María José Lista

Rodrigo Prado Martins

Marie-Astrid Contesse

Sarah Findakly

Pierre Pochard

Chrysoula Daskalogianni

Corinne Guetta

Christophe Jamin

Marie- Paule Teulade-Fichou

Robin Fåhraeus

Cécile Voisset

Marc Blondel
email: blondel@univ-brest.fr>

Prof Marc Blondel
email: marc.blondel@univ-brest.fr

Susann Mönchgesang

Christoph Ruttkies

Hendrik Treutler

Marcus Heisters

Erwin De Genst

Serge Muyldermans

Gaëlle Friocourt

Frédéric Bihel

Jonghoon Choi

Eun Kyu Lee

Jaebum Choo

Junhan Yuh

Jong Wook Hong

Leo Van Der Pol

Michiel Stork

Peter Van Der Ley

Hanna Lindberg

Torleif Härd

John Löfblom

Stefan Ståhl

Anett Patzschke

Matthias G Steiger

Caterina Holz

Christine Lang

Diethard Mattanovich

Michael Sauer

Claudia Kleinhans

Ramani Ramkumar

Gabriele Mohan

Thomas Vacun

Barbara Schwarz

Yang Haller

Alexander Sun

Petra Kahlig

Anna Kluger

Heike Finne-Wistrand

Jan Walles

Hansmann

Alessandro Pellis

Enrique Herrero Acero

Hansjoerg Weber

Michael Obersriebnig

Rolf Breinbauer

Ewald Srebotnik

Georg M Guebitz

Antonio Valle

Gema Cabrera

Howbeer Muhamadali

Drupad K Trivedi

Nicholas J W Ratray

Royston Goodacre

Domingo Cantero

Jorge Bolivar

Ting Tang

Chongli Yuan

Hyun-Tae Hwang

Xuebing Zhao

Doraiswami Ramkrishna

Dehua Liu

Arvind Varma

Michael Melcher

Theresa Scharl

Bernhard Spangl

Markus Luchner

Monika Cserjan

Karl Bayer

Friedrich Leisch

Gerald Striedner

Jing Dai

Sang-Jin Suh

Morgan Hamon

Eszter Majer

José-Antonio Navarro

José-Antonio Daròs

Luis M Larraya

Alicia Fernández-San

María Millán

Inmaculada Ancín

Jon Farran

Veramendi

Frida Ojala

Anton Sellberg

Thomas Budde Hansen

Ernst Broberg Hansen

Bernt Nilsson

Jens Begemann

Antje C Spiess

• Yeast as a model for biomedical research • Yeast model for human diseases

Keywords: human (NCL) and yeast (NSR1) Nucleolin, Epstein-Barr virus (EBV), EBNA1, GAr domain, Saccharomyces cerevisiae, G-quadruplex, translation regulation, immune evasion, PhenDC3 EBV, Epstein-Barr virus, GMP, genome maintenance protein, GAr, glycine-alanine repeat, NCL, human Nucleolin, MHC, major histocompatibility complex, G4 Cancers linked to Epstein-Barr virus (EBV), EBNA1, Epstein-Barr virus (EBV) ARS, autonomous replicating sequence, CDK, cyclin dependent kinase, CEN, centromeric sequence, CHPK, conserved herpesvirus-

The Epstein-Barr Virus (EBV) is the first oncogenic virus described in humans. It is a ubiquitous virus which infects over 90% of the human population.

EBV causes a latent infection of B lymphocytes and remains asymptomatic in most of infected individuals. However, in some conditions like immunosuppression, EBV can cause uncontrolled cell proliferation responsible for some types of cancers like Burkitt lymphoma, Hodgkin lymphoma and nasopharyngeal carcinoma.

During latency, EBNA1 is the only protein expressed in all EBV-infected cells as it is essential for EBV genome replication and maintenance. EBNA1 is highly antigenic and T cells raised against EBNA1 exist; nevertheless EBV-infected cells are not eliminated by the host immune system. This is due to the central GAr (Gly-Ala repeat) domain of EBNA1 which is able to inhibit the translation of its own mRNA in cis, thereby preventing the recognition of EBV-infected cells by the immune system of the host. The mechanisms involved in this GAr-based translation inhibition were unknown when I started my PhD.

During this thesis, we identified the first cellular factor, Nucleolin (NCL), able to affect GAr-based translation inhibition. We showed that NCL overexpression enhances EBNA1 translation inhibition and inversely, that its downregulation decreases EBNA1 translation inhibition effect. Furthermore, we demonstrated that the interaction between NCL and GAr RNA occurs throught NCL binding to GAr-encoding mRNA via a particular secondary structure called G-quadruplex (G4). We showed that disrupting NCL-GAr RNA interaction with small G4 ligands enhances EBNA1 translation and thus antigen presentation, which constitutes a new therapeutic avenue to treat EBV-associated cancers.

Finally, we went deeper into the mechanism involved in this interaction by showing that NCL has a direct effect on the GAr-based translation inhibition and that the nuclear location of NCL-GAr RNA interaction is forced by the nuclear location of NCL in the cells.

Taken together, these results have shed light upon the mechanism involved in the GAr domain translation inhibition and immune evasion, and have revealed a possible therapeutic target to eradicate EBV-associated cancers.

First of all I want to thank all the members of the jury for accepting being part of my PhD jury, as well as for their good and useful comments. In particularly, I want to thank Robin for letting me be part of his lab, for welcoming me in his lab several months, and for showing me that we always have to express what we think. Thank you very much also for all the interesting discussions we had and for your support. I'm so happy I have had the opportunity to meet you and work with you.

I also want to thank the director of the Inserm UMR 1078, Prof. Claude Férec and all the members of the unit; it was a pleasure to be part of this unit during 3 years. I don't find the words to say how much I thank my PhD directors Marc and Cécile. During this three years I learned a lot of things regarding the professional and personal environments. I never learned as much different techniques as I learned with you. You also teached me how to be independent and to pursue my own objectives. Thank you for supporting me (even in the crazy things), and for showing me the good side of everything when I could not see it. To show me that when a Western blot does not work, it doesn't matter because it will probably work tomorrow. Thank you very much Cécile, you were like my mom here, for helping me to move in the new flat, to make all the courses I need and together with Gilles to set up the closet. I will never forget that you were there for me whenever I needed it, either for my personal or professional life. Finally I want to thank you also for helping me until the very last day with this thesis manuscript, to give me the strength to resist when I said I wanted to throw away my computer. But each time you said to me "Allez courage on a presque fini Majo!" for all this THANK YOU, I will never forget all that! I want to thank Rodrigo Prado-Martins (whose nickname is Pierre Curie) for all the moments we shared. Together we managed to find the common point between two different histories. We passed together many moments of happiness and sadness in the lab when the experiments were not working. You gave me the strength to continue every day when nothing worked and you became my friend. So thank you very much for having been part of this adventure on my side until the end.

I also want to thank the other members of Robin lab's: Chrysoula, Sarah and Laurence. Even if I was not formally part of the lab but I've had the opportunity to work with you and I feel I know you for many years. Thank you for the good atmosphere in the lab and all the good comments. I will never forget that we manage to establish "la salle de sport au labo". I want to thank Mónica: if I'm here today it is because of you and all the opportunities you gave me.

You believed in me from the very first time in 2008 when I joined your lab to start the my internship of my second year of license. You teached me everything: to work in a lab, to work in a team and to write. Thank you for all your support, for the opportunity to come in France and for being more than my boss.

Gracias a vos Nacho, a pesar de que nuestros caminos se hayan separado fuiste la persona que más contribuyó a que hoy este acá terminando esta tesis. Juntos vivimos el hecho de estar lejos de casa, de nuestro País, de nuestra familia y amigos. Juntos pasamos por miles de momentos muy felices y algunos tristes. Juntos superamos todas las dificultadas que fueron apareciendo a lo largo de esta "aventura" que al final nos llevó a no estar juntos. Siempre me entendiste, me apoyaste y me ayudaste a crecer, a relativizar las cosas y me mostraste todas las veces que me encandilé sola y hasta por momento me creí Madame Curie. Gracias por todos los consejos que me diste, tanto en el ámbito profesional como en lo personal. Por todo esto y mucho más gracias por haber compartido 5 años de nuestras vidas juntos.

Thank you very much to all my team Levure, the past and the current members. In particular, thanks to Nadège, Hélène, Gaëlle, Vlavie, Alice, Olo, Marie-Astrid, Justina, for the good atmosphere in the lab, for all the times we laughed and enjoyed working (believe it or not). In particular, I want to thank Olo: we survived together to the three years of PhD. We went through many thinks I think: experiments, beer times, hangovers, writing panic, vegetables basket, and celebration. So thank you very much for all of it and also for helping me mounting this manuscript; if not, it wouldn't have been so pretty. Vlavie, ahhh Vlavie….. I have a list of things to thank you for. You were my very first friend here in Brest, when I only spoke English (pretty bad by the way). I remember all the crepes parties, the after's lab and all the things you showed me in the lab. I am so so happy that we passed this three beautiful years together and I hope our friendship will last forever (wherever we are).

Justina my friend! Since I arrived in the lab in 2013 I was sure we would become friends. I think we can write a book of the numerous things we did together. You taught me all my first words in French and we learned English together. I remember the day I moved from the Red lab to the Blue lab just to be next to you! Working there was really fun, even making minipreps! I remember our pajama parties and all the talks we had that lasted forever! We always have something to talk about! So for these and many more things I'd like to thank you very much for having been close to me during these three years and for giving me the opportunity to become your friend. I want to thank my second first friend Popo. You were also the first friend I've had in France; I remember all the things we did together from the beginning when we were also speaking in English.

You were my confidant and also the responsible of my meeting with Huguet and all the faculty group.

I am so happy that I have had the opportunity to be with you during your pregnancy and also to spend time with Maël, and to teach him the Spanish ohhh yeah! I want to thank an excellent group of people that I've had the opportunity during the course of my, PhD and that helped me to "survive" during the last year: Paul, Regi, Nath, MLC, Marjo, Angi, Momo, Kiki, Flo. together we learned how to be "good" PhDs (and make many "bêtises") and to enjoy every day even when things were not going so well, either because the experiments didn't work or because we were not in a good mood. It only met you one year and half ago but it's increadible how many things we went through together. From the famous "pause café", to mardi de Fauvettes, jeudi pizzabière, mercredi 54 or jeudi Arizo. So thank you all for all the moments we spend together and for letting me be part of this "Brest family".

In particular, I want to thank Polo Blue my friend. For being there when I needed you, for all the times you followed the crazies ideas that we had with Flo (allez une vodka red bull, OUII), for supporting me when I started with the photos! and for your good mood every day.

A Mari-lor for being there for me when I needed someone to talk to, for being a great friend and a beautiful person. For all the girls' nights and also for letting me be part of your life. I hope you will visit me in Paris.

A ma Chérie, à mon amour, every person says that it is difficult to find someone that corresponds 100% to you; in my case I found you! I know I can say everything to you and I have the feeling I've knownyou all my life. We can laugh together, cry together and rebuilt the world in one night with "one petite 64 blanche". we went through so many things together in one year… it is amazing and so special: shopping, pajama parties, dancing, dinner, everything chérie. Thank you very much for all that and for letting me be your friend! I wait for you in Paris! Flo, Huguet, Gordi….ula many things to say…merci d'être mon bonheur de tous les jours depuis qu'on est ensemble, pour me montrer que tout est relatif, pour faire de moi la personne la plus heureuse et pour m'aimer comme tu le fais. Je n'aurais jamais cru trouver l'amour de ma vie à 10900km et surtout finir avec un « Franchute », mais depuis que je suis avec toi je connais le vrai amour et la vie de couple. Merci d'être très facile à vivre, de suivre mes folies (de la semaine et du WE), de sortir avec moi quand ce n'est pas du tout une bonne idée, d'apprendre à être grands (un jour peut-être) avec moi, et d'être une personne magnifique avec moi. Pour tout ça mon Gordi un grand merci et j'espère que tu sais combien je t'aime.

