (48; reviewed in refs. 17 and 49). These emergences appear to be random and rare. Consistently, retroviral endogenization events have been identified in a large series of vertebrates, with some of them having occurred over 400 Mya in marine species [START_REF] Aiewsakun | Marine origin of retroviruses in the early Palaeozoic Era[END_REF]. Envelope gene capture in which this gene has been conserved after retrovirus endogenization has recently been characterized in spiny-rayed fishes dating back 100 My [START_REF] Henzy | An intact retroviral gene conserved in spiny-rayed fishes for over 100 My[END_REF]. This indicates that retroviral env capture has most probably taken place across all of vertebrate evolution, and endogenization probably even predates the emergence of vertebrates. Therefore, the stochastic emergence of specific modes of reproduction during vertebrate evolution could, in principle, be accounted for by retroviral capture events, taking into account the ancestry of these elements. One other hint provided by the present investigation is that we not only could demonstrate ret-roviral env capture in the form of the functional fusogenic envelope protein syncytin-Mab1 but also could correlate its capture in time with the emergence in the Mabuya genus of a placental structure ∼25 Mya [START_REF] Pereira | Arrival and diversification of mabuyine skinks (Squamata: Scincidae) in the Neotropics based on a fossil-calibrated timetree[END_REF]. Although we acknowledge that the temporal correlation between the acquisition of a retroviral env gene possessing all the characteristic features of canonical mammalian syncytins and the emergence of the Mabuya placenta is not proof of a causal link, it constitutes a significant hint for a direct involvement (see also the properties of the syncytin-Mab1

+ C o n A -C o n A M a b -E n v 1 S y n c y t in -C a r 1 Empty vector Anti-tubulin γ + C o n A -C o n A M a b -E n v 1 S y n c y t in -C a r 1 Empty vector Anti-Phospho MPZL1 (Y241)
Anti-MPZL1 Fig. 9. Mab-Env1 induces degradation and phosphorylation of MPZL1. MCF10A cells were transduced either with an empty lentiviral vector or with the same vector coding for syncytin-Car1 or Mab-Env1. Con A treatment was performed (or not) 1 h before cell lysis as a positive control. Lysates were deglycosylated using PNGase F, and Western blot analysis was performed using an antibody recognizing both phosphorylated and unphosphorylated MPZL1 (Top) or only phosphorylated MPZL1 (Middle). (Bottom) An anti-tubulin-γ antibody was used to compare total protein levels in each well. Each set of blots represents an independent set of transduced cells.

receptor likely to be relevant to such a role, as discussed above). To be complete, we must also mention that part of the syncytin-Mab1 sequence, although not the complete env, could be identified in two clades closely related to the Mabuya genus, namely the clade containing the Trachylepis and Chioninia genera and the clade containing the Eumecia and Lubuya genera, but not in any other, more distantly related clades (notably Dasia, Toenayar, or Eutropis). This suggests that Mab-Env1 probably entered the Scincidae family slightly before the divergence of the Mabuya genus dated ∼25 Mya, and that the related env gene was not maintained as a protein-coding sequence in Trachylepis and Chioninia or in Eumecia and Lubuya. For the former two, this scenario would be consistent with Trachylepis being oviparous and Chioninia being lecithotrophic [START_REF] Metallinou | A single origin of extreme matrotrophy in African mabuyine skinks[END_REF]. For the latter two, which display matrotrophic placentation (21,[START_REF] Flemming | Extraordinary case of matrotrophy in the African skink Eumecia anchietae[END_REF][START_REF] Metallinou | A single origin of extreme matrotrophy in African mabuyine skinks[END_REF], it must be hypothesized either that the full-length gene could not be amplified for technical reasons or that another capture has taken place later in evolution. Along this line, it is theorized that the emergence of viviparity in American (Mabuya) and African (Eumecia and Lubuya) Scincidae results from two independent emergence events [START_REF] Metallinou | A single origin of extreme matrotrophy in African mabuyine skinks[END_REF].

In any event, the identification in the present paper of both a syncytin and its cognate receptor in a remote nonmammalian species opens the door to further searches for other syncytin captures in distant placental vertebrates and to in-depth investigations of their roles. These could include noncanonical functions, as suggested here, that would add to their fusogenic activity. This could lead to a deeper understanding of the mechanisms underlying the rare and stochastic emergence of placentation, a remarkable example of evolutionary convergence, and provide hints on species evolution in vertebrates.

Methods

Animals and Tissues. Mabuya females were captured in the wild by M.P.R.-P. in the municipality of Curiti, Department of Santander, Colombia. This Colombian population is an as-yet unnamed species described in ref. [START_REF] Pinto-Sánchez | Molecular phylogenetics and biogeography of the Neotropical skink genus Mabuya Fitzinger (Squamata: Scincidae) with emphasis on Colombian populations[END_REF] as Mabuya sp. IV. This species corresponds to the one studied in refs. 19, 24, 25, 27, and 53. Placental and uterine tissues were collected from killed pregnant females and were either stored in RNAlater (Qiagen) or processed for in situ hybridization. Other tissues (ovary, oviduct, liver, intestine, kidney, heart, lung, and brain) were collected from the same killed females. Total RNA was extracted after tissue lysis in TRIzol reagent (Invitrogen), and genomic DNA was extracted by phenol-chloroform extraction. Genomic DNA from Mabuya croizati, Mabuya dominicana, Mabuya nigropalmata, Mabuya macrorhyncha, Mabuya unimarginata (sensu lato), and Mabuya zuliae and from Chalcides mionecton, Chioninia delalandii, Eutropis multifasciata, Heremites vittatus, Trachylepis quinquetaeniata, and Tribolonotus gracilis was obtained by phenol-chloroform extraction from tissue samples provided by A.M. Similarly, genomic DNA was extracted from Dasia olivacea tissues provided by Aaron Bauer (Villanova University, Villanova, PA ) and from Lubuya ivensii tissues (Museum of Comparative Zoology numbers R-193864, R-193868, R-193869, and R-193870) provided by Breda Zimkus (The Louis Agassiz Museum of Comparative Zoology, Cryogenic Collection, Harvard University, Cambridge, MA). Genomic DNA from Eumecia anchietae and Toenayar novemcarinata was also provided by Aaron Bauer.

Mabuya sp. IV Placenta Transcriptome High-Throughput Sequencing and de Novo Assembly. Total RNA was purified from Mabuya sp. IV placenta before sequencing using the Illumina HiSeq. 2000 sequencing system. cDNA library construction, sequencing, and transcripts assembly were performed by the French National Sequencing Center (Genoscope, Evry, France). Briefly, poly(A) + RNA was selected with oligo(dT) beads, chemically fragmented, and converted into single-stranded cDNA using random hexamer priming according to the Illumina TruSeq protocol. The second strand was then generated to create double-stranded cDNA. Next, the paired-end library was prepared following Illumina's protocol: Briefly, fragments were endrepaired and then 3′-adenylated, and Illumina adapters were added by using NEBNext Sample Reagent Set (New England Biolabs); ligation products were purified, and DNA fragments (>200 bp) were PCR-amplified using Illumina adapter-specific primers. After library profile analysis (showing a typical library size of 200-600 bp) by Agilent 2100 Bioanalyzer (Agilent Technologies) and qPCR quantification (MxPro; Agilent Technologies), the library was sequenced using 100-base-length read chemistry in a paired-end flow cell on the Illumina HiSeq2000 instrument (Illumina). Nucleotide sequence information has been deposited at the European Nucleotide Archive (ENA; https://www.ebi.ac.uk/ena) under the accession number ERA1116158. To maximize the number of reconstructed transcripts, several independent assemblies were performed using two different de novo assembly software programs (Oases and Trinity) as well as a range of k-mer lengths (55, 59, 63, 67, 71, 75 and 79), and only transcripts of >100 bp were conserved. We then used the EvidentialGene pipeline to remove duplicates and concatenate fragmented transcripts from each individual assembly [START_REF] Gilbert | Gene-omes built from mRNA-seq not genome DNA[END_REF]. Reads were mapped on the nonredundant assembly using Bowtie2 [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF], and transcript abundance was quantified using Express [START_REF] Roberts | Streaming fragment assignment for real-time analysis of sequencing experiments[END_REF]. To identify the transcripts, we used TBLASTX (word_size 3, gapopen 10, gapextend 3, BLOSUM 45) with an e-value of 10 -3 to compare them with the nonredundant protein database of vertebrate genomes (RefSeq database Vertebrates January 5, 2015), and used the best hit as the transcript identifier for downstream analysis. Gene identifiers were converted to gene symbols using the bioDBnet (https://biodbnet-abcc. ncifcrf.gov) website for comparison with transcriptomes of other species [human and mouse placenta [START_REF] Hou | Elephant transcriptome provides insights into the evolution of eutherian placentation[END_REF], Chalcides ocellatus pregnant uterus [START_REF] Brandley | Uterine gene expression in the live-bearing lizard, Chalcides ocellatus, reveals convergence of squamate reptile and mammalian pregnancy mechanisms[END_REF], and Eutherian uterine decidua [START_REF] Lynch | Ancient transposable elements transformed the uterine regulatory landscape and transcriptome during the evolution of mammalian pregnancy[END_REF]]. For functional annotations and enrichment, housekeeping genes were first removed [START_REF] Eisenberg | Human housekeeping genes, revisited[END_REF], and functional annotations [GO Term, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, and Mouse Genome Informatics Mammalian Phenotype] were performed using the Enrichr website (amp.pharm.mssm.edu/Enrichr/).

Database Screening and Sequence Analyses. Retroviral endogenous env gene sequences were searched in each transcriptome assembly. Sequences containing an ORF longer than 400 amino acids (from start to stop codons) were extracted from the transcriptomes using the getorf program of the EMBOSS package (emboss.sourceforge.net/apps/cvs/emboss/apps/getorf.html) and were translated into amino acid sequences. These sequences were compared with the TM subunit amino acid sequences of 35 retroviral envelope glycoproteins from representative endogenous retroviruses, among which are known syncytins, and infectious retroviruses using the BLASTp program (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Putative envelope protein sequences were then selected based on the presence of a hydrophobic domain (transmembrane domain) located 3′ to a highly conserved C-X 5-7 -C motif. Multiple alignments of amino acid sequences were carried out using the MUSCLE algorithm in AliView [START_REF] Larsson | AliView: A fast and lightweight alignment viewer and editor for large datasets[END_REF]. Maximum likelihood phylogenetic amino acid trees were constructed with PhyML 3.0 [START_REF] Guindon | New algorithms and methods to estimate maximumlikelihood phylogenies: Assessing the performance of PhyML 3.0[END_REF], with bootstrap percentages computed after 1,000 replicates. Substitution models were selected using the SMS program ( 63): LG+Γ+I for TM sequences and RtREV+ Γ for RT sequences.

Ethics Statement. This study was carried out in strict accordance with the French and European laws and regulations regarding animal experimentation (Directive 86/609/EEC regarding the protection of animals used for experimental and other scientific purposes). Mabuya were collected under the permit issued by the Ministerio de Ambiente y Desarrollo Sostenible (Colombia) and handled in accordance with government guidelines on the ethical treatment of animals and all applicable regulations (Estatuto Nacional de Proteccion de los Animales, Ley 84, December 27, 1989) and following the considerations of the Herpetological Animal Care and Use Committee (HACC, 2004).

Other methods are detailed in Supporting Informaton.

ACKNOWLEDGMENTS. We thank Adriana Alberti, Corinne Da Silva, and Jean Weissenbach (Genoscope) for Mabuya transcriptome sequencing and assembly; Jean Weissenbach for the gift of the GeneBridge4 RH clones; Aaron Bauer and Breda Zimkus for providing Scincidae samples; Olivia Bawa (Gustave Roussy Preclinical Evaluation Platform) for contributions to the histological analyses; Mélanie Polrot (Gustave Roussy Animal Care Facilities) for assistance in mouse handling; David Ribet for help in GeneBridge4 panel data analysis; Cécile Lemaitre, Jhen Tsang, and Marie Dewannieux for help with the MPZL1 phosphorylation assay; and Christian Lavialle for discussion and critical reading of the manuscript. This work was supported by the CNRS, by a grant from the Ligue Nationale contre Le Cancer (Equipe Labellisée) (to T.H.), and by Agence Nationale de la Recherche Grant ANR RETRO-PLACENTA (to T.H.).

SI Methods

Conservation of Mab-Env1 in Scincidae. Amplification of the Mab-Env1 ORF or an internal fragment was performed using touchdown PCR with AccuPrime polymerase (Thermo Fisher) on 200 ng of genomic DNA. The following program was used: 10 cycles of 10 s at 94 °C, 30 s at 60 °C, -1 °C per cycle, and 2 min at 68 °C, followed by 40 cycles of 10 s at 94 °C, 30 s at 50 °C, and 2 min at 68 °C with a final elongation step of 7 min at 68 °C. Amplified fragments were purified and sequenced directly when possible or were cloned in pGEM-T Easy (Promega) before sequencing. Primers are described in Table S1.

Real-Time RT-qPCR. Mab-Env mRNA expression was determined by RT-qPCR. Reverse transcription was performed with 500 ng of DNase-treated RNA as in ref. [START_REF] De Parseval | Survey of human genes of retroviral origin: Identification and transcriptome of the genes with coding capacity for complete envelope proteins[END_REF]. PCR was carried out with 5 μL of diluted (1:20) cDNA in a final volume of 25 μL using the Fast SYBR Green PCR Master Mix (Qiagen) in an ABI PRISM 7000 sequence detection system. Primers are listed in Table S1. Primer couple efficiency was tested using a standard curve obtained with serially diluted RT samples (and proved to be >95%), and primer specificity was verified with single-peak dissociation curves.

Transcript levels were normalized relative to the amount of the housekeeping RPL19 gene (ribosomal protein L19), using the ΔCt method ð2 -ðCtenv-Ct RPL19 Þ Þ. Samples (three individuals for each organ) were each assayed in duplicate for each env and for the housekeeping gene.

In Situ Hybridization. Freshly collected Mabuya sp. IV placentas were fixed in 4% paraformaldehyde at 4 °C and were embedded in paraffin. Serial sections (7 μm) were either stained with H&E or were used for in situ hybridization. Three PCR-amplified fragments of Mab-Env1 (391 bp, 396 bp, and 482 bp) and two fragments of Mabuya MPZL1 (268 bp and 262 bp) (primers are listed in Table S1) were cloned into pGEM-T Easy (Promega) for in vitro synthesis of the antisense and sense riboprobes generated with SP6 RNA polymerase and digoxigenin 11-UTP (Roche Applied Science) after cDNA template amplification. Sections were processed, hybridized at 42 °C overnight with the pooled riboprobes, and incubated further at room temperature for 2 h with alkaline phosphatase-conjugated anti-digoxigenin antibody fragments (Roche Applied Science). Staining was revealed with nitroblue tetrazolium and 5-bromo-4-chloro-3indoyl phosphate phosphatase alkaline substrates, as indicated by the manufacturer (Roche Applied Science).

Antibody Production and Immunohistochemistry. A DNA fragment containing 201 amino acids of the Mab-Env1 envelope SU subunit (amino acids 30-231; for primers see Table S1) was inserted into the pET28b (Novagen) prokaryotic expression vector and expressed in BL21(DE3) bacteria. The recombinant C-terminally Histagged protein was purified from bacteria lysates by nickel affinity chromatography. Mouse immunization was performed in accordance with standard procedures. Sera containing polyclonal antibodies were recovered independently from 10 mice and were tested by Western blot analyses using lysates of 293T cells transiently transfected with a Mab-Env1 expression vector. The most specific sera of the Western blot experiment were then tested for signal strength and absence of background staining by performing immunohistochemistry on 293T cells transfected with an empty vector or a Mab-Env1 expression vector. For immunohistochemistry, paraffin sections were processed for heat-induced antigen retrieval (Tris EDTA, pH9; Abcam) and incubated overnight with anti-Mab-Env1 serum or the corresponding preimmune serum.

Staining was visualized using the peroxidase/diaminobenzidine Mouse PowerVision kit (ImmunoVision Technologies). All slides were immunostained in cover plates on the same day to obtain a standardized intensity of staining.

Mab-Env and MPZL1 Expression Vectors. The Mab-Env1 to Mab-Env4 fragments that were PCR-amplified from placental cDNA of Mabuya sp. IV using the cloning primers listed in Table S1 were digested with EcoRI and MluI, and the PCR products were cloned into the phCMV vector (GenBank accession no. AJ318514, a gift from F.-L. Cosset, Ecole Normale Supérieure de Lyon, France). Similarly, Mabuya MPZL1 was amplified from placental cDNA using the cloning primers listed in Table S1 and was cloned into the phCMV vector. For the lentiviral expression vector, Mab-Env1 and syncytin-Car1 ORFs were PCR amplified from phCMV vectors containing the respective ORF (from the present study and ref. 10) using the lentiviral vector primers listed in Table S1 and were cloned into a modified CSGW lentiviral vector [START_REF] Perro | Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer[END_REF].

Pseudotyping and Fusion Assays. Mab-Env-pseudotyped MLV particles were produced by cotransfecting 10 6 293T cells with 1 μg CMVi (a vector expressing MLV gag-pol), 1.5 μg MFG-nlsLacZ (a retroviral vector expressing nlsLacZ), and 0.5 μg Mab-Env1, Mab-Env2, A-MLV, or human Syncytin-1 (HERV-W Env) expression vectors using the jetPRIME transfection kit (Polyplus). Supernatants were harvested 48 h after transfection, filtered (PVDF membranes, pore size 0.45 μm; Millipore), supplemented with Polybrene (8 μg/mL), transferred to target cells (5-8 × 10 4 cells per well), and spinoculated at 1,200 × g for 2.5 h. X-gal staining was performed 3 d postinfection. Cell-cell fusion assays were performed by cotransfecting 2 × 10 6 cells with 2.75 μg of nlsLacZ expression vector and 2.75 μg of Mab-Env1 or MMTV Env expression vector (or empty vector as control), using Lipofectamine LTX reagent (Invitrogen). Forty-eight hours after transfection, cells were treated with preheated (37 °C) DPBS•HCl at pH 4 or 7 for 5 min at 37 °C. DPBS was then replaced by preheated (37 °C) normal supplemented growth medium, and cells were incubated at 37 °C for 6 h before X-gal staining. All cell lines are described in ref. 11 and were grown in DMEM supplemented with 10% FCS (Invitrogen), 100 mg/mL streptomycin, and 100 U/mL penicillin, at 37 °C and 6% CO 2 .

MPZL1 Phosphorylation Assay. MCF10A cells were obtained from ATCC (CRL-10317) and grown in DMEM/Ham's F12 medium (1:1), supplemented with 5% horse serum (Invitrogen), 100 mg/ mL streptomycin, 100 U/mL penicillin, 10 μg/mL insulin, 20 ng/mL EGF, 0.5 μg/mL hydrocortisone, and 100 ng/mL cholera toxin, at 37 °C and 6% CO 2 . Populations were obtained by transduction using VSV-G-pseudotyped HIV particles and a modified CSGW lentiviral vector [START_REF] Perro | Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer[END_REF] expressing the gene of interest and a hygromycin resistance factor. Cells were selected for a minimum of 1 wk in complete culture medium with 50 μg/mL Hygromycin-B (AppliChem) and were serum-starved for 2 h before being exposed (or not) to 100 μg/mL of Con A (Sigma-Aldrich) for 1 h. Cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher) supplemented with Halt Phosphatase Inhibitor Mixture (Thermo Fisher). Proteins were deglycosylated using PNGase F (New England Biolabs), separated by SDS/ PAGE, and transferred to nitrocellulose membranes. Tris-buffered saline/0.1% Tween/5% BSA buffer was used for blocking and antibody binding. Specific antibodies against MPZL1 (no. 9893) and phospho-MPZL1 Y263 (no. 8088) were purchased from Cell Signaling Technologies. The anti-tubulin-γ antibody was purchased from Sigma-Aldrich (T5326).

GenBridge Screening. A23-derived GeneBridge4 RH clones (a gift from J. Weissenbach, Genoscope, Evry, France) [START_REF] Gyapay | A radiation hybrid map of the human genome[END_REF] were grown in the DMEM medium described above, supplemented with HAT (100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine). Mab-Env1-pseudotyped MLV particles were produced by cotransfecting 10 6 293T cells with 2.25 μg CMVi, 2.25 μg MFG-nlsLacZ, and 0.25 μg of Mab-Env1 expression vector using the Lipofectamine LTX reagent (Invitrogen). Supernatants from the transfected cells were harvested 48 h after transfection, filtered through PVDF membranes (pore size 0.45 μm) (Millipore), supplemented with Polybrene (8 μg/mL), and transferred to the RH clones seeded in 24well plates (2.5 × 10 4 cells per well) the day before infection, followed by spinoculation at 1,200 × g for 2.5 h at 25 °C. X-gal staining was performed 3 d postinfection. A threshold value for positive infection of 100 focus-forming units/mL gave the best logarithm of the odds scores and gene localization results after analysis of the data using the RH map program (41) (+4 if the presence of the marker correlates with infection or the absence of the marker correlates with absence of infection, and -2 if it does not correlate).

SI Transcriptome Analysis

Placental transcripts were assembled using de novo assembly methods in the absence of a reference genome (Methods). Concatenated assemblies led to the identification of 4,020,383 highly redundant transcripts (Fig. S1A). Reduction of the redundancy of the transcriptome led to the identification of 72,763 transcripts ranging from 123 bp to 24.4 kb (N50 = 2,869 bp; median size = 1.1 kb) (Fig. S1A). Of these, 26,690 transcripts (36.7%) could be positively matched to a known protein gene from the Refseq Vertebrate database. A set of 21,253 transcripts shows significant expression [more than two tags per million [START_REF] Wagner | A model based criterion for gene expression calls using RNA-seq data[END_REF]], 16,398 of which were positively annotated (77.2%) and corresponded to 13,189 unique genes (Fig. S1B), of which 10,079 were nonhousekeeping genes. Only this subset of transcripts was conserved for further analysis. This subset of transcripts contains a range of genes annotated as "placental" genes with key biological functions during placenta development, such as core placental transcription factors (e.g., Cdx2, Hand1, Gata3, Stat3, Essrb, Tfa2pc, and Gcm1), placental hormones (e.g., Prl, Crebrf, Ghr, and Ghrh), iron transporters (e.g., Tfr2, Slc11a2, and Fth1), gap junction proteins (e.g., Gja1, Gja4, Gja5, Gja8, and Gjc2), interleukins and interleukin receptors (e.g., Il-15 and Il-11ra), metalloproteinases (e.g., Mmp-2 and Mmp-9), and angiogenesis factors (e.g., Vegf, Vegfb, Il-11ra, Dll4, and Pgf) (Dataset S2).

We further compared the transcriptome of the Mabuya placenta with those of eutherian mammalian placentas [genes expressed in both human and mouse placenta ( 57)] (Dataset S3).

A significant proportion (4,064, 40.3%) of the genes expressed in the Mabuya placenta are also expressed in eutherian placentas. Functional Gene Ontology (GO) annotations highlight biological functions associated with placenta biology, such as embryonic development [embryonic morphogenesis, GO:0048598, adjusted P value (adjp) = 4.5 × 10 -4 ; organ morphogenesis, GO:0009887, adjp = 7.5 × 10 -4 ; in utero embryonic development, GO:0001701, adjp = 1.7 × 10 -3 ; embryo development ending in birth or egg hatching, GO:0009792, adjp = 2.2 × 10 -3 ; chordate embryonic development, GO:0043009, adjp = 2.2 × 10 -3 ]; or regulation of cellular migration (negative regulation of cell motility, GO:2000146, adjp = 1.5 × 10 -4 ; negative regulation of cell migration, GO:0030336, adjp = 1.5 × 10 -4 ; positive regulation of cell motility, GO:2000147, adjp = 3.4 × 10 -4 ; positive regulation of cell migration, GO:0030335, adjp = 3.9 × 10 -4 ; leukocyte migration, GO:0050900, adjp = 4.5 × 10 -4 ). Accordingly, a significant fraction of enriched mouse phenotypes linked to this set of genes is associated with abnormal development and organ morphology (including abnormal extraembryonic tissue, adjp = 1.4 × 10 -17 ; abnormal embryonic tissue, adjp = 1.6 × 10 -14 ; abnormal embryogenesis, adjp = 2.8 × 10 -10 ; and abnormal embryo size, adjp = 5.3 × 10 -13 ) and abnormal immune response (abnormal immune system, adjp = 1.2 × 10 -14 ; abnormal adaptive immunity, adjp = 4.8 × 10 -15 ; and abnormal antigen presenting, adjp = 3.0 × 10 -11 ). Finally, mouse KEGG pathway analysis reveals the specific enrichment of the Rap1 signaling pathway (adjp = 2.7 × 10 -4 ) associated with trophoblast invasion and cell-cell fusion.