I want to thanks my friends from Uruguay, en especial a mis mejores amigas Ceci y Maru que mas que amigas son mis hermanas. Gracias por haber compartido toda esta aventura conmigo, por apoyarme en los momentos difíciles cuando estábamos lejos. A pesar de la distancia siempre estuvieron ahí cuando las necesité, apoyándome en todo y mis confidentes de siempre. Gracias por el inolvidable finde en Colonia, por apoyarme hasta el final en la Maestría, por mostrarme que era capaz de terminar, y por acompañarme en el cambio de vida y en todas las decisiones que tomé aunque no estuvieran de acuerdo. Por todo eso y mucho más GRACIAS de estar siempre a mi lado, las amo muchísimo y siempre las amaré.

Finally I want to thank my family: Lily, Co, Fede, Brother and Mamá. Si hoy estoy acá es gracias a todo el apoyo y amor incondicional que me brindaron siempre. Lily y Co los mejores tíos que una sobrina pueda tener, mis segundos padres, todo! Gracias infinitas por estar siempre y aunque estemos lejos siempre me acompañan y están presentes en mi corazón. Brother y Mamá, nosotros somos el núcleo de una familia hermosa. Solo nosotros sabemos lo hermoso que hemos construido y lo indestructible que somos juntos. Gracias por apoyarme, entenderme y sobre todas las cosas soportarme lo cual los tres sabemos que no es tarea fácil. Así que gracias por ser mi familia, y aunque estemos lejos siempre siempre vamos a ser inseparables.

Chapter 1: ---------------------------------------------------------------------------------------------------------------------------Identification of NCL as the first cellular factor involved in the GAr domain regulation of translation ---Chapter 2: ---------------------------------------------------------------------------------------------------------------------------Unraveling NCL involvement in the GAr-based translation inhibition: nuclear trapping, translation inhibition or both? - -------------------------------------------------------------------------------------------------------------Chapter 3: --------------------------------------------------------------------------------------------------------------------------Yeast as a tool to decipher some of the steps of EBV life cycle - ----------------------------------------------------Discussion ----------------------------------------------------------------------------------------------Bibliography ------------------------------------------------------------------------------------------

Table of figures

Table of tables

Introduction

During my PhD, I studied the mechanism by which the Epstein-Barr virus is able to establish a lifelong infection of memory B cells without being detected by the host immune system. In line with this, I went deeper into the mechanism by which the central Gly-Ala (GAr) domain of EBNA1 protein inhibits the translation of its own mRNA in cis and, in this way escapes from T cell recognition and elimination by the host immune system. In the introduction section, I will first focus on EBV: its discovery, its life cycle, the diseases which are associated to its infection. In the second chapter, I will present EBV essential protein EBNA1: the central function of EBNA1 GAr domain will be deeply addressed as well as the possible mechanisms by which EBNA1 allows EBV infected cells to evade the immune system thanks to its GAr domain. In the third part of the introduction, I will present the yeast model that recapitulates the GAr-based inhibition of translation.

CHAPTER I: The Epstein-Barr virus (EBV)

1. Discovery of the first human oncogenic virus: EBV From the beginning of the 90´s many observations showed evidence in favor of viruses related to human cancers. Nevertheless it was before 1957 that Denis Burkitt made its first observation in Africa of a strange form of lymphoma which was later called Burkitt lymphoma. This pathology was very common in young children in Africa and was fatal after metastasis. D. Burkitt was fascinated by this new type of lymphoma which led him to make a 10 weeks travel during which he determined a specific pattern of distribution of this disease in 12 countries. After analyzing all collected data, he realized that the distribution of this lymphoma corresponded to the location of malaria and yellow fever mosquito. He published his initial results in 1958 6 . In 1961 he was invited to give a lecture in the Middlesex Hospital in London where he had the opportunity to meet Anthony Epstein. A. Epstein was fascinated by D. Burkitt´s results and they started to collaborate. However it was only in 1963 when Yvonne Barr and Burt Achong joined A. Epstein´s laboratory that they started to observe EBV´s particles in biopsies taken from African patients 7 . Just after EBV´s discovery, the Henles couple located in Philadelphia received some culture samples from A. Epstein and observed that antibodies against this new virus were present in children with Burkitt lymphoma but also in healthy children from Africa, demonstrating that EBV was widely distributed 8 . Nowadays we know that more than 90% of the human population is latently infected by EBV and that the infection remains asymptomatic in most infected people.

EBV belongs to the family of herpesviridae

Herpesviruses are DNA double-stranded viruses. Their genomes do not integrate into host DNA but persist in infected cells as extrachromosomal circular episomes. They are ubiquitous in the animal kingdom and they are the causing agents of lymphomas and immunodeficiency in humans. Eight members of this family have been identified in humans so far and all of them share a common origin as their genomes display high amino acid sequence similarity for many of their viral gene products 9 .

Herpesviruses can be classified in three subfamilies: (1) Alphaherpesviruses (α), (2) Betaherpesviruses (β) and ( 3) Gammaherpesviruses (γ) depending on genome organization and sequence similarity.

These three families are presented below:

(1) The Alphaherpesviruses include herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2) and the varicella-zoster virus (VZV). All of them are able to establish latent infection in sensory neurons, but can also infect several other types of human cells like mucosa and skin cells 10 .

(2) The Betaherpesviruses include cytomegalovirus (HCMV or HHV-5) and three human roseolaviruses (HHV-6A, HHV-6B and HHV -7). They are able to latently infect myeloid cells.

(3) The Gammaherpesviruses: In humans there are two γ-herpesviruses, the Epstein-Barr virus (EBV or HHV-4) which corresponds to the lymphocryptovirus genus and the Kaposi-sarcoma associated virus (KSHV or HHV-8) which corresponds to the rhadinovirus genus. Both of them are lymphotropic viruses that are able to latently infect B cells and epithelial cells. They establish a lifelong asymptomatic infection of their host with some intermittent lytic periods 9 . These two oncogenic viruses have been associated to some human cancers: EBV infection can cause Burkitt lymphoma (BL), Hodgkin lymphoma (HL), nasopharyngeal carcinoma (NPC) and some gastric cancers, whereas KSHV has been initially associated to Kaposi sarcoma (KS) and then also to Multicentric Castleman´s disease (ML) and primary effusion lymphoma (PEL) 11 .

EBV-associated diseases

Most EBV infections are lifelong and asymptomatic; however many human diseases have been associated to EBV. In some cases EBV primary infection causes an acute infection described as infectious mononucleosis most commonly in teenagers and young adults. Moreover, several types of cancers have been associated with EBV, the most frequent being Burkitt and Hodgkin lymphomas and the epithelial nasopharyngeal carcinoma (Table 1).

Infectious mononucleosis (IM):

IM was first described in 1920 by TVP. Sprunt and FA. Evans as a new syndrome characterized by an acute infection with abnormal peripheral blood lymphocytes morphology, which were later shown to correspond to activated T CD8 + cells responding against EBV infected cells 12 . This disease is characterized by a global expansion of T CD8 + cells raised against EBV-derived antigens.

The most common symptoms are fever, fatigue, cervical lymph node enlargement and sore throat.

IM is transmissible via saliva explaining why it is commonly called the "kissing disease". Nevertheless, it can also be transmitted by blood transfusion, solid organ or hematopoietic cell transplantation.

Burkitt lymphoma (BL):

It is the first cancer that has been linked to EBV infection. It was first described in 1958 as a lymphosarcoma present in young children in tropical Africa 6 . After EBV discovery, it was observed that most BL cases in equatorial Africa carry this virus 13 . Burkitt lymphomas are classified in three different groups: the endemic, the sporadic and the HIV-associated forms. More than 90% of endemic BL are EBV-positive while only 5-15% of BL are sporadic and 40% are associated to HIV infection. All BL are characterized by the chromosomal translocation of the c-Myc locus from its normal localization on chromosome 8 to one of the immunoglobulin (Ig) heavy or κ and λ light chain loci located on chromosomes 14, 2 or 22, respectively. Due to these translocations, the oncogene c-Myc finds itself under the transcriptional control of the Ig promoter causing its overexpression, hence leading to the high BL cells proliferation observed 14 . Most of BL present a latency I EBV profile, and some of them present a latency III profile (see section 4.2 below).

Hodgkin´s lymphoma (HL):

This type of lymphoma is more common in undeveloped countries like Peru or Kenya (57-59% of incidence) than in developed countries like North America where it represents between 20-50% of the total lymphomas 15 . HL are classified as classic HL (HLC) or nodular lymphocyte predominant HL (NLPHL) based mostly on the morphology and immunophenotype of the malignant cells. HLC accounts for at least 95% of HL cases and is characterized by the presence of only 0,1-10% of malignant Hodgkin-Reed Sternberg (HRS) cells and the disruption of the lymph nodes 16 . NLPHL cases represent less than 5% of HL cases and are characterized by the presence of lymphocytes predominant cells (LP). HRS and LP cells present a deregulation of B cell specific genes that can be either very weakly expressed or not expressed at all. This is the case of key genes as B cell receptor BCR, CD19 and CD20. Both cell types also show a strong constitutive expression of NF-κB which contributes to the survival of cancer cells 17 . In general, HL shows a latency II profile of EBV infection (see on section 4.2 below).

Nasopharyngeal carcinoma (NPC):

NPC is a rare epithelial head and neck cancer with an incidence in most part of the world of 1-100.000 cases per year. Nevertheless, NPC is very common in Southern China and in Tunisia, where its prevalence is 25-30 cases per 100.000 people every year and represents one of the most predominant cancers among men 18 . All NPC are associated with EBV. It mostly occurs in males of about 40-60 years old. NPC is highly aggressive with an extensible local infiltration and early metastasis. There are three types of NPC: keratinizing squamous cell carcinoma, nonkeratinizing and basaloid carcinomas. The nonkeratinizing carcinoma is the most common one and is fully associated with EBV infection. It seems to be due to the proliferation of a single EBVinfected cell clone in the nasopharyngeal epithelium 19 . NPC carcinomas show a latency II profile of EBV infection (see on section 4.2 below).

3.5 Gastric carcinomas: between 2 and 20% of all gastric cancers are associated with EBV infection in epithelial cells with a world average of 10%. It is most common in males and the predominant localization is the proximal stomach. This type of cancer is associated to a latency II program (see on section 4.2 below). The most common factor associated to gastric cancers is infection by Helicobacter pylori. However, H. pylori infection is not a risk factor for gastric carcinoma associated to EBV which means that both pathogens are involved in different carcinogenic pathways 20 .

NK/T-cell lymphomas:

they are rare aggressive non B cell tumors which are mostly common in Asia and Latin America. EBV expression pattern corresponds to a latency II program (see on section 4.2 below) with variable LMP1 expression, which contributes to the excessive production of proinflammatory cytokines mediated by NF-κB activation 21 .