In addition, since the Mabuya placenta is composed of both maternal and fetal tissues, in Dataset S4 we compared the transcriptome of the Mabuya placenta with those of the pregnant uterus of another live-bearing lizard (C. ocellatus) [START_REF] Brandley | Uterine gene expression in the live-bearing lizard, Chalcides ocellatus, reveals convergence of squamate reptile and mammalian pregnancy mechanisms[END_REF] and mammalian endometrial tissues [START_REF] Lynch | Ancient transposable elements transformed the uterine regulatory landscape and transcriptome during the evolution of mammalian pregnancy[END_REF]. Five hundred twelve genes were expressed in all three tissues [i.e., mammalian endometrium, pregnant (but not nonpregnant) C. ocellatus uterus, and Mabuya placenta]. Again, functional annotations reveal specific enrichments of genes involved in the regulation of cell migration (positive regulation of cell migration, GO:0030335, adjp = 5.8 × 10 -3 ; regulation of endothelial cell migration, GO:0010594, adjp = 3.8 × 10 -3 ; leukocyte migration, GO:0050900, adjp = 7.7 × 10 -4 ), blood coagulation (blood coagulation, GO:0007596, adjp = 2.9 × 10 -4 ; coagulation, GO:0050817, adjp = 2.9 × 10 -4 ; hemostasis, GO:0007599, adjp = 2.9 × 10 -4 ), and response to hormone stimulus (cellular response to hormone stimulus, GO:0032870, adjp = 2.9 × 10 -4 ; response to steroid hormone, GO:0048545, adjp = 2.9 × 10 -4 ; cellular response to steroid hormone stimulus, GO:0071383, adjp = 6.2 × 10 -4 ).

Taken together, these strongly argue for the convergent evolution of specific transcriptomes within both maternal and extraembryonic fetal tissues associated with live-bearing and embryonic development in the distantly related mammalian and Mabuya species. Fig. S2. Recovered Mab-Env internal fragments from non-Mabuya species, as listed in Fig. 5. Functional domains are indicated using the color scheme in Fig. 2, and stop codons or frameshift-inducing deletions are highlighted in red. The corresponding fragment from Mabuya sp. IV is provided as reference; asterisks indicate nucleotide identities; identity percentages range from 79 to 89%. . Each cell clone was infected in duplicate and examined for the number of lacZ+ foci. The resulting matrix was derived, and data were compared with a known matrix of specific markers situated across the human genome (SI Methods). (C) Localization of the identified high-scoring genetic markers (nos. 1-8) on chromosome 1, showing the positions of the genes present within 3 Mb, centered on the best-scoring marker, as determined using the University of California, Santa Cruz (UCSC) Genome Browser. The transmembrane candidate receptor genes are highlighted in red.

Implication of ERV env genes in placental structural transitions in Hyaenidae 2.1 Summary

Like all other carnivorans, all extant members of the Hyaenidae species present the shared carnivoran Syncytin-Car1, but their placental organization is very different from that of other carnivorans. Hyaenidae present a highly invasive hemochorial placenta in which the maternal tissues are completely disrupted at term and maternal blood is in direct contact with placental tissues, whereas all other carnivorans present the less invasive endotheliochorial phenotype in which maternal vessels preserve their integrity. This structural transition from an endotheliochorial to a hemochorial placenta must have occurred recently as Hyaenidae split from the closest carnivoran family about 30 Mya.

To investigate the involvement of ERV env genes in this structural transition, and since there is no sequenced genome available, we performed high-throughput RNA sequencing of placental Crocuta crocuta (spotted hyena) transcripts. The placental transcriptome was then screened in silico leading to the identification of three expressed full-length env ORFs, which contain all major characteristic Env domains. As could be expected, one of these three expressed env was identified as the previously described hyena syncytin-Car1. The other two, Hyena-Env2 and Hyena-Env3 are distinct from previously described syncytins, although Hyena-Env3 closely resembles the non-syncytin Canis-Env3 described previously in dog. Phylogenetic analyses of these two new envelopes identifies them as gamma-type Env.

Since the investigated placental structure transition occurred only in Hyaenidae, we studied the conservation state of the two new Hyena-Env among carnivorans (syncytin-Car1 having already been shown to be conserved under purifying selection). PCR amplification of an internal fragment or the whole Hyena-Env2 ORF was possible only using genomic DNA from any of the four extant species of hyena, no amplification was observer outside of Hyaenidae indicating that Hyena-Env2 is hyena-specific. Additionally, sequence conservation among hyenas was high (>98.4% nucleotide identity) with a low ratio of non-synonymous/synonymous mutations, suggesting that Hyena-Env2 is under purifying selection in this group. Hyena-Env3 on the other hand was shown both by PCR and in silico analysis to be present in many other carnivorans,

2.1

mainly Caniformia but also a non-hyena Feliformia. Closer comparison of Hyena-Env3 and the numerous related coding and non-coding copies present in Caniformia genomes revealed that while Canis-Env3 is related to Hyena-Env3, the former is a non-functional copy, having lost the signal peptide necessary for membrane addressing during Env synthesis. A dog provirus associated to Canis-Env3 displays two LTRs with 100% sequence identity, suggesting that the Hyena-Env3/Canis-Env3 associated retrovirus was actively endogenizing until recently, or might even still be active. Since only Hyena-Env2 was shown to be hyena specific, we focused on this gene for our study.

Using an inverse PCR approach we identified the 3' flanking sequence of the inserted Hyena-Env2 containing provirus and using the sequence of the corresponding region in the cat genome we designed primers leading to the amplification of the complete 9 kb proviral genome. The target site duplication is preserved on both sides of the provirus and the LTRs display 89% sequence identity, in accordance with a relatively recent integration. The gag and pol ORFs are disrupted by several frameshift or non-sense mutation events. The conserved RT domain associated to Hyena-Env2 could be identified and indicates a gammaretroviral origin. In silico analysis and PCR amplification of the orthologous locus in non-hyena genomes confirmed that it is empty, while the amplification of the locus in hyena genomes confirmed that it is present at the same position in all four extant hyena, having therefore been acquire before radiation of modern day Hyaenidae. Characterization of the placental Hyena-Env2 transcript shows that it presents the canonical retroviral splicing but is not initiated in the 5'LTR and instead presents an additional splicing event. Initiation occurs in a host genome region that presents a CpG island in the orthologous cat genome region, suggesting that transcription of Hyena-Env2 is driven by a host promoter. Accordingly, in the cat genome this CpG island drives transcription of a host gene, albeit in the opposite orientation.

Placental expression of the three hyena genes was studied by in situ hybridization and revealed that syncytin-Car1 is expressed in the ST, with a similar expression profile to that described previously in cat and dog. Hyena-Env3 hybridization only led to very faint staining, potentially due to its low level of expression, that hinted at expression mainly in the ST. For Hyena-Env2, the sense RNA probe used as a negative control displayed very strong staining, revealing the presence of a Hyena-Env2 containing anti-sense transcript. Still, the anti-sense RNA probe shows a specific staining of the cell layer underlying the fused ST and containing the unfused CT cells. This was also confirmed by immunohistochemistry using anti-Hyena-Env2 antibodies (obtained by inoculating mice with Hyena-Env2 SU peptides). An expression of Hyena-Env2 in the CT specifically is in accordance with a role in placental physiology.

Finally, we investigated the fusogenic potential of all three hyena genes. Syncytin-Car1 behaved as was shown previously, being able to mediate cell infection by pseudotyped virions with a strong tropism towards carnivoran cells. Hyena-Env2 displayed no fusogenic properties in the pseudotyping or cell-cell fusion assay. Hyena-Env3 on the other hand was able to mediate infection of pseudotyped MLV particles, at levels comparable to those achieved with Syncytin-Car1. Hyena-Env3 presents a strict carnivoran tropism, being unable to mediate infection of any non-carnivoran cell lines tested. Interestingly, analysis of the Hyena-Env3 SU sequence shows that is shares conserved domains with the SUs of Env previously described as part of the SLC1A5 interference group and on an SU based phylogenetic tree, Hyena-Env3 clusters with these other Env. Cloning of human, hyena, cat and dog SCL1A5 confirmed that only the latter three allowed infection of restrictive cell lines. Use of SLC1A5 as cognate receptor usually confers a very broad tropism to the envelope and it is unclear why this is not the case with Hyena-Env3, though alignment of its SU with that of other SLC1A5 interference group members shows that Hyena-Env3 presents several mutations and shifted residues in highly conserved domains previously described to be implicated in receptor binding.

In conclusion, we have identified a newly captured syncytin-like gene that is specific to hyenas and conserved among all species of this family. The expression profile of Hyena-Env2 at the maternofetal interface is in accordance with a potential role in the transition from an endotheliochorial to a hemochorial placenta that occurred in the same timeframe as env capture.

Hyena-Env2 is not fusogenic in our assay, but fusogenicity might not be the role it was selected for in hyena placentation as the placental fusogen Syncytin-Car1 seems to fulfill this role. Hyena Syncytin-Car1 does not seem to differ from that of other carnivorans, be it in expression or fusogenicity, and is therefore unlikely to be the reason of the observed placental structural transition. An implication of Hyena-Env2 in cell migration/invasion processes is possible and identification of its cognate receptor might provide a potential mechanism, as was the case with Syncytin-Mab1/MPZL1. For now Hyena-Env2 joins the several previously described conserved syncytin-like non-fusogenic Envs. We have also identified a fusogenic ERV env that is not hyena-specific and belongs to the well-known SLC1A5 interference group, although it displays a peculiar infection profile that is much more specific than that of other Env in this group. The variations observed in the conserved domains of Hyena-Env3 SU might shed light on the receptor specificity of the envelope and contribute to the understanding of Env/SLC1A5 receptor binding.

Abstract

Capture of retroviral envelope genes from endogenous retroviruses has played a role in the evolution of mammals, with evidence for the involvement of these genes in the formation of the materno-fetal interface of the placenta. It has been shown that the diversity of captured genes is likely to be responsible for the diversity of placental structures, ranging from poorly invasive (epitheliochorial) to highly invasive (hemochorial) with an intermediate state (endotheliochorial) as found in carnivorans. The latter recapitulate part of this evolution, with the hyena being the sole carnivoran with a hemochorial placenta. Here we performed RNAsequencing on hyena placental transcripts, and searched for endogenous retroviral envelope genes that have been captured specifically in the Hyaenidae clade and are not found in any other carnivoran. We identified an envelope gene that is expressed in the placenta at the level of the materno-fetal interface, as evidenced by in situ hybridization/immunohistochemistry. The gene entry is coincidental with the emergence of the Hyaenidae clade 30 Mya, being found at the same genomic locus in all 4 extant hyena species: Crocuta crocuta, Hyaena hyaena, Parahyaena brunnea and Proteles cristatus. Its coding sequence has further been maintained -under purifying selection-during all of Hyaenidae evolution. It is not found in any of the 30 other carnivorans -both Felidae and Canidae-that we screened. This envelope protein does not disclose any fusogenic activity in a series of cell-cell fusion or pseudotyping assays, at variance with the Syncytin-Car1 gene that is found in all carnivorans -including hyena where it is still present, transcriptionally active in the placenta, and fusogenic. Altogether, the present results illustrate the permanent renewal of placenta-specific genes by retroviral captures, and de facto provide a candidate gene for the endotheliochorial to hemochorial transition of Hyaenidae among carnivorans.

Author Summary. The placenta is the most diverse organ among mammals in terms of structural organization, especially at the level of the maternofetal interface where the exchange of gas, waste and nutrients between maternal and fetal blood takes place. Carnivorans represent a rather unique clade, where capture and exaptation of a retroviral envelope, syncytin-Car1, took place about 80 Mya at the time of their radiation. It is conserved among all species, under purifying selection, expressed at the maternofetal interface and fusogenic, consistent with the syncytial structure of the interface and the "endotheliochorial" architecture of all carnivoran placenta -except of hyenas. Remarkably, the latter disclose a highly invasive hemochorial placenta, as found in humans, where a breakdown of maternal blood vessel walls results in maternal blood bathing the syncytial maternofetal interface. Here we identified a retroviral envelope gene capture and exaptation that took place about 30 Mya and is coincident with the emergence of the Hyaenidae clade, being conserved in all extant hyena species. It is expressed at the maternofetal interface in addition to the shared syncytin-Car1 gene. This new env gene, not present in any other carnivoran, is a likely candidate to be responsible for the specific structure of the hyena placenta.

Infection by exogenous retroviruses throughout vertebrate evolution has led to a significant portion of the genome (e.g. 8% in human and 10% in mice) being composed of retroviral sequences (reviewed in [START_REF] Lavialle | Paleovirology of 'syncytins', retroviral env genes exapted for a role in placentation[END_REF][START_REF] Mager | Mammalian Endogenous Retroviruses[END_REF]. Over time, these sequences were altered via genetic mutations, indels and recombinations, either passively or in response to host defense mechanisms. Consequently, the majority of integrated retroviral sequences are nonfunctional remnants [START_REF] Lavialle | Paleovirology of 'syncytins', retroviral env genes exapted for a role in placentation[END_REF]. However, among this large pool of highly reiterated endogenous retrovirus (ERV) sequences, a few examples exist of single copy retroviral genes which have been preserved since their integration, and can be considered as bona fide cellular genes contributing to host physiology. This is the case of the syncytin genes, encoding the envelope (Env) protein of ERVs -initially required for intracellular entry of exogenous retroviruses-, that have been independently co-opted in eutherian mammals for a key role in placenta formation (reviewed in [START_REF] Lavialle | Paleovirology of 'syncytins', retroviral env genes exapted for a role in placentation[END_REF]Denner, 2016). Syncytin-1 and syncytin-2 integrated into the genome of primates 25 and >40 Mya respectively and retained their coding capacity during speciation [START_REF] Blond | An Envelope Glycoprotein of the Human Endogenous Retrovirus HERV-W Is Expressed in the Human Placenta and Fuses Cells Expressing the Type D Mammalian Retrovirus Receptor[END_REF][START_REF] Mi | Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis[END_REF]Blaise et al., 2003). They are specifically expressed in the placenta, are fusogenic in ex vivo assays, and one of them, syncytin-2, displays immunosuppressive activity [START_REF] Blond | An Envelope Glycoprotein of the Human Endogenous Retrovirus HERV-W Is Expressed in the Human Placenta and Fuses Cells Expressing the Type D Mammalian Retrovirus Receptor[END_REF][START_REF] Mi | Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis[END_REF]Blaise et al., 2003;[START_REF] Mangeney | Placental syncytins: Genetic disjunction between the fusogenic and immunosuppressive activity of retroviral envelope proteins[END_REF]. Remarkably, homologous counterparts were then identified in all other orders of placental mammals where they have been searched for, namely within Rodentia (syncytin-A and -B [START_REF] Dupressoir | Syncytin-A and syncytin-B, two fusogenic placenta-specific murine envelope genes of retroviral origin conserved in Muridae[END_REF]; [START_REF] Vernochet | The captured retroviral envelope syncytin-A and syncytin-B genes are conserved in the Spalacidae together with hemotrichorial placentation[END_REF] and syncytin-Mar1 [START_REF] Redelsperger | Capture of syncytin-Mar1, a Fusogenic Endogenous Retroviral Envelope Gene Involved in Placentation in the Rodentia Squirrel-Related Clade[END_REF]), Lagomorpha (syncytin-Ory1, Heidmann et al., 2009), Carnivora (syncytin-Car1 Cornelis et al., 2012), Ruminantia (syncytin-Rum1 [START_REF] Cornelis | Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants[END_REF]), Afrotherians (syncytin-Ten1 Cornelis et al., 2014), and even in the distantly related Marsupials (syncytin-Opo1 [START_REF] Cornelis | Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials[END_REF] (Fig. 1). Recently we have also discovered a syncytin capture outside of the mammalian clade, in the viviparous placental lizard Mabuya (syncytin-Mab1 [START_REF] Cornelis | An endogenous retroviral envelope syncytin and its cognate receptor identified in the viviparous placental Mabuya lizard[END_REF]. All identified syncytins share closely related functional properties, although they have a completely distinct origin, are integrated at separate genomic locations and differ in their sequence. Identification of the mouse syncytin-A and -B finally allowed the unambiguous demonstration that such genes are essential for placentation, with syncytin KO mice displaying impaired placental functions with a lack of cell-cell fusion at the level of the syncytiotrophoblast layers that separate maternal and fetal blood spaces, resulting in impaired embryonic survival [START_REF] Dupressoir | Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene[END_REF](Dupressoir et al., , 2011)). One of the major divergent traits between present-day placentas is the number and nature of layers at the maternofetal interface, leading to four major placental types (Fig. 1). In the simplest type, the fetal trophoblast cells are simply apposed to the intact uterine epithelium (non-invasive epitheliochorial placentation), whereas in the more complex types, the trophoblast cells fuse together to form an invasive syncytiotrophoblast layer which is in contact with the endothelial wall of maternal blood vessels (endotheliochorial placenta of carnivorans) or directly bathed in maternal blood (hemochorial placenta of simians, rodents and lagomorphs) (reviewed in Roberts et al., 2016). As distinct exogenous retroviral envelope (env) genes appear to have been independently integrated and adapted for similar essential placental functions during mammalian

2.2

evolution, we proposed the challenging hypothesis that this stochastic acquisition of genes of exogenous origin has been instrumental in establishing the remarkable structural and functional diversity of the mammalian placenta (reviewed in [START_REF] Lavialle | Paleovirology of 'syncytins', retroviral env genes exapted for a role in placentation[END_REF]. In the past years, we have substantiated this hypothesis by demonstrating the concomitant acquisition of a syncytin and the emergence of a distinct placental organization during mammalian radiation. The emergence of the very specific ruminant synepitheliochorial placenta is concomitant with the acquisition of syncytin-Rum1 in this clade. The implication of syncytin-Rum1 in establishing this structure was further substantiated by the observation that the more important syncytium formation in ovine species, when compared to bovine species, is accompanied by a higher level of expression of the captured syncytin [START_REF] Cornelis | Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants[END_REF]. The Carnivora clade presents a remarkable and even more challenging situation. Indeed, the carnivoran structural organization of the placenta is of the endotheliochorial type (Wooding & Burton, 2008), consistent with the capture of syncytin-Car1 being concomitant with the radiation of this clade about 80 Mya [START_REF] Cornelis | Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora[END_REF], but there is a remarkable exception to this rule: the spotted hyena (Crocuta crocuta, hereafter referred to as hyena) which belongs to the Feliformia subclade within Carnivora clearly possesses a hemochorial type placenta [START_REF] Morton | Placentation in the spotted hyena (Crocuta crocuta Erxleben)[END_REF][START_REF] Wynn | Placentation in the Spotted Hyena (Crocuta crocuta), with Particular References to the Circulation[END_REF][START_REF] Oduor Okelo | The chorioallantoic placenta of the spotted hyena (Crocuta crocuta Erxleben): An electron microscopic study[END_REF][START_REF] Enders | Structure of the midterm placenta of the spotted hyena, Crocuta crocuta, with emphasis on the diverse hemophagous regions[END_REF]. Since the hyena presents the same syncytin as all other Carnivora, i.e., syncytin-Car1 [START_REF] Cornelis | Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora[END_REF], we hypothesized that the unique placental structural organization of this species might be associated with a recent de novo capture of a hyena-specific syncytin, which would be responsible for the observed transition. Accordingly, we searched for such a gene in the hyena and since the genome of this species has not yet been sequenced, we first performed a high throughput RNAsequencing of placental transcripts to identify putative env-related syncytins by an in silico search, followed by a structural and functional characterization of the identified candidates. These analyses resulted in the identification of a newly captured gene, specific to hyenas and expressed in the placenta, consistent with a role of this gene in the stochastic emergence of the hyena-specific placental structure. An alternative hypothesis, involving for instance a severely modified expression of the shared syncytin-Car1 in the hyena seems to be ruled out with no significant difference observed between the hyena gene and the other carnivoran orthologs, including that of dog and cat. The results are discussed in the wider context of retroviral gene capture and exaptation as a central driving force generating the observed structural placenta diversity.

Results

High throughput sequencing and in silico search for retroviral env genes within the hyena Crocuta crocuta placenta transcriptome. Since the hyena genome -at variance with that of some of the other carnivorans -has not been sequenced, we first established the transcriptome of the Crocuta crocuta hyena placenta, in collaboration with the French National Sequencing Center (Genoscope, Evry, France), where assembly of the placental transcripts was also performed using de novo assembly methods in the absence of a reference genome (see Methods). (Upper) Mammals comprise the monotremes (e.g., platypus) still laying eggs, and the marsupials and eutherian mammals, which all possess a placenta. For the latter the placental type is indicated using the same color key as the schemes below. All currently described syncytin capture events are indicated by arrowheads in pink, together with its name. Branch length is proportional to time (expressed in My, [START_REF] Cornelis | Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials[END_REF][START_REF] Nyakatura | Updating the evolutionary history of Carnivora (Mammalia): a new species-level supertree complete with divergence time estimates[END_REF], as indicated in the scale below the tree. (Lower) Schemes illustrating the four types of maternofetal interfaces found in mammals, arranged in order of invasiveness of fetal tissues. A line delimits the maternal and fetal sides.

Transcriptome data was deposited at the European Nucleotide Archive (ENA) under the accession number ERA1116685 and is available for further analyses of placental gene expression. The transcriptome was then screened for putative env-derived syncytin genes as described previously for other mammalian genomes [START_REF] Bénit | Identification, Phylogeny, and Evolution of Retroviral Elements Based on Their Envelope Genes[END_REF]. All ORFs of over 400 aa (met codon to stop codon) were compared to a set of selected Env sequences, including previously described syncytins (see Methods), using BLAST. As illustrated in Fig. 2, the search yielded 3 sequences, the previously described syncytin-Car1, which is conserved among all carnivorans, and two novel env sequences that were named Hyena-Env2 and Hyena-Env3. All three contain domains characteristic of canonical gamma-type retroviral Env proteins: a predicted signal peptide at the N-terminus, a putative furin cleavage site (R-X-K-R) delimitating the surface (SU) and transmembrane (TM) subunits, a hydrophobic domain of over 20 aa located downstream of a highly conserved C-X 5-7 -CC motif and a canonical retroviral Env immunosuppressive domain (ISD) (reviewed in Henzy & Johnson, 2013). When placed on a phylogenetic Env tree, does not cluster with previously described syncytins. Hyena-Env3 shares conserved regions with the human syncytin-1 (39% aa identities) as well as with the previously described non-syncytin Canis-Env3 (66% aa identities) [START_REF] Cornelis | Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora[END_REF]). An RT-qPCR analysis of the placental expression of all three envelopes, using primers specific of each gene, discloses that syncytin-Car1 is very strongly expressed in the placenta, its expression being about 5 times higher than that of the ribosomal protein RPL19 housekeeping gene, at levels similar to those observed in cat and dog placentas [START_REF] Cornelis | Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora[END_REF]. The levels of expression of the two newly identified envelopes are much lower than that of syncytin-Car1, namely about 50 fold lower for Hyena-Env2 and 150 fold lower for Hyena-Env3.

Characterization of the identified hyena candidate genes: are they hyena-specific and conserved? Syncytin-Car1 has already been demonstrated to be common to all carnivorans and to be functionally conserved, with evidence for purifying selection. A refined analysis of amino acid identity and non-synonymous vs synonymous mutation rates could not identify any characteristic feature specific to the hyena ortholog [START_REF] Cornelis | Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora[END_REF] and a thorough BLAST search of the transcriptome did not reveal expression of alternative copies in the hyena placenta.

The Hyena-Env2 gene cannot be found by in silico BLAST search within the cat, cheetah, tiger, dog, ferret, walrus, seal, polar bear or panda genome, and as such is a good candidate for being a hyena-specific ERV env gene. To verify this hypothesis on a larger panel of species, a 416 bp internal fragment was tentatively PCR amplified from a panel of genomic DNAs alongside the control amplification of the whole syncytin-Car1 ORF using the primers described in [START_REF] Cornelis | Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora[END_REF] to assay DNA quality (Fig. 3). Amplification of the internal Hyena-Env2 fragment was observed only in the four extant hyena species, confirming the results of our in silico analysis. Finally, we could amplify and sequence the full-length Hyena-Env2 ORF (using primers situated on both sides of the reading frame) from all extant hyenas: Crocuta crocuta, Hyaena hyaena, Parahyaena brunnea and Proteles cristatus (see Fig. 4), revealing that The furin cleavage site (consensus: R/K-X-R/K-R, orange) between the two subunits, the C-X-X-C motif involved in SU-TM interaction, the hydrophobic signal peptide (purple), the fusion peptide (green), the transmembrane domain (red), and the putative immunosuppressive domain (ISD, blue) along with the conserved C-X5-7-C (CC) motif are indicated. (B)(Left) Characterization of the candidate Crocuta crocuta Env proteins: the hydrophobicity profile for each candidate is shown with the canonical structural features highlighted using the same colors as (A). (Right) RT-qPCR analysis of env gene expression in the Crocuta crocuta placenta. Transcript levels are shown as the ratio between expression levels of each env gene and those of the RPL19 control gene (see Methods).

2.2

they share a very high sequence similarity (over 98.4% nucleotide identities). The ratio of nonsynonymous to synonymous mutations (dN/dS) is well below unity (between 0.20 and 0.67), strongly suggesting that the gene is under purifying selection, even though the small dataset and low number of variable sites may be a statistical limitation. Altogether these results indicate that Hyena-Env2 endogenization occurred after the separation of the Hyaenidae family from the rest of carnivorans about 30 Mya, and that has been conserved since the emergence of this clade.

Interestingly, sequences bearing high similarity to Hyena-Env3 can be found in silico within many Caniformia genomes (e.g., Canis familiaris (dog), Ailuropoda melanoleuca (panda), Ursus maritimus (polar bear), Mustela putorius furo (ferret)), at a variable number of copies, but not Feliformia genomes (e.g., Felis catus (cat), Acinonyx jubatus (cheetah), Panthera tigris (tiger)). The dog genome presents a full-length protein-coding copy, previously identified as Canis-Env3 [START_REF] Cornelis | Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora[END_REF], alongside a large number of non-coding copies. However, in Canis-Env3 the methionine which precedes the signal peptide in the Hyena-Env3 ORF is not present, suggesting that Canis-Env3 is not a functional copy of this gene, lacking a membrane addressing signal. A dog provirus (canFam3 genome assembly, chr3: 82,194,219-82,202,071) associated with a non-coding Canis-Env3 copy presents two 100% identical LTRs and almost intact gag, pol and env ORFs suggesting that this family was active until very recently. In addition, most conserved copies in the dog genome are not present in the orthologous regions of the panda or ferret genomes, indicating that numerous insertions occurred after separation of the three lineages, about 60 Mya (Fig. 3). To assay whether this family of env genes could have entered the Caniformia branch after the Feliformia/Caniformia split about 65 Mya, and still be specific to hyenas within Feliformia, we attempted to amplify a 674 bp fragment internal to the Hyena-Env3 ORF from genomic DNA of the species in our panel. As indicated in Fig. 3, PCR fragments of the expected size were obtained in many Caniformia species tested and sequencing confirmed them being analogous to Hyena-Env3. In Feliformia the PCR assay revealed presence of Hyena-Env3 in all four extant hyena species but also, unexpectedly, in Arctictis binturong. This env gene seems therefore to have been captured on multiple occasions and at different time points in evolution, but is clearly not specific to hyenas.

Structure of the Hyena-Env2 associated provirus and placental transcript. To further characterize the hyena-specific Hyena-Env2 gene, we searched for its insertion locus by inverse PCR. Genomic DNA from Crocuta crocuta was digested, using restriction enzymes with only one cutting site in the Hyena-Env2 gene, and self-ligated to generate circular genomic DNA fragments. PCR was performed on these circular fragments using divergent primers located within the Hyena-Env2 gene fragment and the obtained PCR products were sequenced. One of them contained the expected part of the env gene and extended 3' to it for over 1000 bp, with evidence for the presence of an LTR and flanking genomic sequence. The identified flanking sequence mapped to a locus of the cat genome (the closest sequenced carnivoran genome), most probably corresponding to the ortholog of the integration site of the Hyena-Env2 associated provirus (Fig. 5A). Primers were designed, using the cat genome sequence for the 5' side, to tentatively amplify the whole provirus from Crocuta crocuta genomic DNA and to assay its presence in closely related species, as well as in cat and dog as controls. A large fragment of about 9 kb could be amplified from Crocuta crocuta genomic DNA and, once sequenced, allowed characterization of the Hyena-Env2 containing provirus, bordered by clearly recognizable 5' and 3' LTRs (89% identity) and inserted within the locus identified above (Fig. 5B,C). At the junctions between LTRs and genomic DNA we observed a 4 bp target site duplication, as expected for a proviral integration. The provirus contains the full-length Hyena-Env2 gene, and recognizable gag and pol genes, although with severely altered non-coding sequences. A putative tRNA primer binding site (PBS) can be identified by BLAST using a tRNA database, the sequence matching a glycine tRNA (Fig. 5B,C). Comparing the catalytic RT domain of pol with a set of other, endogenous as well as exogenous, retroviral catalytic RT domains classifies this provirus as a gammaretrovirus (Fig. 6).

PCR amplification and sequencing using the same set of primers and genomic DNA from cat, dog, and the four species closest to Hyaenidae (Arctictis binturong, Cryptoprocta ferox, Suricata suricatta or Helogale parvula) show that the ortholog sites in these species are indeed empty, confirming the results obtained with primers internal to Hyena-Env2. This was further confirmed by in silico analyses of the ortholog site in all sequenced carnivoran genomes (Fig. 5B), which discloses an empty site in all cases. Of note the dog genome presents a 2 kb deletion in this region.

In order to characterize further the sequence of the placental Hyena-Env2 transcript, we performed Rapid Amplification of cDNA Ends (RACE) using total placenta RNA samples, followed by PCR to amplify the 5' or 3' ends of the cDNA. A single major band was obtained and sequenced for each end, providing the complete sequence of the Hyena-Env2 placental mRNA. By aligning the obtained transcript with the Hyena-Env2 associated provirus, we were able to identify the canonical retroviral envelope transcript splicing sites, with a donor site just after the 5' LTR and an acceptor site before the envelope start codon (Fig. 5C). The transcript ends in the 3' LTR with a polyadenylation signal, as classically observed for retroviral transcripts. However, initiation does not occur in the 5' LTR as expected for retroviral transcripts, instead another splicing event takes place since the 5' end of the transcript does not match the sequence of the proviral LTR. Since no hyena genome has been published, we used the BLAT algorithm to try to position the 5' end of the transcript in the cat genome (felCat8 assembly). The sequence matches a region about 1 kb upstream of the site orthologous to the insertion locus of the Hyena-Env2 associated provirus. In the cat genome this region presents a predicted splice donor site at the expected position and appears to be in a CpG island. Several other transcripts are predicted to be initiated nearby (e.g., CHERP, SLC35E1), while others seem to present this region as an intron (e.g., ap1m1) (Fig. 5A). This suggests that expression of the placental Hyena-Env2 transcript, a gene of retroviral origin, is driven by a promoter of cellular origin.

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In situ hybridization of the identified Hyena-Env genes on placental sections. The hyena is the only carnivoran with a placenta of the hemochorial type [START_REF] Morton | Placentation in the spotted hyena (Crocuta crocuta Erxleben)[END_REF][START_REF] Wynn | Placentation in the Spotted Hyena (Crocuta crocuta), with Particular References to the Circulation[END_REF][START_REF] Oduor Okelo | The chorioallantoic placenta of the spotted hyena (Crocuta crocuta Erxleben): An electron microscopic study[END_REF]. In all other carnivorans, the placenta is of the less invasive endotheliochorial type, where the fetal tissues break down the maternal epithelium but leave the maternal blood vessels intact (Fig. 7 top, Wooding & Burton, 2008). In the hemochorial hyena placenta, maternal blood vessels have been destroyed and the fetal tissues form villi that are in direct contact with maternal blood (Fig. 7 bottom, [START_REF] Morton | Placentation in the spotted hyena (Crocuta crocuta Erxleben)[END_REF][START_REF] Wynn | Placentation in the Spotted Hyena (Crocuta crocuta), with Particular References to the Circulation[END_REF][START_REF] Oduor Okelo | The chorioallantoic placenta of the spotted hyena (Crocuta crocuta Erxleben): An electron microscopic study[END_REF]. Each primary villus contains fetal blood vessels and is covered by a layer of fused cells: the syncytiotrophoblast [START_REF] Oduor Okelo | The chorioallantoic placenta of the spotted hyena (Crocuta crocuta Erxleben): An electron microscopic study[END_REF][START_REF] Enders | Structure of the midterm placenta of the spotted hyena, Crocuta crocuta, with emphasis on the diverse hemophagous regions[END_REF]. Under this layer are the cytotrophoblasts, which do not form a continuous layer and have a cytoplasm filled with numerous lipid droplets [START_REF] Oduor Okelo | The chorioallantoic placenta of the spotted hyena (Crocuta crocuta Erxleben): An electron microscopic study[END_REF][START_REF] Enders | Structure of the midterm placenta of the spotted hyena, Crocuta crocuta, with emphasis on the diverse hemophagous regions[END_REF]. The villi present frequent ramifications (not schematized in Fig. 7) forming a complex labyrinthine organization with numerous linked maternal blood spaces. The surface of these ramifications is also covered in syncytiotrophoblast [START_REF] Enders | Structure of the midterm placenta of the spotted hyena, Crocuta crocuta, with emphasis on the diverse hemophagous regions[END_REF]. Maternal blood is pumped into the placental blood spaces at the fetal side and flows down through the labyrinth towards the maternal side where it drains into maternal veins, thus establishing an efficient counter-current exchange system between maternal and fetal blood [START_REF] Wynn | Placentation in the Spotted Hyena (Crocuta crocuta), with Particular References to the Circulation[END_REF]. At the point of contact between the fetal villi and the maternal endometrium, the syncytiotrophoblast layer peels off and the tip of the villus expands and is covered by tall columnar cytotrophoblasts that often contain phagocytized erythrocytes [START_REF] Enders | Structure of the midterm placenta of the spotted hyena, Crocuta crocuta, with emphasis on the diverse hemophagous regions[END_REF]. To determine the expression pattern of the three hyena envelopes in the placenta we performed in situ hybridization (ISH) on placenta tissue sections (gestation day 62, about mid-pregnancy) using three specific digoxigenin-labeled antisense RNA probes for each envelope, as well as the corresponding sense probes as negative controls.

As previously described for the cat and dog placenta [START_REF] Cornelis | Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora[END_REF], hyena syncytin-Car1 is expressed specifically in the syncytiotrophoblast and not in the underlying cytotrophoblast, indicating that the hyena syncytin-Car1 expression locus does not differ from that observed in other carnivorans.

Unexpectedly, for Hyena-Env2 the sense probe used as a negative control resulted in a much stronger signal than the antisense probe detecting the sense envelope transcript. Sense/antisense specific RT-PCR on placental RNA with the primer pairs used to synthesize ISH probes revealed the existence of an antisense transcript that covered at least all 3 regions detected by the probes, since all pairs allowed amplification from sense and antisense cDNA. This antisense transcript most probably initiates downstream from the proviral insertion site, at a CpG island (by analogy with the cat genome, see Fig. 5A) that could drive antisense transcription. Nevertheless, the signal observed using the antisense probes is differential between the different placental cell layers: staining is observed only in the layer that contains the cytotrophoblasts, just below the syncytiotrophoblast. This localization of expression is clearly different from that of hyena syncytin-Car1 and suggests a distinct role for Hyena-Env2. To confirm this ISH result, we gen- erated anti-Hyena-Env2 antibodies by immunizing mice with bacteria-synthesized polypeptides derived from the Hyena-Env2 SU and performed immunohistochemistry with the post immunization sera, using the corresponding pre-immune sera as negative controls (see Methods). As illustrated in Fig. 8 and consistent with the ISH, a strong staining is observed at the level of the cytotrophoblasts -and not of the syncytiotrophoblast.

Of note although no conclusive result could be obtained by ISH for Hyena-Env3 due to low signal strength (consistent with its low expression as measured by RT-qPCR, see above), a specific faint labeling of the syncytiotrophoblast was observed.

Fusogenicity of the identified Hyena-Env in ex vivo assays. Fusogenicity of the identified hyena envelopes was first assayed using a pseudotyping assay, which tested whether the envelope proteins could render recombinant env-less MLV-derived viral particles infectious. 293T cells where co-transfected with a plasmid coding for the env gene under control of a CMV promoter, a plasmid expressing the MLV gag-pol genes and a plasmid expressing a defective MLV genome containing an nls-LacZ reporter gene. Viral particles were then used to infect various target cell lines and infection was assayed by counting X-Gal stained infection foci (Fig. 9A).

Hyena Syncytin-Car1 has been shown previously to behave like other Feliformia Syncytin-Car1 genes in this test: it is able to mediate viral infection and displays a strong tropism for carnivoran cell lines (Cornelis et al., 2012, Fig. 9B and 10).

Hyena-Env2 tested negative in the pseudotyping assay (see Fig. 9B and 10). It is unknown whether this is due to the absence of the proper receptor in all cell lines tested, though we tested several cell lines from cat and dog which should present proteins closely related to hyena proteins, or if an intrinsic loss of fusogenic activity is the cause of this result. Of note, similar results were obtained with HIV-and SIV-pseudotyped particles. These negative results might also be due to a lack of incorporation of Hyena-Env2 into the viral particles. We therefore performed a more straightforward cell-cell fusion assay: 293T cells were transfected with a Hyena-Env2 expressing and plasmid put into contact with a diverse panel of non-transfected target cells 24 h post-transfection. No syncytium formation was observed for any of the target cells used in this assay (cat, dog, human or hamster cells, see Fig. 9B).

Finally, Hyena-Env3 was found to confer infectivity to MLV-derived viral particles for some target cell lines. Among tested cells, only carnivoran cell lines were permissive to viral infection and presented significant numbers of infection foci 72 hours post infection, reaching approx. 10 4 focus forming units/mL (see Fig. 10). These results suggest that Hyena-Env3 confers a carnivoran-specific tropism to the pseudotyped particles. Sequence comparison further showed that the SU of Hyena-Env3 seems to be related to the SU of human Syncytin-1, rabbit Syncytin-Ory1 and of other Env from retroviruses of the SLC1A5 interference group (Fig. 11A), a group of viruses all using the membrane transporter SLC1A5, previously known as ASCT2 or RDR, as a common receptor [START_REF] Malicorne | Genome-Wide Screening of Retroviral Envelope Genes in the Nine-Banded Armadillo (Dasypus novemcinctus, Xenarthra) Reveals an Unfixed Chimeric Endogenous Betaretrovirus Using the ASCT2 Receptor[END_REF][START_REF] Sinha | Retroviruses of the RDR superinfection interference group: ancient origins and broad host distribution of a promiscuous Env gene[END_REF]. Alignments reveal that Hyena-Env3 and SLC1A5 group Envs share conserved domains that are essential for interaction with the receptor [START_REF] Cheynet | Identification of the hASCT2-binding domain of the Env ERVWE1/syncytin-1 fusogenic glycoprotein[END_REF]. Transfection of non-permissive human HEK293T cells with expression vectors for SLC1A5 that we cloned from cat, dog or hyena placental cDNA (see Methods) rendered the cells permissive to infection by Hyena-Env3 pseudotyped MLV particles, while transfection with human SLC1A5 did not allow infection, confirming both the role of SLC1A5 as receptor and the tropism of Hyena-Env3 observed in the pseudotyping assay (Fig. 11B). Some of the amino acids conserved among the different envelopes of the RDR interference group and implicated in protein conformation or receptor binding [START_REF] Cheynet | Identification of the hASCT2-binding domain of the Env ERVWE1/syncytin-1 fusogenic glycoprotein[END_REF] appear to be missing or shifted by one or two positions in Hyena-Env3, which might explain the peculiar carnivoran tropism (Fig. 11C).

Discussion

Here we have performed a high throughput analysis of the hyena placenta transcriptome, which revealed a newly captured gene of retroviral origin that is unique to the Hyaenidae family. It is present in all hyena species, and is not found in any other carnivoran. Its entry date is coincident with the emergence of the Hyaenidae within Feliformia, about 30 Mya. It is present at the same integration site in all hyena species, with an intact coding sequence. This time-resolved retroviral env gene capture is an additional illustration that endogenization of retroviruses is a permanent process, that accounts for the presence of remnant proviral structures in up to 10% of the vertebrate genome (reviewed in [START_REF] Mager | Mammalian Endogenous Retroviruses[END_REF]. Such captures are still ongoing, with for instance KoRV, the koala leukemia retrovirus which is presently being endogenized in the Australian koala population after the infectious exogenous retrovirus has spread across the whole country [START_REF] Tarlinton | Retroviral invasion of the koala genome[END_REF][START_REF] Mager | Mammalian Endogenous Retroviruses[END_REF]. In most cases endogenization of retroviruses is "neutral" to the host physiology, but there are a series of exceptions to this rule. They fall into two major categories, exaptation of either i) the retroviral transcription promoters and enhancers (LTR sequences), or ii) the retroviral protein-coding sequences per se (most commonly the env gene). The former has been demonstrated to be responsible for the coordinated expression of genes located near the ERV integration site, with special emphasis on their role in development (reviewed in [START_REF] Chuong | Regulatory activities of transposable elements: from conflicts to benefits[END_REF]. The latter includes the syncytin envelope genes, which proved to be involved in placenta formation (reviewed in [START_REF] Lavialle | Paleovirology of 'syncytins', retroviral env genes exapted for a role in placentation[END_REF]Denner, 2016). Their impact is directly linked to the canonical properties of retroviral envelope genes, which include a fusogenic activity to mediate membrane fusion during virus entry, and an immunosuppressive activity to inhibit the host anti-viral immune response. Fusion activity has unambiguously been exapted through syncytin genes and is directly responsible for cell-cell fusion and formation of the syncytiotrophoblast cell layer at the maternofetal interface in many mammalian species, with an unambiguous demonstration of this role in mice knocked out for these genes [START_REF] Dupressoir | Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene[END_REF](Dupressoir et al., , 2011)). More recently it has been shown that in some rare non-mammalian species with a placenta, the establishment of a fused cell layer at the maternofetal interface is also associated with the capture of a syncytin gene [START_REF] Cornelis | An endogenous retroviral envelope syncytin and its cognate receptor identified in the viviparous placental Mabuya lizard[END_REF]. The immunosuppressive activity also carried by captured syncytins is likely to play a role -at least in part -for the taming of the maternal immune response against the 2.2 The Hyena-Env3 SU is closely related to envelopes previously described as using SLC1A5 (also known as RDR and ASCT2) as receptor. Maximum likelihood tree using SU subunit amino-acid sequences from syncytins and a series of endogenous and infectious retroviruses. Previously described syncytin genes are highlighted in blue and the RDR interference group is highlighted in orange. Branch length represents the average number of substitutions per site (scale bar top left), and the percent bootstrap values obtained from 1,000 replicates are indicated on the branches. (B) Carnivoran but not human SLC1A5 allow Hyena-Env3 mediated infection of non-permissive cells. 293T cells were transfected with either an empty vector or expression vectors for SLC1A5 from human, cat, hyena or dog. Transfected target cells were then infected using Hyena-Env3-pseudotyped MLV particles and viral titer in focus forming units per mL were quantified after LacZ staining. Arrows indicate titers below background, values shown are the mean of three independent experiments ± SD. (C) Sequence alignment of SU protein subunits, from the methionine to the putative furin cleavage site, of endogenous and exogenous envelopes using SLC1A5 as a receptor as well as Hyena-Env3 (in bold). Variable regions are represented by the number of omitted residues; dashes correspond to deletions/insertions. The conserved motif shown to be essential for the Syncytin-1/SLC1A5 interaction (SDGGGX 2 DX 2 R) is highlighted in orange, and the three cysteinecontaining motifs (PCXC, CYX5C, and CX 7-9 CW) and amino acids that are highly conserved among retroviral members of the RDR interference group are highlighted in yellow [START_REF] Cheynet | Identification of the hASCT2-binding domain of the Env ERVWE1/syncytin-1 fusogenic glycoprotein[END_REF]. Amino acid mismatches in conserved regions are highlighted in red, asterisks indicate amino acid identities.

semi-allogenic graft of the fetus and placenta during pregnancy (reviewed in [START_REF] Lavialle | Paleovirology of 'syncytins', retroviral env genes exapted for a role in placentation[END_REF]. Along these lines, the Hyena-Env2 capture falls within the now well-established set of data showing that the present-day syncytin genes, found in all placental mammals where they have been searched for, simply correspond to independent retroviral gene captures that have taken place over the course of mammalian evolution, resulting in different syncytin genes present in the major clades of placental mammals. The scheme also proposes -to account for the observed convergent evolution of such gene captures-that emergence of placental mammals was initiated by the primitive capture of an ancestral retroviral envelope gene, about 150 Mya, which was followed by reiterated captures of new retroviral env genes that, depending on their "fitness" with regards to fulfilling placental functions, were selected in place of the previously captured envelopes, replacing them following a classical Darwinian selection process (reviewed in [START_REF] Lavialle | Paleovirology of 'syncytins', retroviral env genes exapted for a role in placentation[END_REF]. This model, first proposed by our group [START_REF] Saib | Endogenous retroviruses: Thierry Heidmann wins the 2009 Retrovirology prize[END_REF]Dupressoir & Heidmann, 2011), accounts for the diversity of the syncytin genes found today in a simple manner. It was recently further sustained by the discovery that a primate Env-V gene that discloses all the characteristic features of a bona fide syncytin in Old World monkeys, has lost its fusogenic activity in higher primates -including humans-where two other syncytins are present and active [START_REF] Esnault | Differential Evolutionary Fate of an Ancestral Primate Endogenous Retrovirus Envelope Gene, the EnvV Syncytin, Captured for a Function in Placentation[END_REF]. However, this env gene is still under purifying selection, thus suggesting that its fusogenic activity might not be the sole property for which it has been and/or still is selected for (see below). Along this line, we had previously identified a captured retroviral env in the Cavia genus that discloses only part of the features characteristic of syncytins -and thus named it syncytin-like env-Cav1 [START_REF] Vernochet | A syncytin-like endogenous retrovirus envelope gene of the guinea pig specifically expressed in the placenta junctional zone and conserved in Caviomorpha[END_REF]. This envelope is conserved under purifying selection and expressed specifically in the "syncytial streamer" of the Cavia placenta, suggesting it plays a role in placentation, but we could not demonstrate any fusogenic activity in vitro. Two additional examples of retroviral gene captures with strong expression in the placenta and conservation for over 100 Mya of evolution, with evidence for strong purifying selection, are worth mentioning, as they clearly cannot be fusogenic either since they are lacking cell membrane attachment (due to a conserved premature stop codon [START_REF] Cornelis | Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials[END_REF][START_REF] Lavialle | Paleovirology of 'syncytins', retroviral env genes exapted for a role in placentation[END_REF] or are actively shed after they are exported to the cell membrane [START_REF] Heidmann | HEMO, an ancestral endogenous retroviral envelope protein shed in the blood of pregnant women and expressed in pluripotent stem cells and tumors[END_REF]. Altogether, these data strongly suggest that fusion activity is likely not the unique function "exapted" through captured env genes with placental expression, and the presently identified Hyena-Env2 gene can be added to the above-mentioned list of "orphan" genes, for which the biological role still needs to be elucidated but does not seem to imply cell-cell fusion. In this specific case, acquisition of Hyena-Env2 is not associated with loss of the shared syncytin-Car1 gene, indeed the hyena orthologue does not disclose any deviation from the normal behavior of a gene under purifying selection [START_REF] Cornelis | Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora[END_REF], and we could show here that its fusogenic activity and pattern of placental expression are similar to those in cat or dog.

Accordingly, the Hyena-Env2 gene does not seem to fulfil the same function as syncytin-Car1, being non-fusogenic, but rather seems to be a more recently captured add-on to hyena

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placentation. The absence of a demonstrated fusogenic activity of Hyena Env-2 associated with its conservation during Hyaenidae evolution, suggests another role. We therefore propose that Hyena-Env2 is directly involved in the transition from an endotheliochorial carnivoran placenta to the hemochorial hyena placenta, that differs from the former mainly by the disruption of maternal blood vessel walls by invading fetal cells, resulting in maternal blood bathing the syncytial maternofetal interface, as observed in all hemochorial placentas, including human (Wooding & Burton, 2008). In this respect it is worth mentioning that a recent analysis of the properties of the envelope protein of the HERV-K family of human endogenous retroviruses has provided hints that this envelope could promote an epithelio-mesenchymal phenotypic transition as well as cellular invasion (EMT) [START_REF] Lemaître | A human endogenous retrovirus-derived gene that can contribute to oncogenesis by activating the ERK pathway and inducing migration and invasion[END_REF] and that the recently described syncytin-Mab1 might be able to modulate cell migration phenomena through interaction with its cognate receptor [START_REF] Cornelis | An endogenous retroviral envelope syncytin and its cognate receptor identified in the viviparous placental Mabuya lizard[END_REF]. Although preliminary assays carried out with Hyena-Env2 could not demonstrate a similar effect, a more refined experimental setup, or even better, identification of the Hyena-Env2 receptor, should now be carried out to understand in molecular terms how the captured envelope contributes to the transition in placentation type. tissue samples of Crocuta crocuta placenta were provided by A. Conley and S. Glickman (University of California Veterinary School, USA). This study was carried out in strict accordance with the French and European laws and regulations regarding animal experimentation (Directive 86/609/EEC regarding the protection of animals used for experimental and other scientific purposes).

Crocuta crocuta placentome transcriptome high throughput sequencing and de novo assembly. Total RNA was purified from Crocuta crocuta placenta before sequencing using the Illumina HiSeq 2000 sequencing system. cDNA library construction, sequencing, and transcripts assembly were performed by the French National Sequencing Center (Genoscope, Evry, France). Briefly, poly(A)+ RNA was selected with oligo(dT) beads, chemically fragmented, and converted into single-stranded cDNA using random hexamer priming according to the Illumina TruSeq protocol. The second strand was then generated to create double-stranded cDNA. Next, the paired-end library was prepared following Illumina's protocol: fragments were end-repaired and then 3'-adenylated, and Illumina adapters were added by using NEBNext Sample Reagent Set (New England Biolabs); ligation products were purified, and DNA fragments (>200 bp) were PCR-amplified using Illumina adapter-specific primers. After library profile analysis (showing a typical library size of 200-600 bp) by Agilent 2100 Bioanalyzer (Agilent Technologies) and qPCR quantification (MxPro; Agilent Technologies), the library was sequenced using 100-baselength read chemistry in a paired-end flow cell on the Illumina HiSeq2000 instrument (Illumina). Nucleotide sequence information has been deposited at the European Nucleotide Archive (ENA; https://www.ebi.ac.uk/ena) under the accession number ERA1116685. To maximize the number of reconstructed transcripts, several independent assemblies were performed using two different de novo assembly software programs (Oases and Trinity) as well as two k-mer lengths [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF][START_REF] Lynch | Ancient transposable elements transformed the uterine regulatory landscape and transcriptome during the evolution of mammalian pregnancy[END_REF], and only transcripts of >100 bp were conserved.