EBV life cycle

EBV was the first described human oncogenic virus and the first virus to have its genome totally cloned and sequenced. Its genome is composed of a double stranded DNA of approximately 180 kb which encodes approximately 80 proteins, from which only few have been characterized yet (Figure 1b-c). EBV genome is enclosed in an icosahedral nucleocapsid surrounded by a lipid envelope derived from the nuclear membrane (Figure 1a). EBV has the property to immortalize B cells in culture, and cell lines derived from this immortalization process are called lymphoblastoid cell lines (LCLs). As of today, EBV infection remains one of the most efficient mean to immortalize B cells in vitro. EBV´s life cycle can be divided in three different steps: virus entry and primary infection, latent phase (composed by latency I, latency II and latency III) and lytic phase.

Virus entry and primary infection:

EBV is able to infect B lymphocytes and epithelial cells causing a latent infection in most of the cases.

In order to latently infect resting memory B cells, the virus uses the normal pathway of B cell differentiation into antibody-secreting B cells (Figure 2). This means that mature, naive or memory B cells present in the lymphoid tissue has to be activated and consequently to proliferate in order to become a resting memory B cell which will be able to circulate in the peripheral blood. This mechanism was demonstrated in vivo by the group of DA. Thorley-Lawson in 1998 23 . They proved that resting memory B cells circulating through the peripheral blood are the only cells able to undergo spontaneous outgrowth, thus having the possibility of being immortalized.

The entry of EBV into resting B cells or epithelial cells involves the joint action of at least five viral proteins. First, the envelope protein gp350 binds to CD21 (complement receptor) which is present on B cells membrane or to an alternative receptor on epithelial cells which anchors the virus to the target cells. This action allows the binding of gp42 to the corresponding HLA class II molecule, which then mediates the membrane fusion that is carried out by the viral proteins gB, gH and gL 24 . gH and gL glycoproteins are present in all herpesviruses and are necessary for virus penetration into target cells. gH is essential for membrane fusion while gL acts as a chaperone for the proper folding of gH.

Virions that contain gH-gL-gp42 complexes infect B lymphocytes whereas virions containing only gH-gL complexes infect epithelial cells.

The group of JM. Palefsky demonstrated that primary infection of epithelial cells from the oropharynx is a complex mechanism that can occur in two different ways: either EBV-infected cells from saliva infect other cells via cell-to-cell contact involving the apical cell surface, or EBV virions infect directly the basolateral surface of cells with the joint action of cellular integrins 25 .

EBV latent infection:

During latency, EBV has a strict gene expression pattern which includes: six EBV nuclear antigens (EBNAs), two latent membrane proteins (LPMs), EBV-encoded small RNAs (EBERs) and miRNAs BART (BamHI-A region rightward transcribed). Depending on the proteins that are expressed, EBV latent infection can be classified in three different classes: latency I, latency II and latency III (Table 2). All of them have in common the expression of EBNA1.

EBV latency programs:

-Latency I, the EBNA1-only program:

This latency program is characteristic of the Burkitt lymphoma and implies the expression of a single viral protein, EBNA1, and small non-coding RNAs called EBERs. Nevertheless, there are some controversies because some groups found that the latent membrane proteins 1 and 2 (LMP1, LMP2) are also expressed in latency I cells. This latency I program is the expression pattern of EBV-carrying resting memory B cells which are EBV long-term hosts.

-Latency II, the default program:

The latency II program is characteristic of the Hodgkin's lymphoma and the nasopharyngeal carcinoma. Besides EBNA1, latency II cells also express three additional viral latent antigens: LMP1, 2A and 2B.

-Latency III, the growth program:

The latency III program is characterized by the complete expression of all EBV latent genes including all the genes expressed in the other latency programs as well as EBNA2, EBNA-LP, EBNA3A, EBNA3B and EBNA3C. This type of latency is observed in infectious mononucleosis (primary infection), LCL´s (lymphoblastoid cell lines) and some BL.

EBV latent proteins and gene products

In this paragraph EBV proteins and non-coding RNAs produced during the latent phase will be briefly presented (Figure 3 and Table 2): EBNA1 (EBV nuclear antigen 1) is the only viral protein expressed all along EBV life cycle. During the latent cycle EBNA1 has three essential functions for EBV: the replication and the maintenance of the viral genome 27 as well as the immune evasion of infected cells (see chapter 2). EBNA1 is also able to inhibit the spontaneous virus reactivation by inhibiting ZEBRA transcription, ZEBRA being essential to activate the lytic program 28 . In addition, EBNA1 is able to interact with premyelocytic leukemia proteins (PML) and induce their degradation, thereby preventing p53 activation and thus induction of apoptosis upon DNA damage 29 . Furthermore, EBNA1 contributes to the oncogenic process by inducing the apoptosis suppressor protein Survivin in EBV-associated lymphomas 30 .

LMP1 is a transmembrane protein that is expressed in all forms of HL, NPC and LCL. It contains six transmembrane domains that mediate its association with lipid rafts and signaling from two transformation effector sites that are necessary for EBV-mediated B cell transformation. LMP1 mimics the functions of CD40 receptor in B cells which is involved in the regulation of antibody production 31,32 . The group of E. Kieff demonstrated in 1993 that LMP1 is sufficient for EBV-mediated B cell transformation in vitro 33 . It is the main EBV-encoded oncogene and it is able to activate the signaling pathways of NF-κβ, PI3K, JNK and p38 33,34 . The activation of PI3K kinase pathway promotes cell survival and suppression of apoptosis, while the activation of JNK parthway induces cell proliferation and transformation 35,36 . In epithelial cells, it has been shown that LMP1 activates vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), cyclooxygenase-2 (COX-2) and matrix metalloproteinase 9 (MMP-9). All these factors are involved in the formation of new blood vessels that can feed tumor cells 37,38 .

LMP2A and LMP2B are two closely related EBV-encoded proteins expressed during the latency II and III programs. LMP2A contains 12 transmembrane domains (TM) and an N-terminal domain involved in cytoplasmic signaling. On the other hand, the isoform 2B has the same TM domains but lacks the N-terminal functional domain 39 . In B cells, LMP2A prevents the recruitment of the B cell receptor (BCR) in lipid rafts, which abrogates BCR functions. It also promotes B cell growth and induces lymphoma due to a BCR-like signal through the constitutive activation of the extracellular ERK/MAPK pathway 40 . In epithelial cells LMP2A stimulates cell motility and diffusion and enhances the transition to NPC due to the induction of the metastatic tumor antigen (MTA1) 41 . EBV infects resting B cells inducing a continuous cell proliferation. The resulting lymphoblastoid cells (LCLs) express the full set of latent proteins, which are able to reactivate quiescent B cells to enter into S phase, continue to proliferate and avoid apoptosis. The expression of the two intermediate-early lytic genes BZLF1 and BRLF1 triggers the activation of viral early gene expression involved in DNA replication and metabolism, followed by the expression of the late genes which are mostly involved in the capsid formation.

EBNA2 and EBNA-LP. These two proteins are involved in B cell transformation through the activation of the transcription of viral and cellular genes involved in cell transformation. EBNA2 is a phosphorylated DNA binding protein which is able to activate the transcription of CD23, CD21, c-Myc, c-fgr, BLR2/EBI1 and all latent genes 43 . EBNA2 contributes to cancer progression since it is also able to enhance the expression of the anti-apoptotic proteins Bcl-xL, Bcl-2 and bfl-1. Several potential functions like mediating apoptosis have been attributed to EBNA-LP; nevertheless, the most widely confirmed is its ability to act as a coactivator of EBNA2. E. Kieff et al demonstrated that EBNA-LP plays a critical role in B cell transformation by stimulating EBNA2-mediated transcription activation 44 .

EBNA3A, EBNA3B and EBNA3C. The expression of these three proteins which share a similar structure is under the control of the same promotor. All of them are able to regulate the transcription of EBNA2 and Notch via direct binding to RBPJ transcription factor 45 . EBNA3A and EBNA3C are essential for B cell transformation and immortalization whereas EBNA3B is dispensable. The group of F. Wang demonstrated that EBV-infected cells lacking EBNA3B are still able to stimulate B cell transformation and immortalization in vitro 46 . EBNA3A and 3C inhibit the production of BIM transcripts which impedes the apoptosis process and thus allows the proliferation of cancer cells. It has been demonstrated that EBNA3C can affect p53-mediated activities. For example, EBNA3C is able to interact directly with the N-terminal domain of p53 which is also the binding site for several important cellular factors like pRb and c-Myc. In line with this, EBNA3C recruits MDM2 in order to favor p53 ubiquitination and degradation by the proteasome 47 .

EBERs (Epstein-Barr-encoded small RNAs).

These are EBV-encoded small non-polyadenylated noncoding RNAs which are transcribed by the host RNA polymerase III. EBER1 is 167-nucleotides long whereas EBER2 is 172-nucleotides long. They are the most common non-coding EBV RNAs during the latent phase.

The expression of EBERs enhances tumor formation and growth both in vitro and in vivo 48 . It has been demonstrated that EBERs are able to interact with La protein, the ribosomal protein L22, phosphoribulokinase (PKR) and other cellular proteins. It has been shown that the association of EBERs with La protein causes a reduction of RNA pol III transcripts stability 49,50 . The binding of EBERs to L22 causes the delocalization of L22 to the nucleoplasm which affects the translation of specific RNAs. Finally the interaction of these RNAs with PRK protein, which is involved in interferon regulation, blocks the PRK-mediated phosphorylation of the initiation factor 2α (IF2α), which in turn blocks the translation initiation mediated by IF2α and also the resistance to IFNα-induced apoptosis 51 . BARTs miRNAs are present in all EBV-associated latency phases and their principal function is to prevent B and epithelial cells to trigger apoptosis. For example, miR-BART5 promotes cell survival by targeting the pro-apoptotic gene PUMA and, in NPC, BARTs miRNAs contribute to the establishment of the latent infection.

EBV-encoded miRNAs BARTs

On the other hand BHRF1 expression is restricted mostly to latency III program 52 and accomplishes similar functions. Once a B cell is immortalized it usually maintains EBV in a latent phase. However virus life cycle can switch from latency to replication spontaneously or via induction by external stimuli like 12-Otetradecanoylphorbol-13-acetate (TPA) and butyrate 53 . Later on, it was shown that the expression of the single viral gene BZLF1 (also known as ZEBRA) can trigger the lytic cycle.

EBV lytic cycle

The lytic cycle is the active phase of EBV life cycle in which approximatively 80 viral proteins are expressed in order to produce the maximal quantity of virions that will further infect other cells. The lytic phase is divided in three distinct steps among which various proteins are expressed: the immediate-early, early and late proteins. The expression of ZEBRA, the first lytic protein, triggers the switch from latent to lytic phase (Figure 3). ZEBRA is a transcription factor that controls the expression of the immediate-early gene Rta that consequently, and together with ZEBRA, will control the expression of many early genes 4 . ZEBRA activates the transcription by binding to ZEBRA response elements (ZREs) found in the promoters of EBV lytic cycle target genes. During the early phase EBV encodes genes that are involved in the nucleotide metabolism like the DNA polymerase or the thymidine kinase BXLF1. Finally during the late phase the "core replication" genes are expressed in order to ensure a fast replication of the viral genome. Among these genes are BMRF1 (DNA polymerase processivity factor), BBLF4 (helicase), BALF5 (DNA polymerase), BSLF1 (primase), BALF2 (single-stranded DNA binding protein) and BBLF2/3 (primase accessory protein) 54 .