Database screening and sequence analyses. Retroviral endogenous env gene sequences were searched for in each transcriptome assembly: Sequences containing an ORF longer than 400 amino acids, (from start to stop codon) were extracted from the transcriptomes using the getorf program from the EMBOSS package (http://emboss.sourceforge.net/apps/cvs/emboss/apps/getorf.html) and translated into amino acid sequences. These sequences were compared with the TM subunit amino acid sequences of 35 retroviral envelope glycoproteins (from representative ERVs -among which known syncytins -and infectious retroviruses), using the BLASTp program of the National Center for Biotechnology Information (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Putative envelope protein sequences were then selected based on the presence of a hydrophobic domain (transmembrane domain) located 3' to a highly conserved C-X 5-7 -C motif.

Multiple alignments of amino-acid sequences were carried out by using the AliView program (Larsson, 2014) and the MUSCLE protocol. Maximum likelihood phylogenetic amino

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product was purified and sequenced using primers designed to anneal about every 600 bp.

Characterization of the placental Hyena-Env2 transcript. Rapid amplification of cDNA ends (RACE) was performed using the SMARTer RACE cDNA Amplification Kit (Clontech). cDNA was generated from 100 ng Crocuta crocuta placental RNA and the placental Hyena-Env2 transcript 5' and 3' ends were amplified using the "RACE" primers in Table 1 andthe & Heidmann, 1998). PCR was carried out with 10 µl of diluted (1:10) cDNA in a final volume of 25 µl using the FastSYBR Green PCR Master Mix (Qiagen) in an ABI PRISM 7000 sequence detection system. Transcript levels were normalized relative to the amount of the housekeeping RPL19 gene (ribosomal protein L19). Samples were assayed in triplicate, see Table 1 for primer sequences.

In situ hybridization. Freshly collected Crocuta crocuta placentas were fixed in 4% paraformaldehyde at 4 • C and were embedded in paraffin. Serial sections (4 µm) were either stained with H&E or were used for in situ hybridization. Three PCR-amplified fragments of Hyena-Env2 (452 bp, 416 bp, and 496 bp) and three fragments of Hyena-Env3 (415 bp, 448 bp and 414 bp) (primers are listed in Table 1) were cloned into pGEM-T Easy (Promega) for in vitro synthesis of the antisense and sense riboprobes generated with SP6 RNA polymerase and digoxigenin 11-UTP (Roche Applied Science) after cDNA template amplification. In situ hybridization of hyena syncytin-Car1 transcripts was performed using two sets of sense and antisense riboprobes directed against cat syncytin-Car1 [START_REF] Cornelis | Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora[END_REF] Sections were processed, hybridized at 42 • C overnight with the pooled riboprobes, and incubated further at 4

• C overnight with alkaline phosphatase-conjugated anti-digoxigenin antibody fragments (Roche Applied Science). Staining was revealed with nitroblue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate phosphatase alkaline substrates, as indicated by the manufacturer (Roche Applied Science).

Antibody production and immunohistochemistry. A DNA fragment containing 197 amino acids of the Hyena-Env2 envelope SU subunit (amino acids 240-437; for primers see Table 1) was inserted into the pET28b (Novagen) prokaryotic expression vector and expressed in BL21(DE3) bacteria. The recombinant C-terminally His-tagged protein was purified from bacteria lysates by nickel affinity chromatography. Mouse immunization was performed in accordance with standard procedures, using female 8-week-old BALB/c mice. Pre-immunization sera as well as sera containing polyclonal antibodies were recovered independently from 10 mice and were tested by Western blot analyses using lysates of 293T cells transiently transfected with a Hyena-Env2 expression vector. The most specific sera of the Western blot experiment were then tested for signal strength and absence of background staining by performing immunohistochemistry on 293T cells transfected with an empty vector or a Hyena-Env2 expression vector. For immunohistochemistry, paraffin sections were processed for heat-induced antigen retrieval (Tris EDTA, pH 9; Abcam) and incubated overnight with anti-Hyena-Env2 serum or the corresponding pre-immune serum. Staining was visualized using the peroxidase/diaminobenzidine Mouse PowerVision kit (ImmunoVision Technologies). All slides were immunostained in cover plates on the same day to obtain a standardized intensity of staining.

Expression vectors and fusogenicity assays. PCR fragments containing the Hyena-Env2

and Hyena-Env3 ORFs were amplified from Crocuta crocuta genomic DNA by PCR using the "Cloning" primers (see Table 1). They were digested with EcoRI (Hyena-Env2) or XhoI (Hyena-Env3) and MluI and cloned into the phCMV vector (GenBank accession AJ318514, gift from F.-L. Cosset, Ecole Normale Supérieure de Lyon, France). Cat, dog and Crocuta crocuta SLC1A5 was amplified from placental cDNA using conserved primers (see Table 1) and PCR fragments were digested using EcoRI and MluI and cloned into the same phCMV vector as above. Hyena-Env-pseudotyped MLV viral particles were produced by cotransfecting 106 293T cells with 1 µg CMVi (MLV gag-pol expression vector), 1.5 µg MFG-nlsLacZ (retroviral genome expressing nlsLacZ), and 0.5 µg hyena syncytin-Car1, Hyena-Env2, Hyena-Env 3 or amphotropic murine leukemia virus (A-MLV) Env expression vectors using the JetPRIME transfection kit (Polyplus transfection). 48 h post-transfection, virus containing supernatants were harvested, filtered (PVDF membranes, pore size 0.45 µm; Millipore) and transferred to target cells in 24 well plates (5 -8 × 10 4 cells/well), supplemented with Polybrene (Final concentration 8 µg/mL). Spinoculation was performed at 1200 g for 2.5 h at room temperature. X-Gal staining was performed 72 h post-infection and infection foci counted under microscope. When relevant, target cells were transfected using LTX (Life technologies) 48 h before infection.

For co-culture cell-cell fusion assays, 2 × 10 6 HEK293T cells were co-transfected with 2.75 µg Hyena-Env expression vector and 2.75 µg of R9SA (an nls-LacZ expressing reporter plasmid) using LTX (Life Technologies). 4 h post transfection cells were resuspended and seeded alongside target cell lines in 24 well plates at a ratio of 20:80, 30:70 or 50:50 of transfected:target and at 2 × 10 5 cells/well total. X-Gal staining was performed 24 or 48 h after start of co-culture and presence or absence of syncytia assayed under microscope. All cell lines are described in [START_REF] Cornelis | Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants[END_REF] and were grown in DMEM supplemented with 10% FCS (Invitrogen), 100 mg/mL streptomycin, and 100 U/mL penicillin, at 37 • C and 6% CO 2 .

Two novel examples of ERV Env exapted for a potential role in placentation and a new member of the SLC1A5 interference group

Our investigation into the implication of endogenous retroviral env in placentation of Hyaenidae and Mabuya have led us to the identification of a non-mammalian syncytin, a new mammalian syncytin-like and an endogenous envelope belonging to a well-known interference group. In this section, I will first discuss a few interesting aspects of each of these envelopes, while the next sections will consider their links to placental evolution and potential non-canonical roles in placentation.

Hyena-Env2 is a conserved syncytin-like envelope gene expressed during hyena placentation

Hyena-Env2 is the first of two novel envelopes identified following our high-throughput sequencing of a hyena placental transcriptome. Hyena-Env2 presents all major functional domains of a retroviral gamma-type envelope, including a very well conserved ISD, and we have been able to characterize the entire associated provirus, including the conserved RT sequence which supports a gammaretroviral origin for this gene. Conservation studies show that Hyena-Env2 is a gene present only in hyenas, BLAST analysis in 3 Feliformia and 6 Caniformia genomes yielded no sequence bearing significant similarities and all attempts to amplify the Hyena-Env2 ORF, or parts of it, were negative in these species. The identification of a single insertion locus in Crocuta crocuta and the fact that this locus presents the Hyena-Env2 associated provirus in all extant hyena species (with an identical TSD sequence) confirmed that the single insertion event occurred before radiation of modern day hyenas. Conversely, all investigated syntenic non-hyena loci were empty, with the non-duplicated TSD sequence readily identifiable (except in the case of dog in which this genomic region is deleted).

The exact age of the Hyena-Env2 insertion into the Hyaenidae genome is difficult to pinpoint due to the evolutive history of hyenas. This family split of from other carnivorans about 30 Mya

1.2

. . Hyena-Env is a member of the SLC A interference group with a peculiar tropism [START_REF] Nyakatura | Updating the evolutionary history of Carnivora (Mammalia): a new species-level supertree complete with divergence time estimates[END_REF] and reached the height of its diversity, comprising 20-30 different species, about 10 Mya (de Queiroz & Gauthier, 1992). Nowadays the fossil record of Hyaenidae comprises 70 known species, but only 4 extant representatives of this clade subsist, this family which was once one of the most species-rich carnivoran families is now one of the most species poor [START_REF] De Queiroz | Phylogenetic Taxonomy[END_REF]. The 4 extant hyenas share a last common ancestor about 10 Mya, which puts the age of insertion of Hyena-Env2 at a very imprecise 10-30 Mya. Sadly since the placenta is composed of soft tissue, there is no indication in the fossil record as to the reproductive mode of the 70 extinct hyenas. In any case, the Hyena-Env2 ORF has been conserved in a functional coding state in all extant hyenas and the characterization of the associated provirus in Crocuta crocuta makes it readily apparent that this was not the case of the other canonical ERV ORFs, both gag and pol presenting a number of disruptions.

While Hyena-Env2 is non-fusogenic in our assay (which might be due to the nature of our target cells and not an intrinsic fusion loss), its conservation and expression in the unfused layers of the placenta still hint at a possible role in placenta physiology. Its placental expression as assessed by RT-qPCR is much lower than that of Syncytin-Car1, but it is important to keep in mind that i) the proposed role for Hyena-Env2 is distinct of that of the carnivoran syncytin and may not necessitate a similarly important expression, and ii) the RNA sample used to sequence the placental transcriptome corresponds to a mid-gestation placenta that is already hemochorial. It is possible that Hyena-Env2 expression is variable during gestation and decreases once a hemochorial phenotype is attained. In mice, where placental samples from distinct gestation periods are readily available, it has been shown that both syncytin-A and -B transcripts display highly differential expression depending on gestation day [START_REF] Dupressoir | Syncytin-A and syncytin-B, two fusogenic placenta-specific murine envelope genes of retroviral origin conserved in Muridae[END_REF].

The non-fusiogenicity of Hyena-Env2 obviously prevents it from accomplishing a classical syncytin role in hyena placentation, however there are a number of other placental roles that could be fulfilled by syncytins as well as the conserved non-fusogenic syncytin-likes. The potential roles of Hyena-Env2 and other syncytin-likes will be discussed in the following sections, in particular in 3.2.

Hyena-Env3 is a member of the SLC1A5 interference group with a peculiar tropism

While Hyena-Env3 is not hyena-specific and shows very weak placental expression, it may warrant further investigation. It seems that the Hyena-Env3 associated retrovirus has been active very recently (or is still active) as revealed by the many detectable insertions in almost every published Caniformia genome, the most conserved of which are not present at orthologous loci of different species, suggesting recent endogenizations. The detection of an almost intact provirus with 100% identical LTRs in dog also suggests recent activity of this family. Nevertheless, many of the env ORFs associated with these insertions are non-coding and the one described as

1.3

Canis-Env3 by [START_REF] Cornelis | Ancestral capture of syncytin-Car1, a fusogenic endogenous retroviral envelope gene involved in placentation and conserved in Carnivora[END_REF] has lost its initiation site when compared to the Hyena-Env3 ORF, losing its signal peptide sequence in the process and rendering the envelope non-functional. Hyena-Env3 on the other hand still displays significant fusogenic activity and while it might not serve a role in placentation currently, it is possible that this envelope comes to play a more important role in the future, following the example of the EnvV2/HERV-W substitution and the baton pass model, after all the HERV-W insertion that gave rise to Syncytin-1 also occurred in Old World monkeys, but the envelope was not conserved in that clade.

The relationship between Hyena-Env3 and its cognate receptor SLC1A5 might also prove to be interesting in the context of both endogenous and exogenous retrovirology. Most SLC1A5 using envelopes do not show any particular tropism apart from the group-wide shared difficulty with infecting rodent cells. In the case of Hyena-Env3 the tropism is much more restricted as the Env seems to be able to interact only with carnivoran SLC1A5. Analysis of the sequence of Hyena-Env3 SU and carnivoran SLC1A5 have only revealed that in the former some of the motives conserved among SLC1A5 using Env SUs are absent or, more often, present insertions of one or two amino acids. It is unclear whether these differences are able to explain the particular tropism of Hyena-Env3, but further investigation might shed more light on the receptor specificity of retroviral envelopes and in particular those of the well-known SLC1A5 group, which also contains two previously described mammalian syncytins.

Syncytin-Mab1: characterization of a non-mammalian syncytin and its receptor

Syncytin-Mab1 is the first syncytin identified in a non-mammalian species. This identification suggests that the process of ERV Env exaptation for a role in placentation is not limited to the mammalian clade, but that it might have occurred in some (or all) of the non-mammalian placental species.

Similarly to what is observed for all presently described mammalian syncytins, the expression of syncytin-Mab1 co-localizes with the presence of a fused cell layer in the Mabuya placenta. This observation, alongside the fusogenic potential observed in ex vivo assays, suggests that Syncytin-Mab1 participates in the establishment of the placental syncytium. Interestingly, while Syncytin-Mab1-pseudotyped MLV particles display very high titers in our infection assay, cellcell fusion is observed only in presence of an acidic shock. This is similar to the behavior of Syncytin-Rum1 [START_REF] Cornelis | Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants[END_REF], but here again the physiological relevance of this property is unknown. While it is possible that fusion in vivo is at least partly controlled by a temporary acidification of the placental medium, it is also possible that pH-dependence is an artifact of our assay. Indeed, the ex vivo assays were performed using mammalian cells to express and assay proteins from a distantly related lizard clade. It is possible that the cells we used do not express certain lizard specific factors which might result in differential fusion-impacting 1.3

. . Syncytin-Mab characterization of a non-mammalian syncytin and its receptor post-translational processing of Syncytin-Mab1 or Mabuya MPZL1 in the two cell types.

The placental expression of Syncytin-Mab1 in areas presenting only unfused cell layers would suggest that it might also accomplish another function in placental physiology. Analysis of the expression pattern in the paraplacentome reveals a very particular sub-cellular expression pattern, localized exclusively at the membrane forming the maternofetal interface. This pattern would support a role in maternofetal interaction, but the exact nature of this role is unknown. Even disregarding the remarkable example of convergent evolution it represents, Syncytin-Mab1 presents some very unusual characteristics when compared to all other known retroviral envelopes. At first glance it seems very close to standard gamma-type envelopes, presenting a recognizable if somewhat divergent ISD, being much too short to be an epsilon-or spumaretroviral envelope, and clustering with other gamma-type Env on a TM based phylogenetic tree. Closer inspection however reveals that the CX n CC motif that is conserved in all gamma-type TMs and is essential for establishment of the SU/TM bond does not present the third cysteine, taking the form of CX 7 C instead. The presence of only two cysteines is reminiscent of the situation in beta-type TM in which the SU and TM are not covalently associated. Additionally, Syncytin-Mab1 does not present the CXXC motif that provides the SU partner for formation of the covalent SU/TM bond in gamma-type envelopes. It is unknown whether this particular association of beta-and gamma-type envelope characteristics is common among lizard retroviruses, or if it is specific to Syncytin-Mab1, but its high fusogenic potential in the pseudotyping assay indicates that it is not detrimental to its canonical Env fusion activity.

On a similar note, it is interesting to see that Mab-Env3 and -4 cluster with the avian gammatype RSV TM in our phylogenetic analysis and that they present a shifted predicted fusion peptide that is flanked by cysteine residue, as is commonly observed in avian gamma-type TM. This envelope type is usually found almost exclusively in birds, but our screen has identified two coding examples of lizard avian gamma-type envelopes. The peculiar profile of envelopes found in the Mabuya sp. IV transcriptome is of particular relevance to the screen for syncytins and syncytin-likes in non-mammalian species, as it not only shows that it is necessary to be able to detect Env that differ from those classically observed in mammals, but also that our in silico screening method seems to be able to do so.

Finally, the nature of the cognate receptor of Syncytin-Mab1 is somewhat surprising. Not only is it a single pass transmembrane protein (which is the exception rather than the rule for gamma-type envelopes, supporting the hypothesis that Syncytin-Mab1 is not gamma-type), it is a signal transducer that has been implicated in several important cellular signaling pathways. The implications of this nature for placental physiology will be discussed in more detail below, but use of such a molecule as receptor is rare enough to be noted here.

Implication of syncytins and syncytin-likes in placental evolution

Retroviral insertions have been a constant driving force of vertebrate evolution

In this study, we describe several recent endogenization events, the syncytin-Mab1 containing provirus was first endogenized by an ancestral lizard about 30 Mya and Hyena-Env2 capture occurred between 10 and 30 Mya in the last common ancestor of all extant hyenas. Additional examples of recent and even still ongoing endogenizations can be observed in mice, sheep and koalas (reviewed in [START_REF] Dewannieux | Endogenous retroviruses: acquisition, amplification and taming of genome invaders[END_REF][START_REF] Greenwood | Transmission, Evolution, and Endogenization: Lessons Learned from Recent Retroviral Invasions[END_REF]. On the other hand, the oldest known endogenization took place in early Palaeozoic fishes, over 450 Mya, at about the same time as the apparition of jawed vertebrates and long before the ocean-land transition (Aiewsakun & Katzourakis, 2017). Between these two extremes reside a number of known endogenization events, [START_REF] Hayward | Pan-vertebrate comparative genomics unmasks retrovirus macroevolution[END_REF] identify almost 100,000 ERVs among 65 published vertebrate genomes and suggest that no jawed vertebrate clade has been entirely spared from retroviral endogenizations, though the total amount observed in each clade shows great deal of variability.

Each new retroviral insertion is a source of genetic diversity, both through the retroviral sequences it brings with it and the randomness of the insertion locus. In mice where ERV activity is still high, it has been shown that at least 10% of de novo mutations observed in laboratory mice during the last 30 years are due to new ERV insertions [START_REF] Maksakova | Retroviral elements and their hosts: Insertional mutagenesis in the mouse germ line[END_REF]. It is also important to remember that the 8% of the human and 10% of the mouse genome occupied by sequences of retroviral origin (Lander et al., 2001;Waterston et al., 2002) represent only the fraction of retroviral insertions that have been fixed in the population, and the evolutive impact of all endogenizations was without a doubt more important. Even so and as discussed in the introduction, there are numerous examples of retroviral insertions still affecting cellular gene expression in animals to this day and a growing amount of evidence shows that retroviral gene exaptation is an ongoing and widespread phenomenon. Interestingly, early development and placentation seem particularly affected by these processes. This might be in part due to the fact that compared to many other animal traits, placentation evolved relatively late, allowing for easier comparative analysis between placental and non-placental animals in order to decrypt the necessary changes, and in part to the fact that germline cells and retroviruses share a special relationship, since to be endogenized a retrovirus needs to infect one of these cells. It stands to reason that ERVs expressed in the germline have better odds of copy number amplification and of fixation in the general population. Additionally, early development is associated with demethylation events which allow expression of otherwise repressed ERV elements, allowing them to contribute their functionality. Finally, the placenta is much more short-lived than other organs and deleterious effects of ERV expression might therefore have less dire consequences.

The apparition of a new organ, such as the placenta, necessitates the establishment of new gene expression networks, so as to be able to regulate gene expression in the new tissues. Studies comparing transcriptomes of monotremes with that of placental mammals reveal that thousands of genes had to be recruited for expression in endometrial tissue, most of them originally expressed in the brain, gut and hemolymphoid systems [START_REF] Lynch | Ancient transposable elements transformed the uterine regulatory landscape and transcriptome during the evolution of mammalian pregnancy[END_REF]. Endometrial expression of many of these genes is regulated by tissue-specific promoters from repeated sequences, including ERV sequences, illustrating the major impact of acquisition of new regulatory sequences by endogenization and subsequent amplification of ERV elements on mammalian evolution [START_REF] Lynch | Ancient transposable elements transformed the uterine regulatory landscape and transcriptome during the evolution of mammalian pregnancy[END_REF]. One of the most famous examples is the expression of prolactin in the endometrium of primates, mice and elephants. In these three clades, endometrial expression of this hormone has emerged independently and studies of the underlying regulatory network shows that in all three cases endometrial expression is driven by species-specific repeated element regulatory sequence and more precisely LTRs in mice and primates [START_REF] Emera | Convergent evolution of endometrial prolactin expression in primates, mice, and elephants through the independent recruitment of transposable elements[END_REF]. A non-placental example can be found in the regulation of interferon response in innate-immunity. A number of interferon-inducible promoters have been shown to be derived from lineage-specific LTR insertion events and now constitute an extensive and complex regulation network [START_REF] Chuong | Regulatory evolution of innate immunity through co-option of endogenous retroviruses[END_REF].

A final, more indirect way in which retroviral insertions can generate genetic variation is found in the example of the KRAB-ZFP family of proteins. These proteins have been implicated in the epigenetic regulation of ERV expression, allowing a silencing of newly acquired and potentially pathogenic ERV elements (reviewed in [START_REF] Hurst | Epigenetic control of human endogenous retrovirus expression: Focus on regulation of long-terminal repeats (LTRs)[END_REF]. While it had been assumed that the observed co-evolution of KRAB-ZFP and new retroviral insertions was only reflective of an arms race between genomic invaders and their host, a recent study has shown that some KRAB-ZFP have been conserved under purifying selection long after the corresponding ERV has ceased to actively integrate the genome [START_REF] Imbeault | KRAB zinc-finger proteins contribute to the evolution of gene regulatory networks[END_REF]. Conservation of these KRAB-ZFP proteins seems to be due to the co-option of the KRAB-ZFP/ERV pair for the regulation of host gene expression through epigenetic processes. In this case ERV sequences contribute directly to genetic variability through their insertion and indirectly through generation of new host anti-ERV factors, that can later also be used for the establishment of lineage specific gene expression networks [START_REF] Imbeault | KRAB zinc-finger proteins contribute to the evolution of gene regulatory networks[END_REF].

In summary, retroviruses and vertebrates have co-existed for about 450 My and retroviral

2.2

insertions have been a constant on-going phenomenon in that timespan, contributing to genetic diversity and potentially enabling major evolutive transitions, notably in the domain of reproduction. The insertion of the lineage specific syncytin-Mab1 and Hyena-Env2 containing proviruses are yet more examples of this phenomenon.

Syncytin-Mab1 and convergent evolution of animal placentas 2.2.1 Convergent use of ERV env genes in placentation

Viviparous matrotrophy has evolved several times independently in animals. The association of these two modes of reproduction has emerged repeatedly in fishes, once in mammals and at least 6 times in squamates (Blackburn, 2015). In all cases structures involved in gas and nutrient transfer have been co-opted to serve a new purpose at the maternofetal interface and often derive from modified oviduct or uterine tissues (Blackburn, 2015). The reasons behind the emergence of this reproductive mode are supposed to be diverse, ranging from thermal advantages in the case of squamata to increased control over fetal development in eutherians (Blackburn, 2015;[START_REF] Griffith | The placenta as a model for understanding the origin and evolution of vertebrate organs[END_REF], but some of the resulting structures show high functional and sometimes structural homology. In placental viviparous matrotrophic species, the membranes of mother and fetus have evolved to be in contact and often display specializations for maternofetal exchange.

The apparition of this organ specialized for maternofetal exchanges in clades as disparate as fishes, lizards and mammals is a noteworthy case of evolutive convergence. In this study we have focused on the placenta of a particular group of lizards, the neotropical genus Mabuya. The Mabuya placenta is a recent emergence (about 25 Mya) and presents many specialized structures that are also found in mammals: areola, absorptive plaques and a region of strong maternofetal interdigitation that also presents a fused cell layer. The existence of this fused cell layer was of particular interest to us, as it seemed to originate through cell-cell fusion events in the maternal epithelium (Leal & Ramírez-Pinilla, 2008), reminiscent of the cell-cell fusion events observed at the maternofetal interface in many mammalian placentas. Such fusion events need presence of a fusogen and in many cases animal fusogens are derived from viral proteins. In the context of mammalian placentas in particular, it has now been well-established that ERV derived fusogenic syncytins are necessary for establishment of fused cell layers at the maternofetal interface [START_REF] Dupressoir | Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene[END_REF](Dupressoir et al., , 2011)). Syncytins are env genes that have been acquired stochastically, are expressed in the placenta and are fusogens. They are under purifying selection, as can be expected for a gene with an important role in placentation. Here we have shown that the Mabuya placenta expresses such a captured retroviral envelope, notably in the fused cell layer, and that this envelope presents fusogenic properties. The discovery of this first non-mammalian syncytin, Syncytin-Mab1, indicates that the emergence of a complex placenta in the Mabuya genus was accompanied by the capture of a retroviral Env, as is thought to be the case in mammals. Syncytin-Mab1 is an unusual ERV Env, presenting the association of a conserved ISD with CX 7 C motif, setting it apart from the gamma-or betaretroviral Env implicated in mammalian placentation, but its expression in the fused cell layer of the placenta suggests that it could nevertheless accomplish the fusogenic function assigned to mammalian syncytins.

Syncytin-Mab1 as the first example of a founding syncytin

While the potential involvement of a syncytin in the placenta of this remote species is already a remarkable example of convergent evolution, it is also the first description of a potential founding syncytin. It has been proposed for a long time now that the emergence of the mammalian placenta was associated with the capture of an ancestral founding syncytin, that provided its host with new functionalities allowing the transition from matrotrophic oviparity (as is observed in monotremes) to matrotrophic viviparity. The founding syncytin associated with mammalian placentation has not been identified to date and it is probable that it has degenerated during the 175 My since apparition of the mammalian placenta, to the point of being unidentifiable, as it has since been replaced by other syncytins in accordance with the baton pass model. The recent emergence of placentation in Mabuya allowed us to find a single syncytin gene present in all tested species of this genus and acquired at the time of placental emergence. Further characterization of this founding syncytin might yield new clues towards the properties of such genes and might shed some light on the processes associated with the apparition of the mammalian placenta.

Complex placentas are also present in Scincidae genera closely related with Mabuya. The Eumecia and Lubuya genera are part of the same branch and each contain a single species that is highly matrotrophic. While the complexity of the Eumecia placenta is poorly known, the placenta of Lubuya presents a highly invasive fetal syncytium. This clade would be of paramount interest for the continued investigation of non-mammalian syncytins. It is probable that viviparity emerged once in this clade, about 30 Mya, but the current placentas seem to present marked differences in organization. This situation could not only lead to the identification of a founding syncytin, but also potentially to that of differentially acquired syncytins explaining the particularities of the placentas of each genus.

Syncytins and syncytin-likes and divergent evolution of the mammalian placenta

While the evolution of placentas shows high amounts of convergence when seen at a high phylogenetic level, among mammals placentas are highly divergent. The mammalian placenta is assumed to be monophyletic, having emerged in the last common ancestor of all mammalian species about 175 Mya. From this ancestral placenta have spawned a number of different placental organizations and have turned the placenta into the organ showing the highest structural diversity among mammals. It is not entirely clear why placental organization is this divergent and there does not seem to be a clear evolutive sequence to the transition. While the epitheliochorial placenta of pigs is much less complex than the hemochorial placenta of mice, both allow appropriate provision of nutrients to the embryo over the course of gestation, and some indicators even suggest that the much simpler pig placenta is more efficient than more complex ones. Interestingly, the repartition of placental types among mammals is clearly paraphyletic, major organizational categories being found in several independent branches. This presents a problem when trying to reconstitute the evolutionary history of mammalian placentation, and parsimonious reconstructions result in the counter-intuitive hypothesis that ancestral mammalian placentation was of the highly invasive hemochorial type, while the derived state is the epitheliochorial type in which placental membranes are simply apposed. There are several major limitations to this method however. First, classification of mammalian placentas is a highly complex process as even within the great categories of epithelio-, synepithelio-, endothelio-, and hemochorial placentation there exists a great amount of variability, both in terms of the structural organization of placental tissue layers and in terms of placental invasiveness and actually proximity of maternal and fetal tissues. These finer details are too cumbersome to be used in a large-scale parsimony analysis and thus get lost in the reductive simplification of mammalian placental categories. Second, analyses based only on the structure of current day definitive mammalian placentas do not take into account the impact of stochastic gene acquisition reflected by syncytins and syncytin-likes. The acquisition of different new genes implicated in placental phylogeny in the different branches of mammals is extremely likely to have an important impact on the emergence of different modes of placentation. The random nature of ERV acquisition and exaptation by different branches is in stark opposition to the concept behind parsimony phylogeny. Of note, the newly emerged placentas of squamates are closer to the less invasive epitheliochorial type, with only the placenta of Lubuya ivensii presenting a significant degradation of maternal tissue. While it is risky to infer the status of the original mammalian placenta based on these recent non-mammalian placentas, they hint at a smoother transition from two epithelia separated by an egg-shell to two apposed epithelia, instead of jumping from separated epithelia to direct trophoblast-maternal blood contact.

The description in the present study of two new env acquired specifically at the time of placental emergence (Mabuya placenta) or a major structural transition (hemochorial hyena placenta) are two more examples of the potential impact the acquisition of such genes can have on placental physiology. The transition from an endotheliochorial to hemochorial phenotype in hyenas would represent a reversion in the hemo-to epitheliochorial model, the reason of which would be unclear. Evolution of the endotheliochorial placenta of carnivorans seems to be associated with the acquisition of the shared Syncytin-Car1, which has been conserved under purifying selection in all of these species for a remarkable 75 My. This gene is still present in hyenas and presents no discernible functional differences that could explain reversion to a previously present placental type. On the contrary, the Hyeanidae family presents the newly acquired Hyena-Env2 with placental expression. This gene is conserved among all extant hyenas and its expression seems to be mainly driven by a cellular promoter, suggesting that it has been exapted, a process usually associated with gain, rather than loss, of function. As such it seems like the most probable hypothesis to explain the transition from an endotheliochorial to a hemochorial placenta in hyenas is through a gain of function, potentially associated with the capture of a new env with placental expression such as Hyena-Env2. This new gene could serve as add-on to hyena placentation, its functions adding to those already provided by Syncytin-Car1 and resulting in a new placental phenotype. There have been a number of syncytins described to-date whose capture is associated in time with the emergence of hemochorial placentation, in mice it has even been shown that each fused layer of the hemotrichorial placenta necessitates the presence of a different syncytin and that the absence of these genes leads to major structural deficiencies and potential death of all embryos [START_REF] Dupressoir | Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene[END_REF](Dupressoir et al., , 2011)).

The acquisition of Hyena-Env2 is a case of particular interest as the structural transition observed in hyenas does not include a novel fused cell layer. Cell-cell fusion is most probably still dependent on the shared Syncytin-Car1 gene, but the major difference in this placenta is the breaching of maternal blood vessels so as to create the large maternal blood spaces characteristic of hemochorial placentas. The potential implication of Hyena-Env2 would therefore be very different from the canonical role of syncytin genes in mammalian placentation, and would be more in line with the alternative roles proposed over the years for syncytins and syncytin-likes in mammalian placentation.

Syncytin-likes and alternative envelope functions in the placenta

Growing evidence of conservation of non-fusogenic Env

Even though it is non-fusogenic and thus cannot fulfill a classical syncytin role in placentation, Hyena-Env2 shows signs of conservation and continued expression in hyena placentas. It is therefore part of the growing list of somewhat orphan genes that can be referred to as syncytinlikes. This group of env also comprises previously identified genes such as HEMO, pan-Mars-Env2, Env-Cav1, ERV3 and EnvV2 in humans. All of these genes are ERV env genes expressed in the placenta and conserved as coding ORFs for 30-80 My. None of them are fusogenic, either due to a probable intrinsic loss of fusogenicity (Hyena-Env2, Env-Cav1, EnvV2) or loss of membrane attachement (HEMO, pan-Mars-Env2, ERV3). Evolutive models predict that ERV sequences that do not fulfill a role in host physiology should quickly degenerate to non-coding sequences or solo LTRs. Analyses in the human genome show that in most ERV groups 90% of integrations degenerate to solo LTRs within 5-10 My [START_REF] Gemmell | Phylogenetic Analysis Reveals That ERVs "Die Young" but HERV-H Is Unusually Conserved[END_REF]. Conservation of coding ORFs with low non-synonymous to synonymous mutation rates therefore strongly suggest that these non-fusogenic syncytin-likes fulfill an important role in host physiology. Examples illustrating the loss of non-exapted env genes can be found in Old World monkey in which the HERV-W Env was not exapted to serve as syncytin and was instead lost during evolution.

While the actual nature of the roles of non-fusogenic syncytin-likes in placentation (or other processes) are still unknown, there are hints available based on their nature as retroviral Env, their pattern of expression, and the implication of some of them in physiological processes.

Alternative placental roles for Env

Immunosuppression

Since most syncytins and syncytin-likes are derived from gamma-type Env containing retroviruses, many present a conserved ISD sequence and could present immunosuppressive properties in vivo. In humans and mice it has been shown that in each case one of the two syncytins displays immunosuppressive properties in the in vivo mouse tumor rejection assay (Syncytin-1 and Syncytin-B respectively, [START_REF] Mangeney | Placental syncytins: Genetic disjunction between the fusogenic and immunosuppressive activity of retroviral envelope proteins[END_REF]. The same study showed that the syncytinlikes ERV3 and EnvV2 were also able to reduce immune responses against tumor cells in the in vivo assay, demonstrating that this property is independent of fusogenic potential [START_REF] Mangeney | Placental syncytins: Genetic disjunction between the fusogenic and immunosuppressive activity of retroviral envelope proteins[END_REF].

In the context of placentation, regulation of the maternal immune system plays a key role, as there is direct contact of tissues from different genetic backgrounds. In more invasive placentation this is particularly true, in the hemochorial hyena placenta for example, the maternal blood is in direct contact with genetically foreign fetal tissue. It is therefore not complicated to imagine that env genes with placental expression play a role in protecting the placenta from attack by the maternal immune system, just as they are thought to suppress immune responses to viral particles in the case of infectious retroviruses. Since this function is not linked to fusogenicity, it is a potential role of syncytin-like proteins. In the case of Hyena-Env2, the ISD shows only two mutations compared to the conserved residues of the canonical CKS-17 sequence, while the ISD of Syncytin-Car1 is slighly more divergent, though it is important to mention that the EnvV2 ISD shows strong divergence from the consensus sequence but is nevertheless functional.

An immunosuppressive role is particularly intriguing in the case of the truncated soluble syncytin-likes. Since the ISD (or part of the ISD for HEMO) is part of the released peptide, they could theoretically act as secreted diffusible factors contributing to a dampening of maternal immune responses in the vicinity of the placenta.

It is probable that immunosuppression was the major function provided by the founding syncytin during emergence of the mammalian placenta. Matrotrophy was very probably present in the common ancestor of monotremes and placental mammals, and as such the logistics of nutrient transfer between mother and fetus had already been solved (at least in part) in the lead-up to the matrotrophic oviparous mode of reproduction, as is still observed in monotremes. The probable major distinction between the early mammalian placenta and the previous oviparous mode of reproduction is therefore the direct contact of maternal tissues with tissues of foreign origin, instead of an inert egg-shell barrier. To accommodate this contact, a particular immunosuppressive context needed to be established, potentially through the expression of the ancestral syncytin. In current day mammalian placentas there are a number of processes involved in tolerance of the fetus and while syncytins such as Syncytin-2 and -B could contribute to this phenomenon, they are not the only actors of maternofetal tolerance.

In addition, current day epitheliochorial placentas do not present cell-cell fusion events, indicating that the presence of a syncytialized layer is not an absolute prerequisite for placentation. Indeed, if one suscribes to the epitheliochorial to hemochorial pathway for placental evolution, it is even probable that the ancestral mammalian placenta did not present substantial cell-cell fusion events, strengthening the hypothesis that the founding syncytin was not selected for its fusogenicity. In this context it would be extremely interesting to try and assay the immunosuppressive properties of the founding Syncytin-Mab1, though its nature of lizard retroviral Env might prevent the use of a mouse based assay. Additionally, lizard placentation does not seem to

3.2

present the same focus on creation of an immunosuppressive context as observed in mammalian placentas [START_REF] Griffith | The placenta as a model for understanding the origin and evolution of vertebrate organs[END_REF]. The exact mechanisms of fetal protection from the maternal immune system in squamates are not known, but they might implicate the presence of retroviral ISD sequences.

Differentiation and proliferation

Another proposed role of ERV derived genes in placental physiology concerns differentiation of trophoblast cells. Creation of new cell differentiation pathways is instrumental in the evolution of a new organ and the placenta presents a number of specific differentiated cell populations, both on the maternal and fetal side. Human trophoblasts for example can persist as isolated unfused CT, presenting certain stem-like properties such as continued proliferation during gestation, can fuse into the ST, a very active secretory tissue, or can differentiate into invasive EVT cells which then split into the endovascular and insterstitial sub-populations. It has been shown that Syncytin-1 is implicated in some of these differentiation pathways [START_REF] Frendo | Direct Involvement of HERV-W Env Glycoprotein in Human Trophoblast Cell Fusion and Differentiation[END_REF], but once again a role in differentiation does not seem to be restricted to syncytins sensu stricto. Among syncytin-likes, the human ERV3 Env has been shown to be implicated in trophoblast cell differentiation ex vivo [START_REF] Lin | Expression of endogenous retrovirus ERV-3 induces differentiation in BeWo, a choriocarcinoma model of human placental trophoblast[END_REF][START_REF] Lin | The cellular mechanism by which the human endogenous retrovirus ERV-3 env gene affects proliferation and differentiation in a human placental trophoblast model, BeWo[END_REF] and sheep enJSRV env seem to play an important role in the formation and differentiation of BNC cells [START_REF] Dunlap | Sheep Endogenous Betaretroviruses (enJSRVs) and the Hyaluronidase 2 (HYAL2) Receptor in the Ovine Uterus and Conceptus[END_REF]. Knock-down of enJSRV transcripts prevents implantation of the sheep conceptus and leads to embryo death [START_REF] Dunlap | Endogenous retroviruses regulate periimplantation placental growth and differentiation[END_REF]. Contrarily to fusogenic and immunosuppressive functions, roles in differentiation are difficult to predict as there is no particular retroviral Env motif or domain associated with them whose absence or presence can be examined. Of note, expression of the HEMO syncytin-like was on the contrary associated with an undifferentiated stem-cell phenotype, in a way reminiscent of that observed for HERV-H expression [START_REF] Heidmann | HEMO, an ancestral endogenous retroviral envelope protein shed in the blood of pregnant women and expressed in pluripotent stem cells and tumors[END_REF]. It is possible that this Env contributes to a maintenance of an undifferentiated state. The presence of undifferentiated and proliferating trophoblast cells is an important aspect of placentation as the surface area of the organ expands massively in a relatively short time, necessitating a constant supply of new cells. In human placentas both the amount of nuclei in the ST and that in the CT layer show a constant increase during gestation [START_REF] Simpson | From 13 weeks to term, the trophoblast of human placenta grows by the continuous recruitment of new proliferative units: A study of nuclear number using the disector[END_REF][START_REF] Mayhew | Proliferative versus hypertrophic growth in tissue subcompartments of human placental villi during gestation[END_REF].

It is unknown if Syncytin-Mab1 or Hyena-Env2 play a role in cell differentiation, as mentionned above such a role is hard to predict and to investigate. The expression of Hyena-Env2 in the CT layer would be in accordance with a potential role in CT differentiation or proliferation.

Invasion

Invasion of maternal tissues by invasive fetal cells is a common phenomenon in placentation. It is observed both in mammals, where it ranges from invasion by a few specialized cells to invasion of the whole conceptus and degradation of all maternal tissue layers, and in non-mammalian placentas. In the Mabuya placenta a subset of fetal cells send out projections that cross the uterine epithelium and in the more invasive Lubuya ivensii placenta invasive syncytialized layers detach the entire maternal epithelium. Even in the placentation of non-vertebrate salps, invasion plays a major role as the whole conceptus breaks through the wall of the maternal ovary to implant itself in the jet chamber. With the exception of this last, somewhat particular case, placental invasion seems to occur with two main objectives. In a few cases it plays a role in allowing fetal secretions a more direct access to the maternal circulatory system, in these cases the opposite is not true as only a few specialized cells are concerned by these processes. This is observed in horse placentation in which BNC migrate into the endometrium where they secrete placental hormones, but also in synepitheliochorial placentas in which the fusion of BNC with the maternal epithelium allows them to secrete their contents on the other side of this barrier. The other type of invasion reduces the distance between maternal blood and the placenta as a whole. This is the case in all placentas in which at least one of the maternal cell layers at term is degraded, i.e., endotheliochorial and hemochorial placentation. Often these more invasive placentas also present subsets of cells that show increase invasion and migrate into maternal tissues independently of the main placental cell mass. While the placenta of hyenas falls squarely into the second category, that of Mabuya presents a curious intermediate state in which all maternal tissues layers subsist until term and only a few cells send out invasive projections that establish direct contact with maternal blood vessels. Contrary to what is observed in epitheliochorial or synepitheliochorial placentation however, the body of these invasive cells remains in contact with the placental epithelium at all times. While the exact purpose of these cells and the extensive membrane network they establish inside the uterine syncytium is unknown, it seems likely that they are involved in the formation of a localized endothelial phenotype, reducing the interhemal distance.

The involvement of retroviral env in invasive processes has not been studied extensively so far, though a few examples of such properties exist. The envelope of exogenous JSRV for example has been shown to be sufficient to induce ongogenic transformation of cells [START_REF] Wootton | Sheep retrovirus structural protein induces lung tumours[END_REF], a process associated with increased migratory and invasive ability. The related endogenous HERV-K Env was shown to also possess oncogenic properties and to be able to enhance cell migration and invasion ex vivo by using the same pathway as that used by JSRV [START_REF] Lemaître | A human endogenous retrovirus-derived gene that can contribute to oncogenesis by activating the ERK pathway and inducing migration and invasion[END_REF]. Interestingly, tumor and trophoblast cells share a number of similarities and it has been argued that the placenta as a whole is more like a cancer than an allograft from the mother's perspective [START_REF] Beaman | Pregnancy is a model for tumors, not transplantation[END_REF][START_REF] Macaulay | The Genes of Life and Death: A Potential Role for Placental-Specific Genes in Cancer[END_REF]. Several of the pathways implicated in cancer cell migration and invasion have been shown to be also implicated in placental invasive processes [START_REF] Ferretti | Molecular circuits shared by placental and cancer cells, and their implications in the proliferative, invasive and migratory capacities of trophoblasts[END_REF]. Expression of syncytin and syncytin-like Env in the placenta could therefore contribute to the invasiveness of these tissues. This has been suggested as role of Env-Cav1 for example, which is non-fusogenic but strongly expressed in invasive syncytial tissues of the guinea pig placenta [START_REF] Vernochet | A syncytin-like endogenous retrovirus envelope gene of the guinea pig specifically expressed in the placenta junctional zone and conserved in Caviomorpha[END_REF].

Similarly, we propose that Hyena-Env2 and Syncytin-Mab1 could contribute to the fetal invasion observed in their respective placentas. The hyena-specific expression of Hyena-Env2 at the maternofetal interface would be an elegant explanation to the hyena-specific invasive phenotype displayed by this organ, even though our preliminary transwell migration and invasion assays performed with Hyena-Env2 transduced untransformed MCF10A cells proved inconclusive. It is possible that this type of assay with this type of cell is not adapted to the probably limited transformative power of a syncytin-like Env, this function seeming to be lessened in endogenous Env when compared to their exogenous counterpart. Neither HERV-K nor enJSRV Env were able to induce oncogenic transformation in 208F cell ex vivo for example, while the exogenous JSRV led to the formation of transformed foci [START_REF] Lemaître | A human endogenous retrovirus-derived gene that can contribute to oncogenesis by activating the ERK pathway and inducing migration and invasion[END_REF]. The invasive potential of syncytin-likes such as Hyena-Env2 will therefore most probably need to be studied using highly sensitive migration and invasion assays. An additional way of studying a potential role of an env gene in invasion is the identification of its cognate receptor (see discussion of Syncytin-Mab1/MPZL1 below). Search for the Hyena-Env2 receptor is currently ongoing, using cDNA library based screening assays similar to those described in [START_REF] Bianchi | Juno is the egg Izumo receptor and is essential for mammalian fertilization[END_REF] and [START_REF] Bacquin | A Cell Fusion-Based Screening Method Identifies Glycosylphosphatidylinositol-Anchored Protein Ly6e as the Receptor for Mouse Endogenous Retroviral Envelope Syncytin-A[END_REF].

In the Mabuya placenta, the invasive cells represent a small sub-population that sends out thin processes into the uterine syncytium. These complex but small structures are difficult to observe using optical microscopy, develop only very late during gestation, and seem to be very labile during sample preparation (Leal & Ramírez-Pinilla, 2008). Accordingly we have not been able to conclusively investigate the expression of Syncytin-Mab1 in this cell population, though its expression profile would certainly allow it to play a role in the invasive fetal cells. The identification of the MPZL1 as the Syncytin-Mab1 receptor even provides a possible mechanism for an invasive potential of this syncytin. Indeed, MPZL1 has been previously shown to increase cell invasion and migration in mouse (with a potential role in early embryo formation, Roubelakis et al., 2007) and in human hepatocellular carcinoma [START_REF] Jia | Amplification of MPZL1/PZR promotes tumor cell migration through Src-mediated phosphorylation of cortactin in hepatocellular carcinoma[END_REF]. The activity of MPZL1 as a signal transducer is mediated by its two intracellular immunoreceptor tyrosine-based inhibitory motives (ITIM). Dimerization of MPZL1 induces phosphorylation of Y241 and Y263 (one in each ITIM) which leads to signal transduction through the SHP-2/Src pathway (Zhao & Zhao, 1998;Zhao et al., 2002). Interestingly SHP-2 is also implicated in the ERK1/2 pathway which has been shown to be to be important for HERV-K/JSRV oncogenicity [START_REF] Lemaître | A human endogenous retrovirus-derived gene that can contribute to oncogenesis by activating the ERK pathway and inducing migration and invasion[END_REF]. Transduction experiments have shown that expression of Syncytin-Mab1 is sufficient to trigger MPZL1 phosphorylation and activation. This activation can also be achieved using concanavalin A, which binds to several MPZL1 monomers and brings them into close contact, triggering activation (Zhao et al., 2002), or simply overexpression of MPZL1, increasing the odds of dimer formation [START_REF] Jia | Amplification of MPZL1/PZR promotes tumor cell migration through Src-mediated phosphorylation of cortactin in hepatocellular carcinoma[END_REF]. We therefore propose that interaction of a Syncytin-Mab1 Env trimer with MPZL1 might trigger its activation in a similar fashion, presenting three SU subunits with MPZL1 binding sites. This mechanism could occur at the membrane surface (within the same cell membrane or two adjacent membranes) or within the cell, though the latter seems to also lead to a decrease in the total amount of MPZL1 as observed in our transduction experiment, possibly through a mechanism similar to receptor interference. Syncytin-Mab1 expressing cells did not

3.3

. . Syncytin-likes in epitheliochorial placentation show increased activation of molecules situated downstream from MPZL1 (Src, ERK1/2). This might be due to a relatively small contribution of MPZL1 signaling to the total amount of phosphoryated effector [START_REF] Jia | Amplification of MPZL1/PZR promotes tumor cell migration through Src-mediated phosphorylation of cortactin in hepatocellular carcinoma[END_REF] report that Src phosphorylation increase by as little as 15% in their assays), or to the fact that expression of Syncytin-Mab1 and MPZL1 in the same cell leads to degradation of the latter following activation, lessening its potential downstream impact. Similarly, transwell migration and invasion assays using transduced MCF10A cells failed to reveal a significant effect of MPZL1, for potentially the same reasons.

The case of Syncytin-Mab1 and MPZL1 is of particular interest in the general study of syncytins and their effects on host physiology, suggesting that the role of these genes can depend as much on their intrinsic properties as on those of their host partner. Accordingly, large-scale efforts to identify the cognate receptors of syncytins are underway and have already led to the identification of the Syncytin-A receptor Ly6E by [START_REF] Bacquin | A Cell Fusion-Based Screening Method Identifies Glycosylphosphatidylinositol-Anchored Protein Ly6e as the Receptor for Mouse Endogenous Retroviral Envelope Syncytin-A[END_REF]. The identification of more receptors might shed additional light on the non-fusogenic involvement of previously described syncytins and syncytin-likes. Since the activation of MPZL1 by Syncytin-Mab1 is probably dependent on receptor aggregation and not fusion, even soluble syncytin-likes might affect host physiology through receptor interaction. It has to be kept in mind however that most of these envelopes are of the gamma-type, which mostly use multiple pass transporters as receptors, probably playing a less direct role in cell signaling processes. Still, Env-receptor interaction is often accompanied by down-regulation of the latter at the cell membrane, having a direct impact on cell physiology by modifying transport capabilities.

Syncytin-likes in epitheliochorial placentation

Among mammalian placentas, epitheliochorial placentation is the only type in which no syncytialized tissue is observed. This type of placenta is observed in a number of species with a high economic importance such a pigs and horses, and represents the last mammalian placentation type in which the involvement of syncytins has not been investigated. Additionally, many of the simpler non-mammalian placentas most closely resemble the epitheliochorial type, with its relatively low amount of invasion and comparatively simple structure.

While the absence of fused cell layers might suggest that syncytins do not play a role in this type of placenta, ERV Env could theoretically play a number of different roles, and the ancestral mammalian syncytin was probably not selected for fusogenicity, as discussed above. It is therefore not unreasonable to propose that syncytin-like genes might play an important role in epitheliochorial placentation, as they probably do in all other types of mammalian placentation.

Being vertebrate, epitheliochorial species have certainly not been spared from retroviral insertion events, and in silico screens of the published horse and pig genome reveal a number of potential candidates for syncytin-like functions, some of which even seem related to previously described syncytin genes. The fact that syncytins can be expressed in fused but also nonfused layers (as is the case of the presently described Syncytin-Mab1, but also Syncytin-1

3.3

for example) indicates that absence of cell-cell fusion does not necessarily imply absence of syncytins, and some epitheliochorial placentas, like that of horse, present differentiated and invasive cell subpopulations, two processes that implicate syncytins and syncytin-likes in other mammalian placentas.

While the investigation of the implication of retroviral envelopes in these placentas will probably be much more complicated, since the non-fusogenic roles proposed for these envelopes are harder to demonstrate and less well-known, these studies nevertheless seem crucially important in order to really understand the potential contributions of ERVs to all of mammalian and non-mammalian placentation.