CHAPTER II: EBV, a master in the art of hiding

The presentation of antigenic peptides by Major Histocompatibility Complex (MHC) class I and II molecules allows the immune system to distinguish between self and non-self. Thanks to this mechanism, the immune system is able to detect bacterial, parasite or viral infections. This process involves the degradation of polypeptides into peptides and their later association with MHC molecules in the endoplasmic reticulum (ER). These loaded MHC complexes are then transported to the cell surface of the antigen presenting cell (APC) and recognized by a specific T cell receptor (TCR) which is exposed at the surface of T cells. There are two different sources of antigenic peptides: intracellular antigens and extracellular antigens. The presentation of intracellular peptides (produced by the cell machinery) is driven by MHC class I molecules that are recognized by cytotoxic T lymphocytes (CTL, T CD8 + ). On the other hand, extracellular peptides (not produced by the cell) are presented to T-helper lymphocytes (T CD4 + ) by the MHC class II molecules.

In normal conditions MHC class I molecules present peptides derived from endogenous proteins in all nucleated cells. Nevertheless, if a viral infection occurs, viral antigens produced by the infected cells will be processed by the 26S proteasome and small peptides of about 8-10 residues will be shuttled by the transporter associated with antigen processing (TAP, 1 and 2) into the ER where the assembly with the MHC class I molecule will take place 55 .

EBV has evolved several strategies to avoid immune recognition and destruction of infected cells during both lytic and latent phases. These strategies are detailed below.

EBV immune evasion strategies during lytic phase

Gammaherpesviruses have evolved several strategies so that infected cells can evade the host immune system. In this section, EBV strategies that allow infected cells to evade T cell recognition during the lytic phase /virion production phase will be described.

Strategies against the activation of the IFN-γ pathway

The interferon γ (IFN-γ) is one of the most important cytokines released in response to all viral infections and to some bacterial and protozoal infections. It is expressed during the innate response by natural killer cells (NK) and by T CD8 + and T CD4 + Th1 cells during the adaptive response. This cytokine induces a cascade of signaling that leads to anti-viral responses targeting all steps of the life cycle of the virus. It is an activator of macrophages and it also enhances the expression of MHC class II molecules. Because of this, γ-herpesviruses have developed several strategies in order to inhibit either IFN-γ production or its signaling pathways. In particular, during the lytic phase, EBV expresses several antigens like BCRF1 (which is a homolog of IL-10), BZLF1 and BARF1 (vCSF). BCRF1 encodes vIL-10, a virokine that mimics the immune suppressive action of IL-10. BCRF1 is expressed during the late lytic phase and causes the inhibition of IFN-γ and IL-2 synthesis and also inhibits the maturation of dendritic cells 56 . BZLF1 is expressed during the intermediate early lytic phase and encodes ZEBRA which is the trigger of the lytic phase, as described above. BZLF1 blocks tyrosine phosphorylation and nuclear translocation of STAT1 (a transcription factor which is activated by IFN-γ), decreases IFN-γ expression and also inhibits the MHC class II surface expression which is stimulated by IFN-γ 57 . Finally, BARF1 is expressed during the early lytic phase and prevents the proliferation of macrophages and also inhibits IFN-γ production by monocytes 58 .

Strategies against MHC class I antigen presentation pathway

γ-herpesviruses also encode other proteins which can affect several steps of the MHC class I antigen presentation pathway. In the case of EBV, the BCRF1 gene which is expressed during the lytic phase is able to downregulate the expression of the proteasome subunit LMP2 and of TAP1, thus preventing the production and presentation of virus-derived antigenic peptides 59 . Moreover, BGLF5, an EBVencoded alkaline exonuclease, can promote host mRNA degradation by retaining mRNAs in the nucleus and thereby shutting off protein synthesis. The resulting inhibition of cellular protein synthesis serves to ensure maximal viral gene expression and evasion from host immune response, as it induces host MHC class I expression shutoff 60 . The early lytic expressed BNLF2α gene is able to interact with the TAP transporter and to inhibit the binding capacity of TAP to both the antigenic peptides and to ATP. Given that ATP hydrolysis is essential to ensure the opening of the transmembrane pore and the translocation of the peptides to the ER this also participates to immune evasion of γ-herpesviruses 61 . Finally, during the early stage of the lytic cycle a third gene BILF1 also contributes to the attenuation of MHC class I responses. BILF1 is a G-protein-coupled receptor (GPCR) which is able to increase MHC class I molecules turnover by inducing MHC class I internalization from the cell surface and its subsequent degradation by the lysosomal compartment 62 .

Strategies against MHC class II antigen presentation pathway

The antigenic presentation by the MHC class II molecules involves the loading of free MHC class II molecules inside the MHC II loading compartment (MIIC) with antigenic peptides produced from the endocytic pathway. Once loaded, MHC class II complexes are translocated to the surface of APC cells.

In contrast to the MHC class I antigen presentation pathway, little is known about how γherpesviruses manage to escape T CD4 + response.

The intermediate early lytic BZLF1 gene has the ability to inhibit the transcription of the gene encoding MHC class II molecules by binding and repressing CIITA PIII. CIITA gene is a master regulator of the MHC class II pathway that promotes the expression of MHC class II molecules. This gene is under the control of several promoters, the number I and III being the most important in B cells in order to ensure the constitutive expression of CIITA [START_REF] Li | Down-regulation of MHC class II expression through inhibition of CIITA transcription by lytic transactivator Zta during Epstein-Barr virus reactivation[END_REF] .

Interestingly, EBV uses the MHC class II complex as a cofactor of CD21 in order to infect B cells [START_REF] Li | Epstein-Barr virus uses HLA class II as a cofactor for infection of B lymphocytes[END_REF] . EBV attachment to B cell membrane occurs via the envelope protein gp350 which binds to the CD21 receptor. In order to achieve B cell infection, the interaction of the envelope protein gp42 encoded by BZLF2 is required 24,[START_REF] Wang | Epstein-Barr virus lacking glycoprotein gp42 can bind to B cells but is not able to infect[END_REF] . gp42 associates to MHC class II molecules in the ER and once at the APC surface it can block the interaction of the TCR with the peptide-MHC class II complexes, thereby inhibiting antigenic recognition by CD4+ T helper cells [START_REF] Ressing | Interference with T cell receptor-HLA-DR interactions by Epstein-Barr virus gp42 results in reduced T helper cell recognition[END_REF] . In addition, the early lytic gene BGLF5, which encodes the EBV endonuclease, catalyzes the host protein synthesis shutoff by inducing the mRNA degradation of several host genes, among which the MHC class II molecules 60 . M. Rowe et al demonstrated that cotransfection of BGLF5 and GFP vectors in 293 cells reduced the levels of GFP mRNA. Moreover, the half-life of GFP mRNA decreased in the presence of BGLF5 demonstrating that it affects mRNA turnover.

Immune evasion strategies during latent phase

Human γ-herpesviruses have evolved a sophisticated strategy to escape T CD8 + recognition. This strategy is mainly based on the control of the expression of a single viral protein: the genome maintenance protein (GMP). GMP can be considered as the Achilles' heel of these viruses because it is essential for viral genome replication and maintenance, and at the same time it is highly antigenic and T cells raised against these proteins exist in all infected individuals.

EBNA1 Genome Maintenance Protein (GMP): the Achilles´ heel of EBV

GMPs are expressed by all γ-herpesviruses. They have the crucial role of ensuring the replication and maintenance of the viral genome after each M phase of the cell cycle. Indeed, they are DNA binding proteins that display two pivotal roles: i) they participate in viral genome replication and ii) they tether the viral episome to host cell chromosomes, thereby ensuring the maintenance of the viral genome in each daughter cell during host cell division [START_REF] Blake | Immune evasion by gammaherpesvirus genome maintenance proteins[END_REF] . Like all GMPs, the EBNA1 protein, the GMP of EBV, has two essential functions during the viral life cycle: the replication of the viral episome and its maintenance in daughter cells. In this section I will present a description of EBNA1, with a focus on these two GMP functions.

EBNA1 protein organization

EBNA1 is a multifunctional protein encoded by BKRF1 gene which is located in the BamHI-K fragment of the viral episome (Figure 1). It is a homo-dimeric DNA-binding protein expressed in all EBV-infected cells [START_REF] Kieff | Epstein-Barr virus and its replication[END_REF] . EBNA1 protein from the EBV prototype strain B95.8 is a 641 amino acids protein that contains three different functional domains: a central glycine-alanine rich (GAr) domain, two arginine-glycine domains (RG) and a C-terminal domain 69 (Figure 4). The first 30 amino acids of the N-terminal domain of EBNA1 are not associated with any particular cellular function. The first RG-rich domain is located from residues 33 to 89 and the second between residues 328 and 386 and both are responsible for EBNA1 association with host chromosomes 27 . These RG-rich domains are similar to the RGG motif present in several RNA-binding proteins and J. Norseen et al demonstrated that they are involved in the recruitment of the origin recognition complex (ORC) to the origin of replication (OriP) of EBV genome for replication initiation [START_REF] Norseen | Role for G-quadruplex RNA binding by Epstein-Barr virus nuclear antigen 1 in DNA replication and metaphase chromosome attachment[END_REF] . The central Gly-Ala (GAr) domain of EBNA1 is conserved in all EBV strains and varies in length from 60 to 300 residues 71 . The canonical GAr domain contains 239 residues. This central GAr domain is crucially involved in EBNA1-based EBV immune evasion during the latent phase of viral infection. Of note, this ability of EBV to evade the immune system depends on the length of GAr: the longest GAr display the most efficient immune evasion. The C-terminal domain of EBNA1 is a DNA binding domain (DBD). EBNA1 DBD binds to the Qp promoter which is responsible for EBNA1 transcription during latency phases I and II 72 (Figure 4). EBNA1 DBD also interacts with the OriP of the viral genome at each M phase of the cell cycle to ensure the viral replication and maintenance. All EBNA1 domains are represented, as well as the amino acid positions. In green are represented the two glycine-arginine (RG) domains, in red the glycine-alanine repeat (GAr), in blue de nuclear localization signal (NLS) and in violet the DNA binding domain (DBD).

EBNA1 function in genome replication and maintenance

As described above, both EBV episome replication and maintenance rely on EBNA1 binding to multiple sites on the OriP. The OriP covers approximatively 1700 bp and contains two different cisacting elements that are both necessary to ensure episomal replication. The first element is a family of 20 tandem copies of a 30 bp sequence (FR) and the second is a 114 bp dyad symmetry (DS) [START_REF] Reisman | A putative origin of replication of plasmids derived from Epstein-Barr virus is composed of two cis-acting components[END_REF][START_REF] Frappier | The Epstein-Barr Virus EBNA1 Protein[END_REF] . FR elements are not directly involved in DNA replication but rather in mitotic segregation and transcriptional activation. The DS element is responsible for DNA replication and it has been shown to be sufficient for EBV replication in the presence of EBNA1 [START_REF] Gahn | The Epstein-Barr virus origin of plasmid replication, oriP, contains both the initiation and termination sites of DNA replication[END_REF] . The crystal structure of EBNA1 bound to DNA has been solved and it shows that its binding site is composed of two different DNA binding domains. The N-terminal DNA binding domain involves the residues 470-503 of EBNA1 and is called the flanking domain, whereas the C-terminal one, called the core domain, includes residues 504-604 [START_REF] Bochkarev | Crystal structure of the DNA-binding domain of the Epstein-Barr virus origin-binding protein, EBNA1, bound to DNA[END_REF] . Structure resolution shows that EBNA1 binds to its binding site as a dimer. The core domain possesses two α-helices that make specific contacts with the DNA. Moreover, EBNA1 assures viral genome maintenance by tethering the viral episome to the host mitotic chromosomes. EBNA1 is the most studied EBV-encoded protein and, at the end of the nineties, the team of L. Frappier used EBNA1 as a bait in a two-hybrid screen to identify human proteins that interact with EBNA1. EBP2 (EBNA1 binding protein 2) was shown to specifically interact with EBNA1 [START_REF] Shire | EBP2, a human protein that interacts with sequences of the Epstein-Barr virus nuclear antigen 1 important for plasmid maintenance[END_REF] . EBNA1 region that binds to EBP2 has been mapped to the small central arginineglycine (RG) rich domain. The deletion of this RG domain in EBNA1 prevents its interaction with EBP2 and consequently the ability of EBNA1 to support the long-term maintenance of a plasmid containing the EBV origin (OriP) in human cells (Figure 5). Resting memory B cells are EBNA1 positive but there is not transcription neither translation of EBNA1. In contrary, mitotic B cells expressed a minimal level of EBNA1 in order to assure episomal maintenance which is low enough to be undetected by the host immune system.