Conclusion

In the present study we have investigated the implication of syncytins and syncytin-like genes in two exceptional placentas. The placenta of hyenas shows an extremely invasive phenotype when compared to that of other carnivorans, while the neotropical Mabuya lizard genus presents one of the few truly complex non-mammalian placentas.

The acquisition of Hyena-Env2 correlates in time with the major structural transition of the placenta of Hyaenidae, while the characteristics and expression of the previously present Syncytin-Car1 do not display any signifcant changes. Hyena-Env2 was shown to be present exclusively in Hyaenidae and conserved in a coding state in all extant members of this family, at a syntenic chromosomal locus. This species-specific acquisition at the same time as a major species-specific structural transition in the placenta suggests that Hyena-Env2 serves as an addon to Hyaenidae placentation, adding its functionality to that of Syncytin-Car1 and leading to the establishment of the invasive hemochorial placenta that is exclusive to this family among carnivorans.

Syncytin-Mab1 is the first identified non-mammalian syncytin, extending this now commonly admitted concept beyond simply mammals, and suggesting that the use of novel genes of retroviral origin might be a wide-spread phenomenon in animals. The use of syncytins for the establishment of placental structures in very distantly related species is a remarkable example of convergent evolution and strengthens the hypothesis that emergence of complex placentas necessitates the capture of a founding syncytins. The presently identified non-mammalian syncytins might be the first example of this group, having been acquired in the same timespan as that of placental emergence. Additionally, Syncytin-Mab1 seems to be a unique envelope associating properties from different retroviral clades and the identification of its receptor suggests an implication in placental invasion as well as cell-cell fusion.

The identification in the present study of several new endogenous retroviral envelopes contributes to the already known diversity and importance of these genes in placentation, but also extends these concepts to new contexts, reinforcing their involvement in placental structural organization and the emergence of new placentas. Les syncytines canoniques sont des gènes conservés au cours de l'évolution, exprimés dans le placenta et capables de faire fusionner les cellules en interagissant avec leur récepteur spécifique (fusiogénicité). Cette fusiogénicité est une propriété clé des syncytines qui sont responsables de la formation du syncytiotrophoblaste (ST), une couche de cellules placentaires fusionnées formant l'interface materno-foetale. Leur présence a été démontrée dans de nombreuses familles de mammifères et leur inactivation chez des souris KO conduit à un défaut de formation des syncytia de l'interface materno-foetale, provoquant un retard de croissance ou la mort des embryons.

Part IV

Bibliography

Les syncytines des différentes familles sont issues d'endogénisations indépendantes, ce qui pourrait contribuer à la diversité des placentas, l'organe le plus variable chez les mammifères. Un des grands critères de variabilité de la structure placentaire est le degré d'invasivité des tissus foetaux. En effet on peut classer les placentas de mammifères en 3 grandes catégories : épithéliochorial/synépithéliochorial, endothéliochorial et hémochorial, du moins invasif au plus invasif. Chez les espèces hémochoriales, comme l'homme par exemple, l'épithélium utérin maternel est entièrement dégradé suite à l'invasion par le tissu placentaire foetal, permettant un contact direct du ST foetal avec le sang maternel, alors que chez les espèces épithéliochoriale comme le cheval, les tissus maternels et foetaux sont simplement apposés.

Recherche de syncytine chez les lézards placentaires Mabuya. À la suite de l'identification de syncytines dans la plupart des clades de mammifères, nous avons étudié l'implication de ces gènes dans la placentation d'espèces non-mammifères. La placentation n'est pas un phénomène restreint aux mammifères, un certain nombre lézards par exemple présentent un placenta. Parmi ces derniers, le genre Mabuya présente un des placentas non-mammifères les plus complexes connus à ce jour. Dû à sa grande similarité structurelle aux placentas des mammifères et notamment à la présence d'une couche de cellule fusionnées à l'interface materno-foetale, nous avons cherché à identifier un gène d'env domestiqué, fusiogène et à expression placentaire comme c'est le cas pour les syncytines de mammifère.

Comme aucun génome de Mabuya n'a été séquencé, nous avons établi un transcriptome par séquençage à haut-débit d'ARN placentaire de Mabuya sp. IV. Un criblage in silico de ce transcriptome nous a permis d'identifier quatre phases ouvertes de lecture (ORFs) codant pour des env rétrovirales que nous avons appelées Mab-Env1 à 4. Ces nouvelles env peuvent être réparties en deux groupes, Mab-Env1 et 2 présentant de fortes similarités, tout comme Mab-Env3 et 4. Les 4 séquences identifiées présentent tous les domaines classiques d'enveloppes rétrovirales et sont proches des enveloppes de type gamma sur un arbre phylogénétique des sous-unités transmembranaires (TM). Mab-Env3/4 sont proches de la TM du virus du sarcome de Rous (RSV) et présentent des caractéristiques d'enveloppes de type gamma aviaires : un peptide de fusion décalé et encadré par deux cystéines. Mab-Env1/2 présentent une séquence plus particulière qui associe la présence d'un domaine immunosupresseur (ISD) de type gamma et un motif CX7C de type beta. Leur position sur l'arbre phylogénétique est proche des TM de type gamma, suggérant que leur phénotype est principalement gamma. Une investigation plus approfondie de Mab-Env1 a permis l'identification du domaine conservé reverse transcriptase (RT) associé à ce gène et a permis de construire un arbre phylogénétique basé sur ce domaine qui place Mab-Env1 dans une position intermédiaire entre les gammarétrovirus et tous les autre genres orthorétroviraux. Des analyses de RT-qPCR ont montré que bien qu'il ne soit pas exprimé exclusivement dans le placenta, Mab-Env1 présente un niveau d'expression significatif dans ce tissu, alors que Mab-Env2 à 4 sont faiblement exprimées dans tous les tissus de notre panel. Pour étudier le profil d'expression de Mab-Env1 au niveau tissulaire, nous avons réalisé des expériences d'hybridation in situ ainsi que d'immunohistochimie (utilisant des anticorps que nous avons produits en souris). Dans le placentome, Mab-Env1 est exprimé dans les deux tissus formant l'interface materno-foetale, mais son expression est plus importante dans le tissus maternel syncytialisé que le tissu foetal, qui est constitué de cellules isolées. Dans le paraplacentome, qui ne présente pas de couche fusionnée, Mab-Env1 est fortement exprimé à l'interface entre les tissus maternels et foetaux. Ce profil d'expression est en accord avec un potentiel rôle physiologique de Mab-Env1 à l'interface materno-foetale.

Pour étudier la conservation de Mab-Env1 au sein du genre Mabuya, nous avons essayé d'amplifier l'ORF entière ainsi qu'un fragment interne de 400 paires de bases par PCR au sein d'autres espèces de Scincidae. L'amplification de l'ORF entière n'a été possible qu'à partir de génomes de Mabuya, suggérant que Mab-Env1 n'a été conservée qu'au sein de ce clade. L'amplification du fragment interne a également été observée à partir des génomes de quatre espèces proches du genre Mabuya, suggérant qu'un provirus contenant Mab-Env1 a eu lieu, mais que l'enveloppe n'a pas été conservé dans ces espèces. Ayant établi que Mab-Env1 est exprimé dans le placenta et conservé chez toutes les espèces de Mabuya testées, nous avons ensuite analysé son pouvoir fusiogène, la troisième caractéristique des syncytines de mammifères. Des tests de fusiogénicité ex vivo ont montré que des virus MLV pseudotypés avec Mab-Env1 étaient hautement infectieux dans de nombreuses cellules mammifères, avec l'exception des cellules de rongeur. Dans des tests de fusion cellule-cellule, Mab-Env1 n'est fusiogène qu'après un choc acide, comme cela avait déjà été observé dans le cas de Syncytin-Rum1.

Le tropisme conféré à des particules virales par Mab-Env1 nous a permis de chercher son récepteur spécifique en utilisant les clones d'hybrides d'irradiation humain/hamster Genbridge4, qui avaient déjà permis l'identification du récepteur spécifique de la Syncytin-2 humaine. Nous avons pu identifier MPZL1 (un transducteur de signal à un domaine transmembranaire) comme récepteur de Mab-Env1. Après clonage du MPZL1 de Mabuya, nous avons pu montrer que son expression à la surface de cellules de rongeur permettait bien leur infection par des particules MLV pseudotypées avec Mab-Env1. L'expression de MPZL1 a été étudiée par RT-qPCR et hybridation in situ. De façon similaire à ce qui avait été observé pour Mab-Env1, MPZL1 montre un niveau d'expression significatif dans le placenta, mais également dans d'autre tissus. L'hybridation in situ montre que MPZL1 est fortement exprimé dans les couches maternelles de l"interface materno-foetale, à la fois dans la couche fusionnée du placentome et la couche non-fusionnée du paraplacentome. Ce profil d'expression est en accord avec une interaction entre Mab-Env1 et MPZL1 in vivo, les deux étant exprimés soit dans la même couche cellulaire (placentome) ou dans deux couches adjacentes mais distinctes (paraplacentome).

Des études précédentes ont montré que chez les mammifère MPZL1 était impliqué dans des phénomènes de migration et d'invasion cellulaires. Pour étudier si l'interaction entre Mab-Env1 et MPZL1 pouvait mener à l'activation de ce dernier, nous avons transduit des cellules non-transformées MCF10A afin qu'elles expriment Mab-Env1. Nous avons pu montrer que l'expression de l'enveloppe était suffisante pour induire la phosphorylation de MPZL1, une étape importante pour la transduction de signal par cette protéine.

Pour conclure, Mab-Env1 est exprimée dans le placenta, conservée au cours de l'évolution et fusiogène, elle présente donc les trois caractéristiques des syncytines mammifère et a été nommée syncytin-Mab1. Syncytin-Mab1 est clairement différente de toutes les syncytine décrites précédemment et a été domestiquée dans un clade très éloigné de celui des mammifères. Cette domestication d'une enveloppe d'ERV pour un potentiel rôle dans le placenta est un exemple remarquable de convergence évolutive et suggère que ce phénomène pourrait avoir lieu dans de nombreux placentas non-mammifères. De plus, l'identification du récepteur de Syncytin-Mab1 suggère une implication de cette dernière dans l'invasion placentaire, un rôle qui a été précédemment suggéré pour les syncytines mammifères ainsi que les gènes de type syncytine.

Recherche d'env impliquées dans l'établissement du placenta atypique de hyène. Comme tous les autres carnivores, les hyènes expriment la syncytine Syncytin-Car1, mais leur placenta présente une structure très différente de celle de ses congénères. Le placenta des Hyaenidae est un placenta fortement invasif du type hémochorial, les tissus maternels étant entièrement dégradés et les tissus foetaux baignant dans le sang maternel. Le placenta de tous les autres carnivores est du type endothéliochorial, dans lequel les vaisseaux foetaux restent intact jusqu'à terme. La transition phénotypique du placenta des hyènes a dû avoir lieux relativement récemment, étant donné que cette famille s'est séparée du reste des carnivores il n'y a que 30 millions d'années.

Pour étudier l'implication d'env d'ERV dans cette transition, et comme aucun génome de hyène n'a été publié, nous avons réalisé un séquençage haut débit des ARN placentaire de la hyène tachetée (Crocuta crocuta). Nous avons ensuite criblé ce transcriptome in silico et identifié trois ORFs d'env entières exprimées dans le placenta. Comme attendu, l'une d'elle s'est avéré être la Syncytin-Car1 précédemment décrite. Les deux autres enveloppes, Hyena-Env2 et Hyena-Env3 sont différentes des syncytines décrites précédemment, bien que Hyena-Env3 ressemble à la non-syncytine Canis-Env3 du chien. L'analyse phylogénétique des deux enveloppes les identifie comme des enveloppes de type gamma.

Comme la transition de structure placentaire investiguée n'a eu lieu qu'au sein de la famille de Hyaenidae, nous avons étudié l'état de conservation de ces deux nouvelles enveloppes au sein des carnivores. L'amplification par PCR de l'ORF ou d'une partie de l'ORF de Hyena-Env2 n'est possible qu'à partir du génome d'une des quatre espèces de hyène, indiquant que ce gène est bien spécifique des hyènes. De plus, la forte conservation de la séquence codante au sein des Hyaenidae (>98.4% d'identité de nucléotides) ainsi que le ratio de mutation non-synonyme sur synonyme faible suggère que Hyena-Env2 est sous sélection purifiante parmi les hyènes. Par opposition, Hyena-Env3 a pu être montrée comme présente dans de nombreux génome de Caniformia, par PCR ainsi que par analyse in silico. Une comparaison plus approfondie de Hyena-Env3 et Canis-Env3 a montré que cette dernière a perdu son peptide signal, un domaine essentiel pour la synthèse des enveloppes rétrovirale, elle peut donc être considérée comme non-fonctionnelle. Un provirus de chien contenant une copie mutée de Canis-Env3 présente deux LTR identiques à 100%, suggérant que le rétrovirus associé à Hyena-Env3/Canis-Env3 a été actif récemment ou est encore actif. Comme Hyena-Env2 est la seule enveloppe identifiée spécifique aux hyènes, nous l'avons privilégié dans notre étude.

Nous avons pu identifier le locus d'insertion du provirus contenant Hyena-Env2 chez Crocuta crocuta et avons réussi à amplifier le provirus entier de 9 kb. La duplication de la cible est apparente des deux côtés du provirus et les LTRs présente une identité de séquence de 89%, en accord avec une intégration relativement récente. Les ORFs de gag et pol sont interrompues pas plusieurs mutations. Le domaine RT conservé associé à Hyena-Env2 a pu être identifié et suggère une origine gammarétrovirale. Des analyses in silico et PCR montrent que ce provirus est bien intégré à ce locus dans toutes les espèces de hyènes et que le locus est vide chez tous les autres carnivores testés. Le séquençage du transcrit placentaire exprimant Hyena-Env2 montre qu'il présente l'épissage canonique des enveloppes rétrovirales, mais qu'un autre épissage a lieu en amont de ce dernier et que l'initiation du transcrit a lieu dans un probable ilot CpG du génome hôte. L'orthologue de cet ilot CpG chez le chat est bien un site d'initiation de transcrits cellulaires.

Le profil d'expression placentaire des trois env de hyène a été étudié par hybridation in situ et a montré que Syncytin-Car1 est bien exprimée de façon spécifique dans le ST, comme c'est le cas dans le chat et chien. Hyena-Env3 semble être également exprimée dans cette couche, mais le faible signal, potentiellement du au faible niveau d'expression de cette enveloppe, rend la conclusion difficile. Pour Hyena-Env2, la sonde sens contrôle présente un signal intense, indiquant la présence d'un transcrit antisens dans le placenta de hyène. Néanmoins, la sonde antisens produit un signal spécifique de la couche non-fusionnée sous-jacente au ST. Ce résultat a également été confirmé par des expériences d'immunohistochimie utilisant des anticorps mifères suggère que la capture d'env est une force évolutive majeure dans ces deux processus. La description d'un nouveau gène de type non-fusiogène conservé au cours de l'évolution renforce la notion que le rôle des env capturées n'est pas uniquement fusiogène, mais qu'elles peuvent également être sélectionnées pour d'autres processus importants dans le cadre de la placentation, notamment l'invasion et la migration cellulaire. En plus de son rôle fusiogène, Syncytin-Mab1 pourrait également être impliquée dans ces processus, notamment par interaction avec son récepteur MPZL1 que nous avons pu identifier, et qui a été impliqué précédemment dans la régulation de la migration et l'invasion cellulaire. Les récepteurs de la plupart des syncytines ne sont pas encore connus et leur identification pourrait, comme cela a été le cas ici, être une façon d'étudier un éventuel rôle non-fusiogène de ces gènes placentaires. En plus d'être la première syncytine non-mammifère décrite à ce jour, Syncytin-Mab1 est potentiellement la première syncytine fondatrice identifiée. L'hypothétique syncytine associée à l'émergence du placenta des mammifères est probablement trop dénaturée pour être identifiée, cette émergence remontant à environ 175 millions d'années. L'émergence bien plus récente du placenta de Mabuya (25 millions d'années) nous a permis d'identifier une syncytine capturée de façon simultanée à cette émergence et qui pourrait donc avoir contribué directement à l'apparition du placenta dans ce clade particulier de lézards.

Université Paris-Saclay Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint-Aubin, France Titre : Identification et caractérisation de deux nouveaux gènes d'enveloppes rétrovirales de type syncytine, capturées pour un possible rôle dans la structure atypique du placenta de hyène et l'émergence du placenta non-mammifère des lézards Mabuya Mots clés : rétrovirus endogène, syncytine, protéine d'enveloppe, placenta, récepteur Résumé : Les syncytines sont des gènes d'enveloppes rétrovirales (env) capturés qui sont essentiels pour l'établissement du placenta chez les mammifères. Il a été proposé que la diversité des syncytines capturées explique pourquoi le placenta est l'organe le plus variable chez les mammifères. Ici nous avons employé deux approches pour étudier le lien entre la capture d'env et l'émergence et la diversité des structures placentaires. D'abord, nous avons étudié la placentation des Hyaenidae, les seuls carnivores à présenter un placenta très invasif hémochorial, comme l'humain. Comme tous les carnivores, les hyènes expriment la syncytin-Car1 précédemment décrite, mais nous avons identifié une nouvelle env, capturée uniquement chez ces dernières, que nous avons nommée Hyena-Env2. Ce nouveau gène est présent au même locus chez toutes les hyènes, ayant été capturé pendant la radiation de la famille. Il est non-fusiogène mais a néanmoins été conservé pendant plus de 10 millions d'années et est exprimé à l'interface materno-foetale du placenta, ce qui en fait un gène candidat pour expliquer le passage à la placentation hémochoriale qui a eu lieu chez les Hyaenidae. Title: Identification and characterization of two novel syncytin-like retroviral envelope genes, captured for a possible role in the atypical structure of the hyena placenta and in the emergence of the non-mammalian Mabuya lizard placenta Keywords : endogenous retrovirus, syncytin, envelope protein, placenta, receptor Abstract: Syncytins are captured retroviral envelope genes (env) that are essential for the establishment of placental structures in mammals. The syncytins present in different mammalian families are highly diverse, resulting from distinct capture events, and it has been suggested that this might play a role in making the placenta the most diverse structure in mammals. Here we used two different approaches to investigate the links between env capture and emergence and diversity of placental structures. First, we investigated placentation in Hyaenidae, the only carnivorans that present a highly invasive hemochorial placenta, as is also found in humans. Hyenas express the previously identified syncytin-Car1 gene, as do all carnivorans, but we identified a new hyena-specific captured env that we named Hyena-Env2. This new gene is present at the same locus in all hyenas, having been captured during the radiation of this family. It is nonfusiogenic but still conserved over at least 10 million years of evolution and expressed at the materno-fetal interface in the hyena placenta, making it a candidate gene for explaining the endotheliochorial to hemochorial placental transition that occurred in Hyeanidae.

Second, we searched for syncytin-like genes in the nonmammalian Mabuya lizards, which are viviparous and present a rare type of highly complex placenta that is very reminiscent of mammalian placentas. We identified an env gene that was captured and conserved in this genus since its radiation 25 million years ago. This gene, that we named syncytin-Mab1, is able to mediate cell-cell fusion in vitro and is expressed in a fused cell layer at the materno-fetal interface of the placenta in vivo, characteristic features of canonical mammalian syncytin genes. We also identified the cellular gene MPZL1 as the cognate receptor of syncytin-Mab1 and showed that their interaction induces activation and phosphorylation of the former. MPZL1 activation has been linked with cell migration and invasion, indicating that this env-receptor interaction could play a role in the placental invasion of maternal tissues observed in Mabuya. In conclusion, the characterization of these two novel env genes indicates that syncytin-like env might have played a role both in the emergence of the Mabuya placenta and the atypical placental structure of hyenas, reinforcing the notion that env capture is a major driving force in evolution.

1 2 2 2 Retroviruses 2 . 1 2 1 . 2 A 2 . 3 3 . 1 3 . 2

 122221212233132 Challenges of viviparity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.1 Maternofetal exchanges . . . . . . . . . . . . . . . . . . . . . . . . . . Placentation and the maternal immune system . . . . . . . . . . . . . . a. Major histocompatibility complex expression b. Uterine natural killer cells c. Adaptive immunity 1.1.3 Maternofetal communication . . . . . . . . . . . . . . . . . . . . . . . a. Placental hormones b. Extracellular vesicles 1.2 Mammalian eutherian placentas . . . . . . . . . . . . . . . . . . . . . . . . 1.2.1 Origin and evolution of the mammalian placenta . . . . . . . . . . . . a. Monophyletic origin b. Placental diversity 1.2.2 Placental types by maternofetal interface . . . . . . . . . . . . . . . . . a. Epitheliochorial placentation b. Synepitheliochorial placentation c. Endotheliochorial placentation d. Hemochorial placentation 1.3 Viviparity in non-mammalian species . . . . . . . . . . . . . . . . . . . . . 1.3.1 Prevalence of viviparity outside of mammals . . . . . . . . . . . . . . Examples of non-mammalian placentas . . . . . . . . . . . . . . . . . a. Non-vertebrates: the placenta of salps b. Fish: placentation in sharks c. Lizards: placentation in Mabuya and other Scincidae Exogenous Retroviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1 Genomic orgarnisation . . . . . . . . . . . . . . . . . . . . . . . . . . a. Non-coding features b. Gag c. Pro and Pol d. Env e. Regulatory and accessory proteins 2.1.2 Retroviral phylogeny . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Subfamily Orthoretrovirinae b. Subfamily Spumaretrovirinae 2.1.3 Viral cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Retroviruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1 Endogenization of retroviral sequences . . . . . . . . . . . . . . . . . . a. Infection of germinal cells b. Intra-genome spreading of ERVs c. Groups of ERVs 2.2.2 Impact of retroviral integration on the host . . . . . . . . . . . . . . . . a. LTR integration can drive ectopic gene expression b. Impact of retroviral gene acquisition on host physiology 2.2.3 Fate of ERVs and control by the host . . . . . . . . . . . . . . . . . . . a. Epigenetic regulation of ERV expression b. Accumulation of mutations and solo LTR formation c. Exaptation and purifying selection 3 Syncytins 3.1 Retroviral envelopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi 3.1.1 Functional domains . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Domains of the SU b. Domains of the TM 3.1.2 Env induced membrane fusion . . . . . . . . . . . . . . . . . . . . . . a. Implication of the cognate receptor in fusion b. Model of viral fusion protein class I induced membrane fusion 3.2 Syncytins in mammals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1 Human syncytins: Syncytin-1 and Syncytin-2 . . . . . . . . . . . . . . Mouse syncytins: Syncytin-A and Syncytin-B . . . . . . . . . . . . . . a. Discovery b. Expression in 2 segregated layers c. Impact of Syncytin knockout on placental physiology 3.2.3 Other mammalian syncytins . . . . . . . . . . . . . . . . . . . . . . . a. Rabbit: Syncytin-Ory1 b. Cat and dog: Syncytin-Car1 c. Old world monkey: EnvV2 d. Cattle and sheep: Syncytin-Rum1 and Fematrin-1 e. Squirrel and marmot: Syncytin-Mar1 f. Tenrec: Syncytin-Ten1 g. Short-tailed opossum: Syncytin-Opo1 3.2.4 Syncytin-like Env . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. Conserved ERV Env with placental expression in human b. Non-human syncytin-like genes 3.3 Syncytins and evolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1 Ancestral syncytin and syncytin replacement . . . . . . . . . . . . . . a. Example of EnvV2, a decaying syncytin in humans b. Baton pass hypothesis 3.3.2 Syncytin diversity and placental diversity . . . . . . . . . . . . . . . . 3.3.3 Other roles of syncytins . . . . . . . . . . . . . . . . . . . . . . . . ERV env genes in non-mammalian Mabuya placentation 1.1 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . syncytin in a placental lizard . . . . . . . . . . . . . . . . . . . . . . . . . 2 Implication of ERV env genes in placental structural transitions in Hyaenidae 2.1 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Hyena-specific retroviral envelope gene capture and placenta evolution . . . III Discussion 1 Two novel Env with a potential placental role and a new SLC1A5 Env168 1.1 Hyena-Env2 is a conserved syncytin-like envelope gene expressed during hyena placentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Hyena-Env3 is a member of the SLC1A5 interference group with a peculiar tropism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Syncytin-Mab1: characterization of a non-mammalian syncytin and its receptor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . and convergent evolution of animal placentas . . . . . . . . 2.2.1 Convergent use of ERV env genes in placentation . . . . . . . . . . . . 2.2.2 Syncytin-Mab1 as the first example of a founding syncytin . . . . . . . Syncytins and syncytin-likes and divergent evolution of the mammalian placenta . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Syncytin-likes and alternative envelope functions in the placenta Growing evidence of conservation of non-fusogenic Env . . . . . . . . . . . Alternative placental roles for Env . . . . . . . . . . . . . . . . . . . . . . . 3.2.1 Immunosuppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2 Differentiation and proliferation . . . . . . . . . . . . . . . . . . . . . 3.2.3 Invasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3 Syncytin-likes in epitheliochorial placentation . . . . . . . . . . . . . . . . .

Figure 1 -

 1 Figure 1 -Dynamics of glucose and amino-acid transport. Schematic representation of placental transport of glucose (top) and amino acids (bottom) and implication of the placental metabolism. Examples of amino-acids involved in placental metabolism are given as one letter codes and based on the example of sheep. Concentration gradients are represented as black wedges.