EBNA1 is thus an essential protein for EBV genome to persist into highly dividing cells. It is also highly antigenic and CD8 + T cells raised against EBNA1 exist in all infected individuals which may compromise EBV persistence in its host. However EBNA1-infected cells are not eliminated by the host immune system. This is due to another property of EBNA1 that allows EBV infected cells to avoid T cell recognition. γ-herpesviruses have indeed developed several strategies in order to decrease the quantity of antigenic peptides derived from their GMPs that are presented at the cell surface of infected cells via the MHC class I complex. Most γ-herpesviruses strategy to evade the immune system recognition during latent phase is based on a tight control of their GMP expression level: it should be high enough to ensure genome replication and maintenance but low enough to avoid the detection of virus-infected cells by T CD8 + cells.

In summary, most EBV strategy to evade the host immune system is to tightly control EBNA1 level to a minimal level to ensure viral genome replication and maintenance and at the same time to minimize presentation of EBNA1-derived antigenic peptides by the MHC class I complex [START_REF] Frappier | The Epstein-Barr Virus EBNA1 Protein[END_REF] . As described in the next section, EBV strategy to evade the immune system lays on the GAr domain of EBNA1.

EBNA1 GAr domain is responsible for EBNA1-based immune evasion

As state above, most EBV´s strategy to evade the immune system is based on a tight control of its GMP (EBNA1) expression level. This tight control actually involves its central GAr domain (Figure 4).

Although the GMPs of all γ-herpesviruses share poor sequence similarities, they all possess a central domain which sequence is highly repetitive. This domain is not essential for genome replication and maintenance functions but it plays a crucial role in the mechanism of immune evasion of infected cells. The function of this central domain on immune evasion has been shown only for human γherpesviruses EBV and KSHV, while for Old / New World Monkeys γ-herpesviruses, this function doesn't seem to exist.

Two mechanisms have been successively explored: GAr was initially thought to act via its ability to inhibit proteasome degradation, thereby limiting the production of EBNA1-derived antigenic peptides [START_REF] Shire | EBP2, a human protein that interacts with sequences of the Epstein-Barr virus nuclear antigen 1 important for plasmid maintenance[END_REF] . It was later demonstrated that its effect rather involves its capacity to self-limit the translation of its own mRNA in cis, a mechanism which also results in the reduction of the production of antigenic peptides [START_REF] Daskalogianni | Gly-Ala repeats induce position-and substrate-specific regulation of 26 S proteasome-dependent partial processing[END_REF] .

Interestingly, the switch from the first model (26S proteasome inhibition) to the second model (translation inhibition in cis) paralleled the evolution of models and knowledge regarding the main source of antigenic peptides [START_REF] Moore | Introduction of soluble protein into the class I pathway of antigen processing and presentation[END_REF][START_REF] Fahraeus | Do peptides control their own birth and death? Nature reviews[END_REF] . These two models will be described below.

EBNA1 GAr domain is responsible for latent EBV-infected immune evasion

In the beginning of the 90´s the group of MG. Masucci observed that latently EBV-infected cells evade the immune system despite the fact that CTLs directed toward EBNA1 are present in all EBV-infected individuals. In addition, the GAr domain represents the major target of T cells raised against EBNA1derived epitopes. For this reason, they wondered if this particular domain could affect the host cell immune response. To answer this question, they constructed several recombinant vaccinia (vacc) viruses expressing the full-length EBNA1 or EBNA1ΔGAr, a form of EBNA1 deleted for its central GAr domain, in which they inserted the antigenic peptide containing the amino acids 416-424 from the EBNA3B protein. Then, they showed that fibroblasts infected with vacc expressing EBNA1 remain undetected from specific CTLs against EBNA3B whereas fibroblasts infected with vacc expressing EBNA1ΔGAr or only EBNA3B epitope were efficiently recognized and eliminated [START_REF] Levitskaya | Inhibition of antigen processing by the internal repeat region of the Epstein-Barr virus nuclear antigen-1[END_REF] . Moreover, they demonstrated that GAr domain acts as a cis-acting element, since the overexpression of EBNA1 alone didn't affect the CTLs recognition of EBNA3B itself. They also determined the ability of EBNA1 GAr domain to act in N-terminal or C-terminal position by introducing the GAr sequence in frame with EBNA3B coding sequence. The results showed similar CTLs recognition of fibroblasts infected with vacc EBNA1 or vacc EBNA3B-GAr, which means that the GAr effect on immune evasion is not significantly affected by its position in N-or C-terminal. The group of MG. Masucci showed that GAr has an inhibitory effect on antigenic recognition but the mechanism involved in this inhibitory effect was still undechiphered. Nevertheless, they proposed two different mechanisms: GAr could interfere with the proteolytic pathway that leads to antigenic peptides production, or GAr-containing proteins could be sequestered in a compartment which is not accessible for antigenic peptides processing 77 .

Origin of the antigenic peptides presented by MHC class I molecules

The source of antigenic peptides for MHC class I molecules has been a subject of controversy.

Peptides where originally through to be produced thought the degradation of full proteins in the cytoplasm by the 26S proteasome. This hypothesis was supported by several observations: i) blocking the proteolysis in the endosomes or lysosomes did not affect the antigenic presentation of MHC class I molecules, while antigenic presentation by class II was affected, ii) drugs able to block the activity of the proteasome also inhibit the antigenic presentation by MHC class I 82 . Moreover, proteins that are specifically targeted to the cytoplasm are also presented by the MHC class I 79 , iii) the last and most important observation was that cells lacking the TAP transporter were not able to present antigenic peptides by MHC class I, since they lack the continual supply of antigenic peptides from the cytoplasm [START_REF] Rock | Degradation of cell proteins and the generation of MHC class Ipresented peptides[END_REF] . However, the observation that viral proteins are in general very stable suggests that the degradation of full-length proteins by the proteasome and the later presentation of derived antigenic peptides by the MHC class I would take many hours or even days. Nevertheless, T CD8 + responses against viral antigens are driven in only one hour. This can be explained only if there is an alternative mechanism that provides antigenic peptides more rapidly. The source of antigenic peptides in the MHC class I pathway has been a subject of controversy. It was thought that the majority of antigenic peptides comes from the degradation of full-length proteins by cytosolic proteasome degradation and their subsequent translocation into the endoplasmic reticulum in which they are subsequently loaded onto MHC class I molecules. Later it was proposed that the most important source of antigenic peptides comes from defective products of the mRNA translation (DRiPs). These DRiPs are produced: a) by alternative initiation or pre-termination, b) by ribosomal frameshift, c) from the 3´non translated region or d) from misfolded proteins.

In 1996 another source of antigenic peptides was proposed. The most important source of antigenic peptides was suggested to be the defective ribosomal products (DRiPs) that are products generated during the first round of translation [START_REF] Yewdell | The DRiP hypothesis decennial: support, controversy, refinement and extension[END_REF] . These DRiPs include defective, misfolded or pre-mature polypeptides as a result of mistakes during the translation process (Figure 6). The first observation that supported this model came from E.A Reits et al in 2000. They were trying to find the source of the antigenic peptides presented by TAP to the MHC class I. Because TAP mobility increases when it is not associated to an antigenic peptide, this can be used to monitor the intracellular peptide content in vivo. They demonstrated that TAP mobility in the ER membrane decreases when it is associated to peptides. Then, they showed an increase in TAP mobility after cycloheximide treatment in cells infected with influenza A virus. This allowed them to conclude that the most important source of antigenic peptides comes from mRNA translation [START_REF] Reits | The major substrates for TAP in vivo are derived from newly synthesized proteins[END_REF] . At the same time, U. Schubert et al demonstrated that cycloheximide treatment inhibits the transport of MHC class I molecules from the ER to the cell surface. They showed that one-third of newly synthetized proteins are rapidly degraded and this seems to be the source of MHC I peptides [START_REF] Schubert | Rapid degradation of a large fraction of newly synthesized proteins by proteasomes[END_REF] .

Most of the experiments that favor the DRiPs hypothesis are based on the observation that presentation by MHC I molecules is markedly reduced when de novo protein synthesis is inhibited. This theory has been shown to be right for most proteins but not all, since it assumes that all mature proteins are accessible for MHC I presentation, and in fact antigens that result from autophagy or lysosomal degradation would escape from proteasome degradation and thus would never achieve MHC class I loading. Moreover, protein synthesis inhibitors, like cycloheximide, not only affect the synthesis of antigenic peptides but also could affect the production of several proteins involved in the MHC class I pathway, and thus cycloheximide-based experiments cannot be used as a fully convincing demonstration that DRiPs are the major source of antigen peptides. J.D Colbert et al challenged the DRiPs model by studying the proportion of mature proteins to MHC I presentation using a tetracycline-inducible system to express antigens that are conditionally stabilized upon ligand binding (Shield-stabilized FKBP antigen). Results showed that at least for this particular antigen the major source of antigenic peptides for MHC I pathway seems to be mature proteins since DRiPs are supposed to be derived from defective non-functional proteins, and in contrast Shield-stabilized Ags bind ligands and are fluorescent. Also, DRiPs are normally and rapidly polyubiquitinated; however polyubiquitination was not observed for this particular antigen [START_REF] Colbert | Substrate-induced protein stabilization reveals a predominant contribution from mature proteins to peptides presented on MHC class I[END_REF] . Finally they showed that inhibition of protein synthesis indeed blocks the synthesis of other components of the MHC I pathway.

Identification of the mechanism by which GAr domain allows immune evasion: proteasome degradation or translation inhibition?

As stated above, the main source of antigenic peptides has been largely debated, between the fulllength protein degradation model and the DRiPs model. Interestingly, the role of GAr in EBNA1 immune evasion followed the same evolution.

It was initially thought that the inhibition of the antigenic presentation by the GAr domain was due to inhibition of protein degradation by the 26S proteasome, which was fully consistent with the model existing at that moment, establishing that the principal source of antigenic peptides comes from degradation of full proteins by the 26S proteasome. Several studies showed that the fusion of the GAr domain to various instable proteins led to the inhibition of their degradation by the 26S proteasome [START_REF] Sharipo | A minimal glycine-alanine repeat prevents the interaction of ubiquitinated I kappaB alpha with the proteasome: a new mechanism for selective inhibition of proteolysis[END_REF][START_REF] Dantuma | Inhibition of proteasomal degradation by the gly-Ala repeat of Epstein-Barr virus is influenced by the length of the repeat and the strength of the degradation signal[END_REF] . In line with this theory, J. Levitskaya et al demonstrated in 1997 that, indeed, in vitro translated EBNA1 was resistant to proteasomal degradation whereas a form of EBNA1 which lacks its GAr domain, EBNA1ΔGAr, was sensitive and efficiently degraded. Also EBNA3B chimeras containing full-length GAr sequences or 17 Gly-Ala residues were not degraded by the 26S proteasome. Nevertheless, they were not able to determine which step of the degradation pathway was affected, since they showed that GAr containing proteins could be efficiently polyubiquitinated [START_REF] Levitskaya | Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein-Barr virus nuclear antigen 1[END_REF] . They thus proposed that the GAr sequences could modify the folding pattern of the resulted peptides and that this may affect their association with components of the ubiquitin-proteasome pathway. This was supported by the observation that GAr sequences inserted in the IκB protein allows the polyubiquitination of the protein but prevents the formation of stable complexes with the 26S proteasome [START_REF] Sharipo | A minimal glycine-alanine repeat prevents the interaction of ubiquitinated I kappaB alpha with the proteasome: a new mechanism for selective inhibition of proteolysis[END_REF] . Based on these results the GAr polypeptide has been proposed to form secondary β-sheets structures which are resistant to unfolding and thus block the binding of the proteasome complex. Another important aspect is the GAr-length dependency: the longer the GAr sequence is, the strongest is its effect on proteasomal inhibition.

Three years later J. Tellam et al demonstrated that forcing EBNA1 to intracellular degradation by combining the N-rule and the ubiquitin (Ub) pathways, dramatically reduces EBNA1 half-life. Ub-EBNA1 constructs were efficiently degraded by the proteasome and the resulting antigenic peptides were actively presented to CTLs. These results were the first demonstration that the GAr domain is not completely able to inhibit proteasomal degradation [START_REF] Tellam | Targeting of EBNA1 for rapid intracellular degradation overrides the inhibitory effects of the Gly-Ala repeat domain and restores CD8+ T cell recognition[END_REF] .

However, it was later demonstrated that, contrary to J. Levistkaya observations, the deletion of the GAr domain doesn't affect EBNA1 turnover, as it is a highly stable protein by itself [START_REF] Daskalogianni | Gly-Ala repeats induce position-and substrate-specific regulation of 26 S proteasome-dependent partial processing[END_REF] . Concomitantly, observations showed that the inhibition of translation of a stable protein decreases the antigenic presentation which supports the idea of an alternative source of antigenic peptides.

At the same time, JW. Yewdell et al indeed demonstrated that the so-called DRiPs (defective ribosomal products) which are abortive products of translation, are the main source of antigenic peptides [START_REF] Yewdell | The DRiP hypothesis decennial: support, controversy, refinement and extension[END_REF][START_REF] Schubert | Rapid degradation of a large fraction of newly synthesized proteins by proteasomes[END_REF] .

Soon after it was proven that the GAr domain effect was in fact due to the inhibition of translation of its own mRNA in cis [START_REF] Fahraeus | Do peptides control their own birth and death? Nature reviews[END_REF][START_REF] Yin | Self-inhibition of synthesis and antigen presentation by Epstein-Barr virus-encoded EBNA1[END_REF] . In line with this demonstration was the observation that the truncated EBNA1ΔGAr protein is normally translated and leads to an efficient presentation of EBNA1-derived antigenic peptides and thereby recognition of EBNA1-expressing cells by specific T cells. This constitutes the proof of principle that the GAr domain of EBNA1 acts by inhibiting the translation of its own mRNA, thereby limiting the production of DRiPs and thus the presentation of EBNA1-derived antigenic peptides. Later, S. Cardinaud et al demonstrated that the effect of the GAr domain was dependent on its position inside the mRNA [START_REF] Cardinaud | The synthesis of truncated polypeptides for immune surveillance and viral evasion[END_REF] . They made two different constructs of EBNA1 mRNA in which they inserted the ovalbumin antigen peptide SIINFEKL (SL8) upstream or downstream of the GAr domain. In vitro translation experiments showed that both constructs had the capacity of inhibiting EBNA1 translation, and that this inhibition was due to an overall translation inhibition of EBNA1 mRNA and not to difficulties in ribosome binding. In addition, they demonstrated that when the SL8 was present upstream the GAr domain, T CD8 + against SL8 normally recognized this antigenic peptide, whereas when it was located downstream the GAr, the translation of SL8 was strongly reduced, as was antigenic presentation [START_REF] Cardinaud | The synthesis of truncated polypeptides for immune surveillance and viral evasion[END_REF] . These data suggest that the GAr domain efficiently inhibits the translation of its own mRNA when it is fused to the N-terminal of the fusion protein.

Mechanism of GAr-based translation inhibition

Nowadays most of the people working in the EBV field acknowledge that most, if not all, of GArbased effect on immune evasion is due to its ability to self-inhibit the translation of its own mRNA in cis. The exact mechanism of this self-inhibition of translation was under investigation and largely debated and was still undechiphered at the time I started this thesis.

Two non-exclusive hypotheses have been proposed: (i) the inhibition of translation by the GAr domain could be due to the GAr polypeptide itself [START_REF] Daskalogianni | Gly-Ala repeats induce position-and substrate-specific regulation of 26 S proteasome-dependent partial processing[END_REF][START_REF] Moore | Introduction of soluble protein into the class I pathway of antigen processing and presentation[END_REF] ; (ii) the particular structure of the GAr domain encoding mRNA could be responsible for its effect [START_REF] Tellam | Messenger RNA sequence rather than protein sequence determines the level of self-synthesis and antigen presentation of the EBV-encoded antigen, EBNA1[END_REF] . These two mechanisms are not mutually exclusive and several observations demonstrated that probably both of them are required for EBV to efficiently self-limit EBNA1 expression and therefore evade the immune system during latent infection (Figure 7).

These two hypotheses will be presented and discussed in the next section. EBNA1 protein is the only protein expressed in all EBV-infected cells. It´s central Gly-Ala domain (GAr) inhibits its own translation in cis which causes a decrease in the production of antigenic peptides and thus a decrease in T CD8 + recognition. The mechanism involved in this translation regulation is still undechiphered, however it has been suggested to involve either 1) the structure of GAr-encoding mRNA or 2) the GAr polypeptide itself, or both. Figure extracted from 4 .

GAr polypeptide is responsible for the translation inhibition of its own mRNA.

This first model establishes that the nascent GAr polypeptide would inhibit the initiation of translation of its own mRNA in cis. S. Apcher et al demonstrated that the insertion of c-myc IRES (internal ribosomal entry site) in the 5´-UTR of EBNA1 mRNA completely abrogates GAr-based inhibition of translation and antigenic presentation [START_REF] Apcher | Epstein Barr virus-encoded EBNA1 interference with MHC class I antigen presentation reveals a close correlation between mRNA translation initiation and antigen presentation[END_REF] . Since this insertion did not modify the mRNA coding sequence nor the protein sequence, this result suggests that the GAr-based inhibition of translation requires cap-dependent translation initiation factors. Nevertheless, this effect was only observed for the c-myc IRES and not for other viral IRES like EMCV (encephalomyocarditis virus). This could be explained since many authors showed that c-myc IRES can activate the cap-independent translation of nuclear mRNAs thanks to a collaboration with some nuclear factors, which allows mRNA export to the cytoplasm. In contrast, EMCV IRES can only activate the translation of cytoplasmic mRNAs [START_REF] Stoneley | Analysis of the c-myc IRES; a potential role for cell-type specific trans-acting factors and the nuclear compartment[END_REF] . This suggests that GAr-carrying mRNAs have a nuclear localization in comparison to cellular mRNAs which are generally located in the cytoplasm, what we recently confirmed for EBNA1 mRNA.

In addition, prematurely terminated EBNA1 products were not detected, which supports the idea that the ribosome has no problem to progress through the GAr sequence, and antibodies directed against the GAr domain can indeed enhance translation, which supports the idea of a direct role for the GAr peptide 5,[START_REF] Apcher | Epstein Barr virus-encoded EBNA1 interference with MHC class I antigen presentation reveals a close correlation between mRNA translation initiation and antigen presentation[END_REF] . This polypeptide hypothesis is also reinforced by the fact that other lymphocryptovirus like Papiine herpesvirus 1 and Macacine herpesvirus 4 contain a GMP (baEBNA1 and rhEBNA1, respectively) which fulfills the same function than EBNA1 in genome replication and maintenance but which has no effect on immune evasion [START_REF] Blake | Immune evasion by gammaherpesvirus genome maintenance proteins[END_REF] . Both GMPs present a GAr-like domain which differs from EBNA1 GAr domain in their amino acid composition. Indeed, their Gly-Ala repeats are disrupted by serines (Ser). In line with these observations, replacing some Gly of EBNA1 GAr domain by Ala or Ser stimulates its translation in vivo 5 . All these observations indicated that the GAr polypeptide seems to be a key factor in GAr-based translation inhibition. A model in which GAr position effect could be explained by the fact that the nascent GAr polypeptide could interfere with the binding of translation initiation factors has been proposed. Hence, according to this model, when GAr is located in a C-terminal position it cannot efficiently interfere with translation inhibition (Figure 8). Moreover, in vitro translation experiments showed that GAr-based inhibition of translation is dependent on the quantity of mRNA to be translated. This observation is in line with the presence of a positive translation factor as at high mRNA concentration the GAr polypeptide / translation factor ratio is high hence allowing an efficient translation inhibition effect. In contrast, if this ratio is low the predominance of the translation factor in comparison to GAr-peptide leads to an inefficient inhibition of translation 5 .

GAr-encoding mRNA sequence is responsible for GAr-based translation inhibition

This theory was first supported by observations that the GMPs of other herpesviruses do contain a central domain which has few or even absolutely no amino acid conservation but high homology in terms of nucleotide sequence and purine enrichment (Table 3) [START_REF] Cristillo | Double-stranded RNA as a not-self alarm signal: to evade, most viruses purine-load their RNAs, but some (HTLV-1, Epstein-Barr) pyrimidine-load[END_REF] . For example, in LANA1 (GMP of Kaposi-sarcoma associated virus) the glutamine-glutamate-aspartate (QED) central domain has no homology with GAr protein sequence, but in terms of nucleotide sequence both are purine-rich and present more than 50% homology. Interestingly, introducing a frameshift in QED central domain produces a new repeat domain which has 65% homology with GAr protein sequence [START_REF] Zaldumbide | In cis inhibition of antigen processing by the latency-associated nuclear antigen I of Kaposi sarcoma herpes virus[END_REF] . This suggests that the nucleotide sequence encoding the central domain of the GMPs rather than the encoding peptide is involved in GAr-based translation inhibition and that the purine-rich region could be A and B panels show the position-dependent effect of the GAr whereas panels C and D explain the dose-dependent effect observed in an in vitro translation assay.

responsible for the immune evasion by causing the stalling of the ribosomes, and thus pretermination of the elongation process [START_REF] Tellam | Regulation of protein translation through mRNA structure influences MHC class I loading and T cell recognition[END_REF] . This was further supported by observations showing that +1 and +2 frameshifts within the GAr mRNA sequence cause changes in the aminoacid composition of the repetitive sequence (+1: glycine/glutamine/glutamic acid; +2: glycine/arginine/serine) but have no effect on EBNA1 translation or antigenic presentation levels. Moreover, substitution of the EBNA1 GAr-encoding mRNA sequence by the G-rich repetitive sequence of Ateline herpesvirus ORF73 (which encodes the GMP of the virus), showed that this domain is also able to inhibit EBNA1 translation and antigen presentation [START_REF] Tellam | Messenger RNA sequence rather than protein sequence determines the level of self-synthesis and antigen presentation of the EBV-encoded antigen, EBNA1[END_REF] . Finally, and further supporting a crucial role for the GAr-encoding mRNA sequence, the group of J.