Figure 2 -

 2 Figure 2 -Timed tree of major mammalian clades. Placental clades are indicated in red, branch length is proportional to time, see scale at the bottom (dos Reis et al., 2012). A red arrow marks the emergence of the mammalian placenta.

Figure 3 -

 3 Figure 3 -Diversity of eutherian placentas as illustrated by several classification systems (A) Classification by gross morphology. In many eutherian placentas (except diffuse) there are distinct regions specialized for maternofetal exchanges . The shape and distribution of these regions can help separate placentas into distinct categories. Areas specialized for maternofetal exchange are indicated in dark red. (B) Classification by interdigitation. While all placentas interdigitate with maternal tissues, the complexity of the interdigitation is variable and four major categories can be established. Orange and F indicate fetal tissues while yellow and M indicate maternal tissues. (C) Classification by vascular arrangement. The relative disposition of maternal and fetal blood vessels has a major impact on the efficiency of maternofetal exchanges. Fetal blood vessels are represented in red, maternal vessels in pink. The qualitative concentration of an imaginary solute is indicated on the schemes when possible and arrows denote transfer of the solute, thickness indicates amount transferred.

Figure 4 -

 4 Figure 4 -Schematic representation of the four types of maternofetal interface in eutherian mammals and their repartition on the species tree. (A) Schematic representations of the four types of maternofetal interfaces found in eutherians, arranged in order of invasiveness of fetal tissues. A line delimits the maternal and fetal side. (B) Placental interface types are distributed paraphyletically.For the latter the placental type is indicated using the same colors as the frames in (A) and the presence or absence of fused cells is indicated by a plus or minus respectively. Branch length is proportional to time as indicated in the scale below the tree[START_REF] Dos Reis | Phylogenomic datasets provide both precision and accuracy in estimating the timescale of placental mammal phylogeny[END_REF].

Figure 5 -

 5 Figure5-Schematic representation of the fusion processes observed between fetal BNC and maternal epithelium in ruminant placentas. (A) Fetal BNC mature between two trophoblast cells before extending cytoplasmic projections towards the maternofetal interface, replacing adherent junctions with their own in the process. Following fusion, the BNC cytoplasm mixes with the maternal epithelial cell cytoplasm and the BNC granules are transferred to the basal membrane where they secrete their contents. The trophoblast absorbs the rest of the BNC membrane and re-establishes adherent junctions. (B) In sheep type epitheliochorial placentation, BNC can fused into an already existing heterologous syncytium to form syncytial plaques. The mechanism is the same as for the formation of trinuclear cells. Adapted from[START_REF] Cornelis | Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants[END_REF]

Figure 6 -

 6 Figure 6 -Remodeling of maternal blood vessels by extravillous trophoblast cells in humans. (A) Schematic representation of maternal uterine blood vessels before remodeling. The vessel is narrow and surrounded by a muscle sheath around its whole length. (B) Maternal blood vessel post-remodeling. The endovascular extravillous trophoblasts (EVT; green) have invaded the opening of the vessel, widening it to alter blood flow and speed. The interstitial EVT (blue) invade the maternal tissues to remove the arterial muscle sheath and reduce vasoreactivity. The EVT are assisted and regulated during this process by the maternal uNK cells (white). (C) Representation of the relative positions of fetus, placenta, maternal tissues and maternal blood vessels during human pregnancy. Adapted from Moffett & Loke (2006); Parham & Moffett (2013).

Figure 7 -

 7 Figure7-Matrotrophic viviparity is a widespread phenomemon among Animalia. Cladogram of Animalia with phyla containing matrotrophic species in red. The number after the taxon name represents the conservative estimate of origins of viviparity, the bar the number of families containing at least one matrotrophic species (truncated for Chordata and Platyhelminthes) and the number following the bar is the number of families containing at least one matrotrophic species compared to the total number of estimated families of the phylum. (inset) Detail of the origins and prevalence of matrotrophy in selected Chordata clades, using the same representation as the main cladogram. Adapted from[START_REF] Ostrovsky | Matrotrophy and placentation in invertebrates: A new paradigm[END_REF].

Figure 8 -

 8 Figure 8 -Structure of the Mabuya sp. IV placenta.(A) Overview of a gravid uterus displaying the apposed maternal and fetal tissues, as well as regions specialized for maternofetal exchanges: the placentome, absorptive plaques and areola. The paraplacentome forms a band around the placentome and is therefore present on both sides. (B) Detailed representation of the maternofetal interface in the placentome and paraplacentome region. In the placentome, maternal and fetal tissues are highly interdigitated, with numerous microvilli between fetal and uterine cells. The fetal epithelium is formed primarily by giant binucleated chorionic cells. The uterine epithelium is replaced by a large syncytial structure formed by the fusion of the uterine cells. In the paraplacentome the syncytium is abruptly replaced by uterine epithelium. Cells still present microvilli, but the tissues are no longer interdigitated. (C) Detail of cellular invasion in the mabuya placenta. (left) Electron micrograph of the complex double membrane system originating from fetal invasive cell projections. (right) Representation of the double membrane projections formed around maternal blood vessels by the fetal invasive cells, using the same color scheme as in (A) and (B). Adapted fromVieira et al. (2007);Wooding et al. (2010).

Figure 9 -

 9 Figure 9 -Organization of retroviral virions and genomes. (A) Micrographs and schematic representation of the immature and mature virion of HIV-1, showing the arrangement of canonical retroviral proteins and the RNA genomes in the particle. Scale bar 50 nm, adapted from Konvalinka et al. (2015). (B) Structural organization of retroviral genomes. For each family the organization of the LTRs and the canonical retroviral ORFs is represented to scale (see bottom), using the indicated type species. The order of subunits is indicated on the first genome and conserved in all others, faded boxes represent spacer or non-canonical subunits, accessory and regulatory genes are not displayed. Arrows with FS represents synthesis of Pro or Pol by frameshift, with R-T by read-through (see2.1.3.d.). Adapted from viralzone.expasy.org

HIV- 1 .Figure 10 -

 110 Figure 10 -Organization and transcripts of the complex HIV-1 genome. (top) Arrangement of canonical and non-canonical ORFs in the HIV-1 genome, using the same color code as Fig. 9, with the addition of accessory and regulatory ORFs in light blue. (bottom) Canonical (a-b) and non-canonical (c-g) retroviral transcripts expressed by the HIV-1 genome, to scale with the latter. Dotted lines represent splicing events and transcripts are sorted into unspliced (a), partially spliced (b-d) or fully spliced (e-g) categories. Adapted from viralzone.expasy.org

Figure 11 -

 11 Figure 11 -Phylogeny of retroviral genera based on RT or TM analysis. (A) Unrooted RT-based PhyML tree using selected retroviral species, with RT-based genera indicated on the tree. The tree was obtained with the PhyML program and the RtREV+Γ+I model selected by the Smart Model Selection program(Lefort et al., 2017). (B) Unrooted TM-based tree using selected retroviral species with RT-based genera indicated on the tree. TM type is indicated in gray alongside the associated CX n C 1-2 motif and the presence or absence of the CKS-17 ISD (see also Fig.22). The tree was obtained with the PhyML program and the WAG+Γ+I+F model, selected by the Smart Model Selection program(Lefort et al., 2017). For both trees, branch length is proportional to the average number of substitutions per site (see scale below each tree), and RT/TM recombinant retroviruses are highlighted in red. BFV: bovine foamy virus, EFV: equine foamy virus, FFV: feline foamy virus, SFV: simian foamy virus, REV: reticuloendotheliosis virus, RD114: RD-114 feline retrovirus, GaLV: gibbon ape leukemia virus, KoRV: koala retrovirus, FLV: feline leukemia virus, MLV: murine leukemia virus, WEHV: walleye epidermal hyperplasia virus, WDSV: walleye dermal sarcoma virus, BLV: bovine leukemia virus, PTLV: primate T-lymphotropic virus, HIV: human immunodeficiency virus, CAEV: caprine arthritis encephalitis virus, EIAV: equine infectious anemia virus, ALV: avian leukosis virus, RSV: Rous sarcoma virus, AMV: avian myeloblastosis virus, MMTV: mouse mammary tumor virus, JSRV: Jaagsiekte sheep retrovirus, MPMV: Mason-Pfizer monkey virus, SMRV: squirrel monkey retrovirus

Figure 12 -

 12 Figure12-Representation of the retroviral replication cycle. The infecting virion attaches to the host cell membrane through Env-receptor interaction which also triggers fusion and viral entry. In the cytoplasm, the RNA genome is reverse transcribed into DNA within the reverse transcription complex (RTC). The DNA genome is uncoated (dissociation of the capsid) forming the pre-integration complex (PIC), which is imported into the nucleus. In the nucleus the viral genome is integrated into the cellular genome and viral transcripts are expressed by the host machinery. Viral proteins are synthesized in the cytoplasm or the endoplasmic reticulum/Golgi (Env only) and are addressed towards the plasmic membrane. At the membrane the Gag containing polyproteins assemble and bind two copies of viral genomic RNA. The assembly buds and takes with it an Env containing fragment of the cellular membrane, releasing an immature virion. Finally, PR provokes virion maturation which yields a new infectious virion. Colors are the same used in Fig.9.

Figure 14 -

 14 Figure 14 -Schematic representation of the retroviral genome integration steps. Plus-strand viral DNA is shown in purple and minus-strand in red, cellular DNA in black, 5' ends of the strands are indicated. Sites of nuclophilic attack are show using arrowheads in the color of the implicated strand, and are shown as faded after the reaction. XXXXXX and YYYYYY indicate any complementary 6 nucleotide sequence. (top) 3' processing: IN binds both ends of the retroviral genome and cleaves a few nucleotides at the 3' ends, leaving a reactive hydroxyl group behind (-OH) (these nucleotides represent the sequence between the PPT and 3'LTR for the plus-strand and PBS and 5' LTR for the minus strand). (bottom) Strand transfer: IN binds the host genome alongside the processed viral DNA. Two bimolecular nucleophilic substitutions are catalyzed by IN, at 4-6 nucleotides from each other(6 here). This binds the 3' ends of the viral genome strands to the host genome. Cellular enzymes then repair the remaining single-strand nicks, cutting the viral 5' flaps (the complementary nucleotides to those lost during 3' processing), and filling the 4-6 bp gap, generating the TSD (gree frames). The representations are not to scale.

Figure 15 -

 15 Figure 15 -Representation of the ESCRT mediated budding model proposed by Lippincott-Schwartz et al. (2017). (A) Viral Gag assembles at the membrane, starting the budding process and recruiting cellular proteins of the ESCRT family. ESCRT-III nucleates and encircles the Gag lattice. (inset) Top view of the Gag/ESCRT assembly (B) Continued assembly of the Gag lattice increases the sphericity of the bud, ESCRT-III continues its nucleation. (C) ESCRT proteins remain associated with Gag and are included in the budding virion from where they regulate a continued assembly of ESCRT-III in the stalk. This assembly occurs in a circular pyramid of tighter and tighter circles, leading to an ESCRT-III mediated narrowing of the stalk. At this stage ESCRT-III can still freely diffuse between the viral and cellular compartments (white arrows). (D) The ESCRT-III assembly reaches it's tightest conformation and forms a closed dome. (E) This restricts the stalk to a diameter small enough for spontaneous membrane fission and release of the virion. Some proteins of the ESCRT system are trapped in the virion. Adapted from Lippincott-Schwartz et al. (2017).

Figure 16 -

 16 Figure 16 -PR mediated maturation of retroviral particles. (A) Examples of immature and mature virions of MPMV (betaretrovirus), HIV-1 (lentivirus) and MLV (gammretrovirus). MPMV maturation is visualized by thin-section electron microscopy, HIV-1 and MLV by cryo-election tomography, all scales bars 50 nm. (B) HIV-1 maturation sequence. (top) representation of the PR cleavage sequence of the HIV-1 Gag and Gag-Pro-Pol polyproteins, using the same colors as in Fig. 9. The size of the arrow is roportional to the rate of cleavage observed in vitro. NC-TF: transframe peptide obtained after frameshifting. (bottom) Schematic representation of the cleavage status of Gag and associated virion morphology. Spacer peptides and non-canonical Gag subunits are represented in dark gray. Adapted from Konvalinka et al. (2015).

Figure 17 -

 17 Figure 17 -Endogenization of exogenous retroviruses. Illustration of the horizontal and vertical transmission pathways of retroviral infection. Infection of somatic cells leads to horizontal transmission through virus production and re-infection of new individuals. This infection is not transmitted horizontally. Infection of germline cells leads to endogenization through horizontal transmission. Following endogenization, every cell contains the viral genome. Through continued reproduction and horizontal transmission events, the endogenous retrovirus can become fixed in the population, i.e., present in all individuals.

Figure 18 -

 18 Figure18-Mechanisms of ERV amplification and fate of ERV insertions. ERVs can amplify their copy number either through an extra-cellular (re-infection, see Fig.12) or intra-cellular pathway (retrotransposition). The latter is often accompanied by re-addressing of Gag and loss of Env. Each new insertion can be deleterious, neutral or advantageous to the host, influencing its chances of being lost or fixed in the populations. Continually vertically transmitted ERV copies face three main fates: solo LTR formation in which a recombination even excises all the viral genome, leaving a single LTR, genetic drift in which the ERV sequence accumulates mutations and indels, and purifying selection in which one or several ERV sequences are maintained functional following exaptation. Same color scheme as Fig.9, faded sequences represent loss of function.

Figure 19 -

 19 Figure 19 -ERVs disproportionately belong to certain retroviral genera. (A) Proportion of ERVs related to each of the major retroviral lineages and corresponding ERV class. Based on the analysis of 35975 ERV genomes in 65 animal species by[START_REF] Hayward | Pan-vertebrate comparative genomics unmasks retrovirus macroevolution[END_REF]. The "other" category contains unclassified retroviral species, often related to spumaviruses. (B) Detailed distribution of ERVs in different host genomes. The trend line indicates the total amount of ERV genomes identified in the species, the bars the proportion each lineage represents among the ERVs of the given genome. A cladogram of retroviral lineages and of the host species is provided as ordinate and abscissa respectively. For simplicity, ERVs not belonging to any of the seven major lineages are not represented. Adapted from[START_REF] Hayward | Pan-vertebrate comparative genomics unmasks retrovirus macroevolution[END_REF].

Figure 20 -

 20 Figure 20 -Mutagenic effects of LTR insertions. (top) Pre-insertion locus. Exon 1 and 2 are part of a transcribed gene, the spliced mRNA is represented in gray, spliced sequences as dotted line. (bottom)Post-insertion locus. In this example the inserted ERV contains LTRs presention a cryptic splice acceptor (sa) site, in addition to the canonical polyadenylation (polyA) and transcript initiation functions. The canonical retroviral splice donor (sd) and acceptor sites are also represented. Cellular transcription can be disrupted through several ways, three are presented here: aberrant splicing leads to addition of ERV sequences into the mRNA (green), premature termination leads to interruption of the transcript and polyadenylation (AAAAA) and abberant initiation leads to formation of incomplete transcripts. Adapted form[START_REF] Maksakova | Retroviral elements and their hosts: Insertional mutagenesis in the mouse germ line[END_REF].

Figure 21 -

 21 Figure 21 -Representation of the organization of gamma-type retroviral Env domains.(left) Relative position of the SU (yellow) and TM (orange) domains, from N-ter to C-ter: signal peptide (purple), receptor binding domain (RBD), proline-rich region (PRR), CXXC motif, furin-like cleavage site (RXKR), fusion peptide (green), heptad repeat 1 (HR1), immunosuppressive domain (ISD, blue), CX n CC motif, heptad repeat 2 (HR2), transmembrane region (red) and cytoplasmic tail (Ct). (right) Schematic representation of Env structure using the same color code. The CX n CC motif forms an intra-molecular disulfur bond and induces formation of a loop. In gamma-type Env this motif also establishes a second disulfide bond with a C of the SU CXXC motif, tethering SU and TM. For a beta-type Env, the CX n CC would take the form of CX n C, the SU CXXC and SU/TM disulfide bond would not exist and the ISD would not have the canonical CKS-17 sequence (see also Fig.22A).

  VL GE QV QS IN FR MK IQ CH AN YK WI C LE EV VL EL GQ DV AN LK TR MS TR CH AN YD FI C LE EA IL HI GN EL QA LK VR LA LS CH AD YR WI C LR QT VI WM GD RL MS LE HR FQ LQ CD WN TS DF C LE AR VA RV EA IT DR IM LY QE LD CW HY HQ Y-C LV IG LK VE AM EK FL YF AM QE LG CN QN QF F-C LQ AR IL AV ER YL KD QQ LL GI WG CS GK LI --C LQ

Figure 22 -Figure 23 -

 2223 Figure 22 -Sequence of the ISD and CX n C 1-2 motif in different retroviral species and rationale of the mice immunosuppression assay. (A, left) ISD sequences of different exogenous and endogenous retroviruses, indicated by brackets on the left, compared to the consensus CKS-17 sequence. Sequences are grouped by their lineage and exogenous/endogenous state, as well as the TM type. Green reflects residue identity for gamma-type TM and yellow the consensus residues for beta-type TM. (right) CX n C 1-2 motif of the retroviral sequences with the cysteines implicated in disulfide bond formation highlighted in orange. (B) Rationale of the tumor cell line immunosuppression assay in mice. Mouse tumor cell lines are transduced with an Env expressing vector, before injection of the Env-expressing cells into mice. Immune responses are quantified by measurement of tumor growth (immunosuppressive tumors grow better) and analysis of anti-Env antibody generation (immunosuppressive Env should elicit a lower antibody response).

Figure 24 -

 24 Figure 24 -Examples of the nature of known retroviral receptors and example of the SLC1A5 RBD (A) Known receptors of exogenous and endogenous retroviral Env, sorted by receptor type. A small scheme illustrates the types of receptor. Gamma-type Env are in black, avian gamma-type in gray, beta-type env in green and syncytin Env in blue. (B) Alignment of the SU sequences of representatives of the SLC1A5 interference group, highlighting the conserved cysteine containing domains in yellow and the major SDGGGXXDXXR motif in orange. The number indicate distance between the residues and mismatched residues in the conserved domains are highlighted in red. Adapted from Malicorne et al. (2016).

Figure 25 -

 25 Figure25-Phylogeny of mammals with known syncytins and syncytin-like genes positioned according to age of exaptation. Mammals contain the viviparous eutherians and marsupials as well as the oviparous monotremes. The supposed age of emergence of the shared mammalian placenta is indicated by a red arrow. All known syncytins (purple) and selected syncytin-like genes (green) have been added to the tree, their arrow corresponding to their approximate age of exaptation and their name indicated in front of the branch. Branch length is proportional to time, see scale at the bottom[START_REF] Dos Reis | Phylogenomic datasets provide both precision and accuracy in estimating the timescale of placental mammal phylogeny[END_REF].

Figure 26 -

 26 Figure 26 -Impact of syncytin-A or -B knockout on mouse placenta. (A) Schematic representation of the impact of syncytin knockout on establishment of the ST layers in the mouse placenta. KO of each syncytin prevents formation of a separate ST layer, which remains individualized. This results in the death of all embryos in the case of syncytin-A or in late growth retardation in the case of syncytin-B. For simplicity, the external fenestrated unfused trophoblast layer is not represented (B, top) Electron micrographs showing the disrupted ST. I and II identify the ST layer, stgc identifies sinusoidal trophoblastic giant cells (the outer non-fused trophoblast layer, see 1.2.2.d.) and ec an endothelial cell. Arrows show desmosomes between unfused ST II cells in the case of syncytin-B knockout. Scale bar: 2 µm on the left, 1 µm on the right. (bottom) Impact of syncytin knockout on the maternal blood spaces. Hematoxylineosin-saffron (left) or thionine (right) stained sections of mouse placenta. Arrows indicate maternal blood spaces and arrowheads fetal vessels. Both maternal blood spaces and fetal vessel diameter are reduced in syncytin-A knockout placentas, as shown by the squeezed red blood cells. In syncytin-B knockout placentas, maternal blood spaces are enlarged while fetal vessels are unaffected. Adapted from Dupressoir et al. (2009, 2011).

Figure 27 -

 27 Figure27-Compared evolutionary fates of HERV-W Env and EnvV2 in simians. Timed phylogenetic tree of major simian clades, with prosimians as an outgroup (TimeTree,[START_REF] Kumar | TimeTree: A Resource for Timelines, Timetrees, and Divergence Times[END_REF].The presence of HERV-FRD Env (FRD), EnvV2 (V) and HERV-W Env (W) as fully functional syncytins is indicated on the tree by colored lines and letters. A fading line and gray letter means loss of at least one syncytin property (fusogenicity for EnvV2, all three for HERV-W Env) in this clade. In the last common ancestor of apes and Old World monkey, all three envelopes were present and probably displayed all syncytin characteristics at the same time. Following split of these clades, either EnvV2 was conserved and HERV-W Env lost (lower arrow), or EnvV2 lost its fusogenic properties and HERV-W Env replaced it as syncytin (upper arrow, baton pass event). Present day syncytins are indicated on the right, using the same colors as in the tree. Adapted from[START_REF] Esnault | Differential Evolutionary Fate of an Ancestral Primate Endogenous Retrovirus Envelope Gene, the EnvV Syncytin, Captured for a Function in Placentation[END_REF].

Figure 29 -

 29 Figure29-Three examples of different methods of syncytin transcript initiation. Depending on the insertion locus and the evolutive history of this locus, syncytin expression can be initiated in different ways. In some cases like Syncytin-Car1, expression is simply driven by its original LTR promoter and the canonical retroviral splice is present. In other cases like that Syncytin-Rum1, the original promoter LTR has been lost during evolution and transcription is initiated by a cryptic promoter present in the otherwise degenerated gag-pol region. Only the splice acceptor site is canonical. Finally, in the case of the syncytin-like HEMO, transcription is initiated by a cellular CpG island promoter and presents multiple splicing events that can yield alternative transcripts containing different parts of the cellular genome 5' of the HEMO Env ORF. Faded out boxes represent non-functional ERV sequences, color coding is the same as Fig.9. The gradient covering the colors of gag, pro and pol indicates that the three cannot be told apart. Green lines represent transcripts and dotted line splicing events.

Guillaume 5 a

 5 Cornelis a,b,1,2 , Mathis Funk a,b,1 , Cécile Vernochet a,b , Francisca Leal c,3 , Oscar Alejandro Tarazona c,4 , Guillaume Meurice d , Odile Heidmann a,b , Anne Dupressoir a,b , Aurélien Miralles e , Martha Patricia Ramirez-Pinilla c , and Thierry Heidmann a,b,Unité Physiologie et Pathologie Moléculaires des Rétrovirus Endogènes et Infectieux, CNRS UMR 9196, Gustave Roussy, Villejuif, F-94805, France; b UMR 9196, Université Paris-Sud, Orsay, F-91405, France; c Laboratorio de Biologia Reproductiva de Vertebrados, Escuela de Biologia, Universidad Industrial de Santander, 680002 Bucaramanga, Colombia; d Plateforme de Bioinformatique, INSERM US23/CNRS UMS3655, Gustave Roussy, Villejuif, F-94805, France; and e Institut de Systématique, Evolution, Biodiversité, Muséum National d'Histoire Naturelle, CNRS UPMC EPHE, Sorbonne Universités, Paris, F-75005, FranceEdited by R. Michael Roberts, University of Missouri-Columbia, Columbia, MO, and approved October 26, 2017 (received for reviewAugust 23, 2017)

Fig. 1 .

 1 Fig. 1. Phylogeny of vertebrates positioning mammals, the Mabuya lizard, and known syncytins. Mammals comprise the monotremes (e.g., platypus) still laying eggs, and the marsupials and eutherian mammals, which all possess a placenta (red font). The lizard Mabuya is shown in red font because it also possesses a placenta. A red arrow indicates the probable time of emergence of the mammalian placenta, which has been proposed to correspond to the primitive capture of an ancestral syncytin, thereafter replaced in evolution by the indicated present-day syncytins (reviewed in ref. 1). All currently described syncytin capture events are indicated by arrowheads in purple, together with the syncytin's name. Branch length is proportional to time [expressed in My(15,[START_REF] Vidal | The phylogeny of squamate reptiles (lizards, snakes, and amphisbaenians) inferred from nine nuclear protein-coding genes[END_REF][START_REF] Kumazawa | Mitochondrial genomes from major lizard families suggest their phylogenetic relationships and ancient radiations[END_REF]], as indicated in the scale below the tree.

Fig. 2 .

 2 Fig. 2. Structure of a canonical retroviral Env protein and characterization of the identified Mabuya candidates. (A) Schematic representation of a retroviral Env protein delineating the SU and TM subunits.The furin cleavage site (consensus: R/K-X-R/K-R) between the two subunits, the C-X-X-C motif involved in SU-TM interaction, the hydrophobic signal peptide (purple), the fusion peptide (green), the transmembrane domain (red), the putative immunosuppressive domain (ISD) (blue), and the conserved C-X 5-7 -C motif (CC) are indicated. (B) Characterization of the candidate Mabuya Env proteins. The hydrophobicity profile for each candidate is shown with the canonical structural features highlighted in A. (C) Retroviral envelope protein-based phylogenetic tree with the identified Mab-Env protein candidates in red. The unrooted PhyML maximum likelihood tree was obtained using TM subunit amino acid sequences (without the cytoplasmic tail) from syncytins (in blue) and a series of endogenous and infectious retroviruses (from the dataset in ref.[START_REF] Henzy | Betaretroviral envelope subunits are noncovalently associated and restricted to the mammalian class[END_REF]. The horizontal scale bar below the tree represents the average number of substitutions per site, and the percent bootstrap values obtained from 1,000 replicates are indicated by circles on each branch (see key in figure). Retroviral families and major envelope types, the associated C-X n -(C)C motif, and the presence or absence of an ISD are indicated on the tree. (D) Amino acid sequences and characteristic structural features of Mab-Env1/Mab-2 and Mab-Env3/ Mab-4 (GenBank accession nos. MG254888-MG254891). Asterisks indicate amino acid identity; colons indicate amino acid similarity.