Tellam demonstrated in vitro that the region of the mRNA encoding the GAr domain forms unusual secondary structures known as G-quadruplexes (G4) [START_REF] Murat | G-quadruplexes regulate Epstein-Barr virus-encoded nuclear antigen 1 mRNA translation[END_REF] . Using circular dichroism (CD) spectroscopy they showed that a G-rich sequence of 18 nucleotides that occurs 13 times all over the GAr sequence of EBNA1 is able to form stable G4s. They proposed that this particular mRNA structure would cause ribosome stalling thereby leading to translation inhibition.

G4 organization

G-quadruplexes are non-canonical intra or intermolecular secondary structures of nucleic acids that form within G-rich DNA or RNA sequences. They are composed of and stabilized by the stacked of guanine tetrads which are assembled in a planar arrangement by Hoogsteen hydrogen bonding (Figure 9). The stability of the structure relies on the presence of a cation, in general K + or Na + , which is located inside or between the tetrads and coordinates the structure 100 . G4 can adopt several topologies depending on the orientation of the DNA or RNA strands; they can be parallel, antiparallel or hybrid. The canonical G4 motif is G≥3NxG≥3NxG≥3NxG≥3 in which the planar quartets are connected by loops containing 1-7 nucleotides (N). Putative G4-forming sequences are frequently present within the human genome which would contain 360.000 of such motifs according to bioinformatics predictions 100 . The yeast Saccharomyces cerevisiae would contain only twenty seven putative G4forming sequences which may reflects the increase significance of these structures during evolution 101 . Some regions of the human genome like the telomeres or the ribosomal DNA (rDNA) are enriched in G4-forming sequences, contrary to coding portions of the genome.

G4 functions in cell biology

G4 may constitute key regulatory elements as they can affect several steps in gene regulation as DNA replication, transcription, mRNA stability and translation. In addition, G4 can also impact mRNA localization.

a) G4 in DNA

When present inside the DNA, G4 can modify chromatin condensation and also gene transcription.

Bioinformatics studies have shown that at least one G4 motif is present in almost 40% of human promoters and located 1000 nt upstream of the TSS (transcription starting site) sequence, which highlights G4 potential in transcription regulation. Depending on their position, G4 can either inhibit transcription if they are in the template strand, or enhance transcription when they are present in the non-template strand. G4 are often present in the promoters of human oncogenes while their frequency in tumor suppressor genes and housekeeping genes is dramatically lower 103 . Because of this, G4 present in promotors of human oncogenes are viewed as therapeutic targets as stabilization of these motifs may represent an attractive new anticancer strategy. In bacteria and yeast Saccharomyces cerevisiae there is also an enrichment in putative G4 motifs present within promoters which also reflects their importance during evolution 104, 105 .

The best studied G4 among those present in human promoters are those present within c-MYC promoter. c-MYC is a transcription factor which expression favors proliferation and as such is often overexpressed in human cancers. A particular nuclease hypersensitive element III (NHE-III) sequence present downstream the c-MYC promoter controls more than 80% of its transcription. In 2002 Most of G4 present in RNA are located in the 5' or 3'-UTR where they can affect translation or polyadenylation, but they can also have an effect on RNA splicing and mRNA turnover. Of note, potential G4 forming sequences within RNA are also present in telomeres where they can impact telomere maintenance.

Due to the ability of GAr to inhibit translation of its own (EBNA1) mRNA, the next section will focus on evidences indicating how G4 present inside mRNAs can affect translation. c) G4 formed in mRNAs can affect the translation rate.

Bioinformatics studies have revealed the presence of G4 forming sequences in at least 8% of 5´-UTR of human genes 108 , which highlights their importance as regulatory elements.

The 5´-UTR is a key element that ensures cap-dependent translation, as the small unit of the ribosome (40S) binds first to this sequence and then starts to scan the mRNA towards the 3´-end until it finds the first initiation (AUG) codon where the association with the large subunit of the ribosome (60S) takes place.

Thus, the presence of G4 in the 5´-UTR of RNA can affect translation initiation by preventing the binding of the initiation complex. G4 can also disturb the scanning of the mRNA by the 40S and affect the recruitment of translation initiation factors (eIFs). Hence, these secondary structures may interfere with cap-dependent translation. As a consequence, they can favor the cap-independent translation by internal ribosomal entry sites (IRES) which are present in many mRNAs 109 . One outstanding example concerns the vascular endothelial growth factor (VEGF) for which a G4 motif present in its 5´-UTR is essential to ensure IRES-mediated translation, which is itself critical under hypoxia 110 .

The first RNA G4 identified that can repress translation was found in the oncogene NRAS. As KRAS, its activation induces cell proliferation, differentiation and apoptosis. Translation control of NRAS occurs via the stabilization or destabilization of a G4 structure present in the 5´-UTR of its mRNA.

Mutations that cause the loose of the G4 motif increase NRAS expression and thus cell proliferation as in cancer cells. This makes NRAS G4 a therapeutic target as G4 ligands able to stabilize G4 will provoke a decrease in NRAS translation and thus apoptosis of cancer cells 111 . The author's also shown that only the G4 motifs of NRAS present near the 5´-UTR (as in wild type conditions) is able to block the formation of the initiation complex whereas the delocalization of the motif (from +14 to +120) completely abolishes the translation inhibition. This observation reflects the importance of G4 position inside the mRNA in order to impede translation.

When found in the 3´-UTR, G4 can cause translation repression. This is the case for the protooncogene PIM1 which encodes a serine/threonine kinase involved in cell growth, transcriptional activation and apoptosis 112 .

G4 binding proteins and their role in G4 stability.

Several proteins have been identified for their ability to bind G4 structures formed in DNA or RNA molecules. These G4 binding proteins can be classified in two groups: G4 binding proteins that destabilize G4 upon binding, and G4 binding proteins that stabilize G4 upon binding. These G4binding proteins can be recruited to G4 in response to various cellular stresses or cell cycle changes for example.

a) G4 binding proteins that destabilize G4 upon binding

In this category 3 main families of proteins can be found: heterogeneous nuclear ribonucleoproteins (hnRNPs), telomere-related G4 binding proteins (shelterin complex) and the transcription factor MAZ.

The heterogeneous nuclear ribonucleoprotein (hnRNPs) are DNA/RNA binding proteins involved in several steps of the nucleic acid metabolism like alternative splicing and translation and also in telomere protection. This is the case of the hnRNP D which is able to bind specifically to the 3' end of the human telomeric sequences and unfold a G4 motif that is able to inhibit telomerase activity 113 . This is also the case for hnRNP A which is also able to unwind the telomeric G4 thereby enhancing telomerase activity. This was demonstrated in vitro showing that depletion of hnRNP A from cells dramatically decreases telomerase activity, and that this can be reverted by addition of recombinant hnRNP A1 114 . hnRNP A1 is also able to affect transcription of various genes. This is the case of KRAS for which hnRNP A1 binds and destabilizes the G4 motif present in the NHE element upstream of the KRAS promoter thereby activating the transcription of this oncogene 115 .

In addition, several proteins from the Shelterin complex have been shown to bind to telomeric G4 sequences. The shelterin complex is formed by 6 proteins (POT1, TRF1, TRF2, RAP1 and TPP1 and TPP2) which are involved in telomere maintenance and genome stability. The group of S. Myong demonstrated in 2012 the mechanism by which POT1, in coordination with TPP1, regulates both G4 folding and unfolding thereby ensuring telomerase processivity 116 . POT1 binds to G4 formed within telomere repeats in a 3´-5´ direction and starts G4 unfolding by a 2 steps process, after which TPP1 is recruited and the complex between these two proteins causes a sliding that leads to a switch from a folded to an unfolded state, which itself affects telomerase processivity. Another well characterized telomeric G4 binding protein is the replication protein A (RPA). It has been demonstrated that RPA is able to regulate telomerase function during cell cycle by causing the unfolding of G4 formed within telomeric DNA, thus allowing telomerase binding 117 .

Finally the zinc-finger transcription factor MAZ is able to bind and destabilize the G4 sequences present in the HRAS promoter thus favoring the formation of a DNA duplex instead of quadruplex, thereby allowing transcription initiation 118 .

b) G4 binding proteins that stabilize G4 upon binding:

In this category, the most studied and well-known G4 binding protein nucleolin (NCL) will be described. Nucleolin was first described in 1973 by the group of H. Busch that called it C23 according to its position in two dimensional gel electrophoresis 119 . NCL is a multifunctional DNA and RNA binding protein widely conserved among eukaryotes. It is mainly localized in the nucleolus and in good agreement with this particular localization it has been involved in RNA metabolism, in particular, ribosomal RNA (rRNA) maturation. Most of its protein domains and functions are conserved from yeast to human. Human NCL is a 713 amino acids protein with a predicted molecular weight of 77 kDa, although it migrates as a 100-110 kDa protein. This discrepancy is attributed to the numerous post-translational modifications (phosphorylation, methylation, ADP-rybosilation, etc.) of the acidic N-terminal domain of the protein 120 . In addition to its acidic N-terminal domain, NCL contains four central RNA-binding domains (RBD) involved in its interaction with mRNAs and pre-mRNAs. Of note, while human NCL contains 4 RBD, yeast Saccharomyces cerevisiae NCL only possesses 2 RBD (Figure 10). These RBD have less sequence homology between them than with the equivalent RBDs from other species 120 . NCL is a RNA-binding protein that binds to its target RNA on a consensus motif (U/GCCCGA) that forms part of a short stem-loop structure of at least 18 nucleotides 121 . Finally the C-terminal domain of NCL is a glycine-arginine rich (RGG or GAR) domain involved in NCL interaction with ribosomal proteins. Of note, this GAR domain should not be mixed up with the GAr domain of EBNA1. As it is also called RGG domain, we will use this nomenclature in order to avoid confusion. The RGG domain has been implicated in the assembly of pre-ribosomal particles and also controls NCL nucleolar localization. Interestingly, this RGG domain is essential for an efficient binding of the RBDs to their target RNA as it is able to modify the binding properties of the RBDs 122 . It was also demonstrated that the RGG domain can bind to mRNA and affect their translation. As stated above, more than 90% of NCL is located in the nucleolar compartment. However it has been shown to be also present in the nucleocytoplasmic fraction and at the cell surface. Within the nucleolus, NCL is mainly present inside the Dense Fibrillar Compartment (DFC), much less in the Granullar Compartment (GC) and sometimes it is also present in the Fibrillar Center (FC). Since NCL has pivotal roles in the rRNA synthesis and ribosome biogenesis it has been proposed that it's shuttling between nucleus and cytoplasm may be involved in the transport of ribosomal components.

Thereby this change of localization plays an important role in ribosomal biogenesis.

NCL has been reported to interact with several DNA and RNA sequences. When binding to DNA it can enhance or repress transcription. For example, NCL has been shown to bind to the NHEIII element present upstream of the promoter of c-MYC gene and to inhibit its transcription by stabilizing its G4forming sequences 123 . NCL can also alter the chromatin condensation thereby affecting DNA replication. It is able to bind RNA sequences in particular in the 5´-UTR of some RNAs which may affect their translation. NCL has also been shown to bind to the 3´-UTR of some mRNAs which may affect their stability. Finally, NCL can also bind to the coding sequence of some mRNAs thereby affecting their translation.

2.3.2.4

Human pathogens using G4 structures in order to evade the immune system recognition.