Fig. 3 .

 3 Fig.3. Real-time RT-qPCR analysis of candidate env gene expression in Mabuya sp. IV. Transcript levels are shown as the ratio between the expression levels of each env gene and those of the RPL19 control gene (SI Methods). Placental tissues and maternal ovary and oviduct tissues were recovered at two gestational stages (defined using ref.33): stage 30, when the uterine syncytium starts to form in the placentome region, and stage 37, when the syncytium is well developed. The results for the four env candidate genes were obtained using the same series of tissues. Values are the means of duplicates from three samples ± SEM.

Fig. 4 .

 4 Fig. 4. Characterization of the Mab-Env1-associated RT-containing pol gene and classification among known retroviral families. (A) ORF map of the identified genomic proviral fragment. Short bars indicate a start codon; tall bars indicate a stop codon. Green horizontal lines represent overlapping RNA-seq transcripts. Similarities to canonical retroviral genes by BLASTp search are colored in yellow for gag and purple for pol. Mab-Env1 is colored red. The conserved RT and TM domains used for the phylogenetic analyses in Figs. 2C and 4B are delineated.(B) Unrooted PhyML tree showing the position of retroviral endogenous and exogenous RT domains using the same dataset as in Fig.2Cwhen available (some syncytin-associated RTs were too degenerate to be identified). The Mab-Env1associated RT is highlighted in red, and those associated with previously described syncytins are shown in blue. Major retroviral families are indicated on the tree. Bootstraps obtained after 1,000 replicates are indicated by circles on each branch (see key in figure). Branch length is proportional to the average number of substitutions per site (see scale). RT sequences are provided as Dataset S1.

Fig. 6 .

 6 Fig. 6. Structure of the Mabuya sp. IV placenta and expression pattern of Mab-Env1. (A) Schematic representation of the late-stage Mabuya sp. IV placenta (see ref. 27). (Left) Overview of a gravid uterus displaying the apposed maternal and fetal tissues.Maternal and fetal tissues are highly interdigitated in the placentome region. In addition to the placentome, the Mabuya placenta develops specialized regions for materno-fetal exchanges such as areolae and absorptive plaques. The brown/orange and gray/yellow areas represent the fetal and maternal tissues, respectively. (Right) Detailed scheme of the materno-fetal interface in the placentome and paraplacentome region. In the placentome, maternal and fetal tissues are highly interdigitated, with numerous microvilli between fetal and uterine cells. The fetal epithelium is formed by giant binucleated chorionic cells. The uterine epithelium is replaced by a large syncytial structure formed by the fusion of the uterine cells. In the paraplacentome the syncytium is abruptly replaced by uterine epithelium. Cells still present microvilli, but the tissues are no longer interdigitated. (B, Left) Hematoxylin eosin saffron (HES)-stained sections of placenta with the maternal uterine syncytium (ms), the maternal epithelium (me), and the fetal chorionic epithelium (fc) delineated; white arrowheads indicate maternal and black arrowheads indicate fetal blood vessels. (B, Right) In situ hybridization (odd rows) or immunohistochemistry (even rows) on serial sections for Mab-Env1 expression using digoxigenin-labeled antisense and sense riboprobes or an anti-Mab-Env1 mouse serum and preimmune serum. (Scale bars: 50 μm.) Areas marked by rectangles are enlarged on the right, and the maternal and fetal domains are delineated.

  Fig. 6. Structure of the Mabuya sp. IV placenta and expression pattern of Mab-Env1. (A) Schematic representation of the late-stage Mabuya sp. IV placenta (see ref. 27). (Left) Overview of a gravid uterus displaying the apposed maternal and fetal tissues.Maternal and fetal tissues are highly interdigitated in the placentome region. In addition to the placentome, the Mabuya placenta develops specialized regions for materno-fetal exchanges such as areolae and absorptive plaques. The brown/orange and gray/yellow areas represent the fetal and maternal tissues, respectively. (Right) Detailed scheme of the materno-fetal interface in the placentome and paraplacentome region. In the placentome, maternal and fetal tissues are highly interdigitated, with numerous microvilli between fetal and uterine cells. The fetal epithelium is formed by giant binucleated chorionic cells. The uterine epithelium is replaced by a large syncytial structure formed by the fusion of the uterine cells. In the paraplacentome the syncytium is abruptly replaced by uterine epithelium. Cells still present microvilli, but the tissues are no longer interdigitated. (B, Left) Hematoxylin eosin saffron (HES)-stained sections of placenta with the maternal uterine syncytium (ms), the maternal epithelium (me), and the fetal chorionic epithelium (fc) delineated; white arrowheads indicate maternal and black arrowheads indicate fetal blood vessels. (B, Right) In situ hybridization (odd rows) or immunohistochemistry (even rows) on serial sections for Mab-Env1 expression using digoxigenin-labeled antisense and sense riboprobes or an anti-Mab-Env1 mouse serum and preimmune serum. (Scale bars: 50 μm.) Areas marked by rectangles are enlarged on the right, and the maternal and fetal domains are delineated.

Fig. 7 .

 7 Fig. 7. Mab-Env1 is a fusogenic retroviral envelope protein. (A) Schematic representation of the cell infection assay with Mab-Env-pseudotyped virus particles. Pseudotypes are produced by cotransfecting 293T cells with expression vectors for the MLV core, a β-galactosidase encoded by a nlsLacZ-containing retroviral transcript, and either a vector expressing Mab-Env proteins or a control vector. The supernatant of the transfected cells is then added to the indicated target cells, which are X-gal stained 3 d postinfection to reveal viral infection. (B) Panel of X-gal-stained target cells infected with particles without Env or pseudotyped with Mab-Env1, Mab-Env2, or the Env protein from A-MLV as positive control. (C) Quantification of viral titers expressed in focus-forming units per milliliter after infection with Mab-Env1-pseudotyped MLV virions. Values shown are the mean of three independent experiments ± SD.(D) Schematic representation of the cell-cell fusion assay. 293T cells were cotransfected with a plasmid expressing a nuclear β-galactosidase and a plasmid expressing either Mab-Env1 or a control plasmid. After 48 h a 5-min acidic shock was performed (or not) using Dulbecco's phosphate-buffered saline (DPBS)•HCl at either pH 4 or 7. Cells were X-gal stained 6 h afterward. (E) Mab-Env1 is able to induce cell-cell fusion after an acidic shock. After exposition to acid medium, cells transfected with the Mab-Env1-expressing plasmid show a degree of fusion equivalent to that of cells transfected with a plasmid expressing the Env protein of mouse mammary tumor virus (MMTV), which was used as a positive control.

Fig. 8 .

 8 Fig. 8. Structure and expression of the Mab-Env1 receptor MPZL1. (A) Alignment of Mabuya MPZL1 (Gen-Bank accession no. MG254887) and human MPZL1 amino acid sequences with major domains and sites indicated. Asterisks indicate amino acid identities, and colons indicate amino acid similarities. (B) To-scale schematic representation of the mature human MPZL1 with major domains indicated. (C) Human and Mabuya MPZL1 act as receptors for Mab-Env1-mediated infection. Target A23 cells were transfected with a plasmid expressing human or Mabuya MPZL1 or an empty vector. Twenty-four hours posttransfection, cells were infected using MLV particles carrying an nlsLacZ reporter gene and pseudotyped with Mab-Env1 or Mab-Env2 or without Env. Cells were X-gal stained 72 h after infection, and infection foci were quantified. Values shown are the mean of three independent experiments ± SD. (D) MPZL1 is expressed in a wide range of tissues. RT-qPCR analysis of MPZL1 expression levels was performed in the same way and on the same series of tissues as in Fig. 3. (E) MPZL1 is expressed at the materno-fetal interface. (Left) HES-stained sections of placenta with the maternal syncytium (ms), the maternal epithelium (me), and the fetal chorionic epithelium (fc) delineated; white arrowheads indicate maternal and black arrowheads indicate fetal blood vessels. (Right) In situ hybridization on serial sections for MPZL1 expression using digoxigeninlabeled antisense and sense riboprobes. (Scale bars: 50 μm.) Areas marked by rectangles are enlarged on the right, and the maternal and fetal domains are delineated.

  Fig.S3. Rationale of the retroviral pseudotyping assay used for screening the GeneBridge4 human/Chinese hamster radiation hybrid panel and mapping of the candidate receptors for Mab-Env1. (A) The 93 hybrid clones of the GeneBridge4 panel generated in ref.[START_REF] Gyapay | A radiation hybrid map of the human genome[END_REF] as schematized on the Left, were seeded and infected with viral pseudotypes produced by cotransfecting 293T cells with expression vectors for the MLV core, the Mab-Env1 protein, and a lacZ-containing retroviral transcript (Right). Viral titers were determined 3 d after infection by X-gal staining of the cells. (B) Viral titers obtained in a representative experiment simultaneously assaying the 93 clones of the panel (and control A23 cells, at the right end of the x axis). Each cell clone was infected in duplicate and examined for the number of lacZ+ foci. The resulting matrix was derived, and data were compared with a known matrix of specific markers situated across the human genome (SI Methods). (C) Localization of the identified high-scoring genetic markers (nos.[START_REF] Lavialle | Paleovirology of 'syncytins', retroviral env genes exapted for a role in placentation[END_REF][START_REF] Denner | Expression and function of endogenous retroviruses in the placenta[END_REF][START_REF] Dupressoir | Syncytin-A knockout mice demonstrate the critical role in placentation of a fusogenic, endogenous retrovirus-derived, envelope gene[END_REF][START_REF] Dupressoir | A pair of co-opted retroviral envelope syncytin genes is required for formation of the two-layered murine placental syncytiotrophoblast[END_REF][START_REF] Mi | Syncytin is a captive retroviral envelope protein involved in human placental morphogenesis[END_REF][START_REF] Blond | An envelope glycoprotein of the human endogenous retrovirus HERV-W is expressed in the human placenta and fuses cells expressing the type D mammalian retrovirus receptor[END_REF][START_REF] Blaise | Genomewide screening for fusogenic human endogenous retrovirus envelopes identifies syncytin 2, a gene conserved on primate evolution[END_REF][8] on chromosome 1, showing the positions of the genes present within 3 Mb, centered on the best-scoring marker, as determined using the University of California, Santa Cruz (UCSC) Genome Browser. The transmembrane candidate receptor genes are highlighted in red.

Figure 1 -

 1 Figure1-Phylogeny of mammals with emphasis on carnivorans, with placental type and known syncytins indicated. (Upper) Mammals comprise the monotremes (e.g., platypus) still laying eggs, and the marsupials and eutherian mammals, which all possess a placenta. For the latter the placental type is indicated using the same color key as the schemes below. All currently described syncytin capture events are indicated by arrowheads in pink, together with its name. Branch length is proportional to time (expressed in My,[START_REF] Cornelis | Retroviral envelope gene captures and syncytin exaptation for placentation in marsupials[END_REF][START_REF] Nyakatura | Updating the evolutionary history of Carnivora (Mammalia): a new species-level supertree complete with divergence time estimates[END_REF], as indicated in the scale below the tree. (Lower) Schemes illustrating the four types of maternofetal interfaces found in mammals, arranged in order of invasiveness of fetal tissues. A line delimits the maternal and fetal sides.

Figure 2 -

 2 Figure 2 -Structure of a canonical retroviral Env protein and characterization of the identified Crocuta crocuta envelope candidates. (A) Schematic representation of a retroviral Env protein, delineating the Surface (SU) and Transmembrane (TM) subunits.The furin cleavage site (consensus: R/K-X-R/K-R, orange) between the two subunits, the C-X-X-C motif involved in SU-TM interaction, the hydrophobic signal peptide (purple), the fusion peptide (green), the transmembrane domain (red), and the putative immunosuppressive domain (ISD, blue) along with the conserved C-X5-7-C (CC) motif are indicated. (B)(Left) Characterization of the candidate Crocuta crocuta Env proteins: the hydrophobicity profile for each candidate is shown with the canonical structural features highlighted using the same colors as (A). (Right) RT-qPCR analysis of env gene expression in the Crocuta crocuta placenta. Transcript levels are shown as the ratio between expression levels of each env gene and those of the RPL19 control gene (see Methods).

Figure 4 -

 4 Figure 4 -Alignment of Hyena-Env2 genes from all four extant hyena species. Functional domains are highlighted using the same color code as in Fig. 2 and the SU and TM subunits are delineated. Asterisks indicate amino acid identities and colons indicate amino acid similarities.

  **** ** * ***** ** dog (C. familiaris)AGAGAT-------------------------------ferret (M.

Figure 5 -

 5 Figure 5 -Characterization of the Hyena-Env2 associated provirus and its genomic location. (A)Hyena-Env2 insertion occurred in a relatively conserved and transcriptionally active region. (Upper) Predicted transcripts at the syntenic cat locus with exons and introns delineated. (Lower) 10 kb region of the cat genome containing the ortholog site to the Hyena-Env2 insertion locus, with repeated sequences (as indicated by RepeatMasker, http://www.repeatmasker.org/) and CpG islands marked, as well as conservation of this region in other carnivorans. The ortholog region to the hyena transcript initiation site is also indicated. See key on the bottom right. (B) The insertion site of the Hyena-Env2 associated provirus is empty in other Carnivora. Timed phylogenetic tree and nucleotide alignment of the syntenic loci of sequenced Carnivora, the four extant Hyaenidae, as well as Cryptoprocta ferox, the closest relative to Hyaenidae. The target site duplication is highlighted by red boxes showing that insertion occurred only in Hyaenidae. For the latter the nucleotide sequence of the extremities of both LTRs as well as the PBS are shown. Asterisks indicate sequence identities in the locus alignment, excluding the dog sequence. (C) Structure of the Hyena-Env2 associated provirus and its placental transcript in Crocuta crocuta. LTRs are indicated in purple, gag in orange, pol in yellow and Hyena-Env2 in red. Vertical bars indicate stop codons or frameshifts in the three reading frames. The spliced placental transcript is drawn to scale below with the exons aligned to their corresponding proviral regions. The sequence of the splicing sites is provided.

Figure 6 -

 6 Figure6-The Hyena-Env2 associated virus is a gammaretrovirus. Unrooted PhyML tree showing the position of retroviral endogenous and exogenous RT domains using the same dataset as in Fig.2Cwhen available. The Hyena-Env2-associated RT is highlighted in red, and those associated with previously described syncytins are shown in blue. Major retroviral genera are indicated on the tree. Bootstraps were calculated after 1,000 replicates and are indicated on each branch (see key in figure). Branch length is proportional to the average number of substitutions per site (see scale).

Figure 7 -

 7 Figure7-Representation of the structural differences of endotheliochorial carnivoran and hemochorial hyena placentas Schematic representation of the mid-gestation carnivoran endotheliochorial placenta (top row) and hemochorial hyena placenta (bottom row). The drawing on the right presents the cellular organization of the syncytial interface. In the hemochorial hyena placenta, the maternal blood vessels have been disrupted resulting in the presence of large maternal blood spaces. Only the primary villi are represented for both placental types.

Figure 8 -Figure 9 -Figure 10 -

 8910 Figure 8 -Expression pattern of syncytin-Car1 and Hyena-Env2 in Crocuta crocuta placenta. (top rows) Hematoxylin eosin (H&E)-staining and in situ hybridizations on serial sections of Crocuta crocuta placenta using specific digoxigenin-labeled antisense probes for syncytin-Car1 and Hyena-Env2 (and corresponding sense probes as a control). Specific staining is observed in the syncytiotrophoblast (ST) for syncytin-Car1 and in the cytotrophoblasts (CT) for Hyena-Env2. Strong staining with the Hyena-Env2 sense probes indicates existence of an antisense transcript. Areas marked by a rectangle are enlarged on the right, all scale bars: 50 µm. (bottom row) H&E-staining and immunohistochemistry on serial sections of Crocuta crocuta placenta using an anti-Hyena-Env2 mouse serum (and the pre-immune serum as control). The region shown is equivalent to that of the Hyena-Env2 ISH. Specific staining is again observed in the cytotrophoblasts (CT). Scale bars: 50 µm.

Figure 11 -

 11 Figure11-Hyena-Env3 belongs to the SLC1A5 interference group of envelopes. (A) The Hyena-Env3 SU is closely related to envelopes previously described as using SLC1A5 (also known as RDR and ASCT2) as receptor. Maximum likelihood tree using SU subunit amino-acid sequences from syncytins and a series of endogenous and infectious retroviruses. Previously described syncytin genes are highlighted in blue and the RDR interference group is highlighted in orange. Branch length represents the average number of substitutions per site (scale bar top left), and the percent bootstrap values obtained from 1,000 replicates are indicated on the branches. (B) Carnivoran but not human SLC1A5 allow Hyena-Env3 mediated infection of non-permissive cells. 293T cells were transfected with either an empty vector or expression vectors for SLC1A5 from human, cat, hyena or dog. Transfected target cells were then infected using Hyena-Env3-pseudotyped MLV particles and viral titer in focus forming units per mL were quantified after LacZ staining. Arrows indicate titers below background, values shown are the mean of three independent experiments ± SD. (C) Sequence alignment of SU protein subunits, from the methionine to the putative furin cleavage site, of endogenous and exogenous envelopes using SLC1A5 as a receptor as well as Hyena-Env3 (in bold). Variable regions are represented by the number of omitted residues; dashes correspond to deletions/insertions. The conserved motif shown to be essential for the Syncytin-1/SLC1A5 interaction (SDGGGX 2 DX 2 R) is highlighted in orange, and the three cysteinecontaining motifs (PCXC, CYX5C, and CX 7-9 CW) and amino acids that are highly conserved among retroviral members of the RDR interference group are highlighted in yellow[START_REF] Cheynet | Identification of the hASCT2-binding domain of the Env ERVWE1/syncytin-1 fusogenic glycoprotein[END_REF]. Amino acid mismatches in conserved regions are highlighted in red, asterisks indicate amino acid identities.

  Advantage 2 Polymerase Kit (Clontech) with the following program: 5 cycles of 30 s at 94 • C and 5 min at 72 • C, followed by 5 cycles of 30 s at 94 • C, 30 s at 70 • C and 5 min at 72 • C and finally 30 cycles of 30 s at 94 • C, 30 s at 68 • C and 5 min at 72 • C. Real-time RT-qPCR. Hyena-env RNA expression was determined by RT-qPCR. Reverse transcription was performed with 100 ng of DNase-treated RNA as in (de Parseval

  Identification et caractérisation de deux nouveaux gènes d'enveloppes rétrovirales de type syncytine, capturées pour un possible rôle dans la structure atypique du placenta de hyène et l'émergence du placenta non-mammifère des lézards MabuyaIntroduction. Les syncytines, une famille de gènes d'enveloppe (env) de rétrovirus endogènes (ERV), jouent un rôle clé dans l'établissement des structures placentaires chez les mammifères.

  Ensuite, nous avons cherché des gènes syncytine dans le genre non-mammifère Mabuya, des lézards vivipares présentant un type rare de placenta très complexe et proche de celui des mammifères. Nous avons identifié une env qui a été capturée et conservée dans ce genre depuis sa radiation, il y a 25 millions d'années. Ce gène, que nous avons appelé syncytin-Mab1, est capable d'induire la fusion cellule-cellule et est exprimé dans une couche de cellules fusionnées à l'interface materno-foetale du placenta, deux propriétés canoniques de syncytine. Nous avons aussi identifié le récepteur de syncytin-Mab1, MPZL1, et avons montré que leur interaction induit son activation et sa phosphorylation. L'activation de MPZL1 a été liée à la migration et à l'invasion cellulaire, indiquant que cette interaction env-récepteur pourrait jouer un rôle dans l'invasion placentaire du tissu maternel observée chez les Mabuya. Pour conclure, la caractérisation de ces deux nouvelles env indique que les gènes de type syncytine ont pu jouer un rôle à la fois dans l'émergence du placenta de Mabuya et dans la structure atypique du placenta des hyènes, supportant la notion que la capture d'env est une force évolutive majeure.

Table 1 -Classification of vertebrate reproductive modes.

 1 The examples provided for each reproductive type are not exhaustive. Adapted fromBlackburn (2015).

Table 1 -List of primers used in this study.

 1 Underlined sequences correspond to the restriction enzyme site indicated in the primer name.

The 5'-U3-R-U5-PBS-3' plus-strand DNA fragment can now bind to the PBS which forms the 5' end of the minus-strand DNA. This forms a DNA/DNA duplex which serves as primer for elongation and completion of both the plus-and minus-DNA strand, leading to formation of the DNA genome.

and variable placental organization has been suggested previously in Ruminants[START_REF] Cornelis | Captured retroviral envelope syncytin gene associated with the unique placental structure of higher ruminants[END_REF], we investigated if the hyena placenta presents a distinct env expression profile when compared to the reference cat and dog placentas, be it through a difference in expression levels of shared genes, or through the acquisition of new hyena-specific genes.

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Acknowledgments

Acknowledgments. We wish to especially thank Adriana Alberti, Corinne Da Silva and Jean Weissenbach (Genoscope, Evry, France) for the Crocuta crocuta transcriptome sequencing and assembly. We also thank Géraldine Véron (MNHN), Frédéric Delsuc (Université de Montpellier, France) and Baptiste Mulot (Zooparc de Beauval, France) for providing us with some of the DNA and/or tissue samples, Olivia Bawa (Gustave Roussy Preclinical Evaluation Platform, France) for contribution to the histological analyses and Mélanie Polrot (Gustave Roussy Animal Care Facilities, France) for assistance in mice handling. We thank Christian Lavialle for discussion and critical reading of the manuscript. This work was supported by the CNRS and by grants to TH from the Ligue Nationale contre Le Cancer (Equipe Labellisée) and the Agence Nationale de la Recherche (ANR "RETRO-PLACENTA"), as well as a grant to MF by the Fondation pour la Recherche Médicale (FDT20170437061).

Material and Methods

2.2

acid trees were constructed with PhyML 3.0 [START_REF] Guindon | New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0[END_REF], with bootstrap percentages computed after 1,000 replicates. Substitution models were selected using the SMS program (Lefort et al., 2017): LG+Γ+I for TM sequences, WAG+Γ+I+F for SU sequences and RtREV+Γ for RT sequences. For all genomes available at http://genome-euro.ucsc.edu/index.html, orthology and other genome wide in silico studies were performed on the same site using the BLAT algorithm. Assemblies used were: Felis catus felCat8 (2014), Canis familiaris canFam3 (2011), Mustela putorius furo musFur1 (2011) and Ailuropoda melanoleuca ailMel1 (2009). The BLASTn algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to screen the genomes of Panthera tigris PanTig1.0, Ursus maritimus UrsMar_1.0, Acinonyx jubatus aciJub1, Odobenus rosmarus divergens Oros_1.0 and Leptonychotes weddellii LepWed1.0, all of them available at http://www.ncbi.nlm.nih.gov/genome/?term=carnivora.

Entry date in the hyena species. For both Hyena-Env2 and 3 presence or absence was assayed by PCR of an internal fragment in all species indicated in the carnivoran family tree. PCR was performed on 100 ng genomic DNA, using conserved primers (see Table ) 1 and Accuprime polymerase (Invitrogen) for 50 cycles (30 s at 94 • C, 30 s at 55 • C, 1 min at 68 • C). For species where a sequenced genome is available BLASTn was used to assay the presence of either of the envelopes. Finally, for Hyena-Env2 the full-length reading frame of all four species of Hyaenidae and the empty locus in cat, dog and the 4 species closest to Hyaenidae according to our tree (Arctictis binturong, Cryptoprocta ferox, Suricata suricatta and Helogale parvula) were amplified by PCR using the above protocol. In all sequenced carnivora the sequence of the empty locus was determined by BLASTn search.

Identification and sequencing of the Hyena-Env2 associated provirus and integration site.

Inverse PCR was performed by digesting 2 µg of Crocuta crocuta genomic DNA with NcoI (New England Biolabs) and inactivating the enzyme at 65 • C for 30 min before DNA purification using the Gel and PCR clean up kit (Macherey-Nagel) and self-ligation using T4 DNA Ligase (New England Biolabs). PCR amplification was performed with divergent primers (see Table 1) and Accuprime DNA Polymerase (Invitrogen) for 50 cycles (30 s at 94 • C, 30 s at 55 • C, 7 min at 68 • C) followed by a nested PCR using the same conditions with nested divergent primers (see Table 1). Fragments of >1 kb were gel purified and cloned into a pGEM-T Easy vector (Promega) before sequencing. Sequences were checked for presence of the Hyena-env gene and BLAT analysis was performed to identify the syntenic region of the Felis catus genome. Locus primers were designed based on the syntenic region (see Table 1) and used to amplify the locus in hyenas and other carnivora.

The full Hyena-Env2 associated provirus was amplified from 100 ng of Crocuta crocuta genomic DNA using Long Expand DNA polymerase (Roche) with Buffer 2, 0.32 µM of each primer and the following program: 10 cycles of 10 s at 94 • C, 30 s at 55 • C and 8 min at 68