G4 structures have been widely described from yeast to human. In addition, in the past few years it has been shown that they can also be formed in the genome or transcripts of several human pathogens, and that they may help those pathogens to evade the immune system (Table 4). The antigenic variation (Av) is the process by which pathogens express different versions of a surface antigen in order to evade the immune system. They possess several variants of this antigen but they express only one of them at a time. The switch between the different variants can be due to sitespecific recombination, epigenetic changes, or homologous recombination, the latter being the most common 124 . In addition, G4 structures can also be implicated in the Av of pathogens. This is the case for Neisseria gonorrhoeae and Neisseria meningitidis. Both of them express three major antigens that are involved in bacterial survival. The pilin proteins are essential for establishing the infection as they are implicated in cellular adherence and bacterial aggregation 125 . pilE is the major component of the pilus and Av occurs by recombination of this locus with several silent variants called pilS. The Av of N. gonorrhoeae is controlled by a DNA G4 motif located upstream the pilE region which controls its transcription rate. The treatment with G4 ligands that stabilize G4 formation inhibits the frequency of Av by decreasing the recombination of pilE with pilS loci 126 . Furthermore, pilE G4 motif recruits N. gonorrhoeae RecA helicase in order to unwind the structure and thus facilitate the recombination, thereby allowing the Av 127 . G4 forming sequences have also been identified in eukaryotic pathogens like Plasmodium falciparum, the parasite causing malaria in humans. P. falciparum is able to enter into erythrocytes and to provoke their attachment to microvascular endothelial cells leading to vascular obstruction. The only protein involved in this process is the P. falciparum erythrocyte membrane protein one (PfEMP1) which is encoded by at least 50 different var genes. Each parasite expresses only one var gene at a time and can switch to other variants to generate the Av. As in N. gonorrhoeae, evidence suggests that some DNA G4 motifs located upstream of the var promoter can control var transcription and thus Av 128 . G4 motifs are also present in some human viruses and are implicated in viral replication and immune evasion. This is the case for the human immunodeficiency virus type 1 (HIV-1), human papillomavirus (HPV) and EBV. In the case of HIV-1, its long terminal repeats (LTR) control transcription of several genes and also of the full-length viral genome of the integrated provirus. The LTR promoter contains two intramolecular G4 sequences whose formation inhibits the transcription initiation. R. Perrone et al demonstrated that a treatment with G4 stabilizing ligands inhibits LTR-mediated transcription thus leading to a decrease in HIV-1 infectivity. Conversely, mutations that abolish G4 formation increase promoter activity 131 . Other G4 forming sequences have been reported in the coding sequence of the nef gene. The Nef protein is expressed during the early phase of HIV-1 life cycle and is essential for viral replication and infectivity. The stabilization of the G4 motifs present within nef coding sequence inhibits its expression and thus decreases HIV-1 infectivity. These observations highlight the importance of G4 structures in viral infection. For these reasons, many efforts were made in order to identify cellular or viral proteins that could interact with these G4 and affect their stability. In this context, E. Tosoni et al used a combination of electromobility assays (EMSA) and mass spectroscopy to identify cellular and viral proteins interacting with G4 within LTR sequences 133 . They found that Nucleolin is able to specifically bind and stabilize G4 present inside the LTR and that this interaction causes a decrease in viral transcription. This interaction could be important for the first switch of the virus life cycle to the latency state as proteins able to attenuate transcription are important in order to consolidate latency. Nevertheless, it has been demonstrated that cell surface NCL is essential to ensure HIV-1 attachment and entry inside the cells since a treatment with AS1411, a G4 oligonucleotide which binds and traps the fraction of NCL localized at the cell surface, leads to a decrease in HIV-1 entry into the cells 134 .

Regarding HPV, several G4 forming sequences have been identified in its genome notably inside the LTR, the minor capsid component L2 and the early genes E1 and E4 130 suggesting that these G4 may have an effect on HPV replication and transcription. The G4 present in GAr mRNA inhibit translation of EBNA1. The treatment with G4 ligands, that stabilize G4 structures, decreases EBNA1 protein level and thus antigen presentation at the cell surface. In contrast antisense oligonucleotides that disrupt the compact G4 motifs enhance translation and antigenic presentation.

CHAPTER III: GAr-based immune evasion is a therapeutic target for cancer: need of a model.

The efficient T cell response against EBV-infected cells in which the GAr domain of EBNA1 has been deleted demonstrates the importance of this domain in the viral strategy to evade antigen presentation [START_REF] Yin | Self-inhibition of synthesis and antigen presentation by Epstein-Barr virus-encoded EBNA1[END_REF] .

The mechanism by which the GAr domain self-limits the translation of its own mRNA in cis clearly represents a relevant therapeutic target to treat EBV-related cancers. Indeed, most tumoral cells of EBV-related cancers are EBV-infected, contrary to the vast majority of non-tumoral cells. Hence, interfering with the GAr-based inhibition of translation should unveil tumoral cells to T CD8 + raised against EBNA1 that exist in every EBV-infected individual. For this purpose, a relevant cell-based assay was required.

In order to create a GAr-based model, yeast looks very appropriate. Indeed, the versatile genetic flexibility of the budding yeast Saccharomyces cerevisiae and the high degree of conservation between yeast and mammalian cellular processes have made S.cerevisiae an invaluable tool for modeling human diseases 135-137 , as well as for identifying and characterizing cellular pathways involved in these disorders and thereby new therapeutic targets 138,139 . Moreover, EBNA1 has been expressed in yeast to study its crucial role in EBV episome maintenance [140][141][142] . These studies showed that a functional EBNA1 protein can be expressed in yeast and indirectly suggested that the GAr domain might affect mRNA translation in this unicellular organism. Therefore, we hypothesized that a broad approach using yeast would be suitable to identify modulators (either drugs or genes) that could interfere with the GAr-mediated inhibition of translation whatever the exact mechanism of this regulation process.

We thus set up a yeast-based assay that recapitulates the effect of the GAr domain on translation regulation 143 . This yeast-based assay consists in the use of a Saccharomyces cerevisiae strain in which the yeast ADE2 gene encoding the phosphoribosylaminoimidazole carboxylase (AIR), involved in the adenine synthesis pathway, was deleted (ade2Δ strain). As a consequence of this deletion, colonies of ade2Δ cells present a red phenotype due to the accumulation of AIR which becomes red when oxidized by active respiration (Figure 12). This ade2Δ strain was used to express an ADE2 construct which restores the normal adenine synthesis thus resulting in a white phenotype of yeast cells as well as several GAr-ADE2 fusions which lead to pink to red phenotypes due to the length-dependent translation inhibition exerted by the GAr domain 143 (Figure 13A). Any compound or gene that would act on the translation inhibition exerted by the GAr domain would cause a modification of the GAr-ADE2 expression level and give rise to a whiter or a redder phenotype (Figure 13B). Yeast colonies from a ade2Δ strain present a redder phenotype due to the accumulation and oxidation of AIR (phosphoribosylaminoimidazole) which is an intermediate metabolite of the adenine synthesis. In contrast, the full expression of ADE2 causes a yeast white phenotype. The color of the yeast colonies is proportionate to ADE2 expression level.

For this purpose, we selected the 43GAr-ADE2 yeast strain that has a pink phenotype which enables the identification of compounds or genes able to affect GAr inhibition of translation. The pink phenotype of this strain should allow to identify compounds (or genes) that cause a redder phenotype, which means that there is an increase in GAr domain inhibition of translation, or a whiter phenotype that signifies an inhibition of GAr effect (Figure 13B). Both phenotypes are equally interesting since a decrease in EBNA1 expression level would be deleterious for the virus genome replication and maintenance due to EBNA1 essential functions; and an increase of EBNA1 expression level would cause the recognition of virus-infected cells by the host immune system.

We used this yeast model to perform a drug screening of FDA-approved compounds and isolated doxorubicin (DXR) which causes a change of color of 43GAr-ADE2 strain from pink to white (Figure 13B). This results were confirmed by western blot showing that DXR treatment increases the expression of 43GAr-ADE2p without affecting ADE2p level. This effect was due to an increase in protein synthesis (Figure 13C) without changing mRNAs levels 143 . After having identified a candidate drug, results were confirmed in mammalian cells by expressing two different types of constructs: EBNA1 / EBNA1ΔGAr and 235GAr-OVA / OVA constructs which are essential in order to further test the capacity of DXR to alter the antigenic presentation (Figure 13D). After validation of DXR activity in mammalian cells the last step was to test if indeed it is able to affect antigen presentation. For that we evaluated the capacity of T CD8 + cells against the SL8 antigenic peptide of OVA to recognize 235GAr-OVA after the treatment with DXR. The treatment with DXR causes a dose-dependent increase in 235GAr-OVA antigen presentation without affecting OVA antigen presentation (Figure 13E-F).

Thus DXR, the drug we isolated thanks to this model, affects the GAr domain-dependent inhibition of translation, both in yeast and in human cells, and also the antigenic presentation 143 . This means that the mechanism involved in the GAr-mediated regulation of translation is conserved from yeast to human. It also validates this yeast-based assay for isolating compounds or cellular factors that could interfere with the EBNA1-dependent immune evasion of EBV. A. GAr domain of EBNA1 responsible for EBNA1 translation inhibition in cis was fused to the reporter gene ADE2. The expression of Ade2p gives white colonies whereas the inhibition of its translation by GAr gives pink colonies (GAr-ADE2 construct). This pink phenotype (B) was used to isolate compounds able to affect this GAr translation inhibition. Drugs able to suppress GAr effect will give white colonies which can be easily monitored in yeast (C) or in human cells (D). Finally, drug effect on antigen presentation is determined using a T cell assay (E-F).

Aim of the PhD

Viruses have evolved with their hosts all over the animal kingdom. Some of them, like the γherpesviruses, are able to establish lifelong infections of their hosts without being detected by the immune system. Their camouflage is based on the control of the expression of a unique viral protein, named GMP (genome maintenance protein), which has two essential missions: first, it ensures the replication of the viral genome during the S phase of the cell cycle; second it acts as a bridge to tether the viral episome to the host chromosomes. One of the most studied GMP is the GMP of the Epstein-Barr virus (EBV): EBNA1. Like all the GMP, EBNA1 is highly antigenic and T cells towards EBNA1 exist in all EBV-infected individuals. In order to hide from the host immune system, EBNA1 is able to control its own mRNA translation and thus the presentation of EBNA1-derived antigenic peptides, a process controlled by its central GAr domain.

Due to the antigenicity of EBNA1 and the mechanism that tightly regulates its own mRNA translation, the EBNA1 protein constitutes a good therapeutic target to treat EBV-associated cancers. Indeed, only few memory B cells are latently infected by EBV thus their destruction by the immune system should not be deleterious for the patients. However, the mechanism of translation inhibition of EBNA1 by its GAr domain is currently undeciphered and the cellular factors involved in this regulation have not been identified yet.

The aim of my PhD was to identify cellular factors that are involved in this GAr domain effect. These cellular factors are potential therapeutic targets that will help to interfere with the GAr-based translation regulation. To identify such cellular factors, we took advantage of the yeast model which recapitulates the effect of GAr in the inhibition of translation (Figure 13), to perform a genetic screening to identify genes which overexpression could affect GAr self-inhibition. Thanks to this screening we identified Nucleolin (NCL). We first demonstrated the involvement of NCL in the GArbased translation inhibition and then we went deeper into the mechanism by which NCL is involved in the GAr domain inhibition of translation.

Results section organization

The results section is organized in three chapters. The first chapter comprises the discovery of Nucleolin (NCL) as the first GAr domain mRNA cellular partner, and also how this interaction can constitute a therapeutic target to treat EBV-associated cancers. These results were the subject of a first publication which has been submitted to Nature Communications in October 2016.

The second chapter describes more deeply the mechanism involved in this NCL-GAr mRNA interaction: how, where and why it takes place. These results will be part of a second manuscript which is currently in preparation.

Finally, the third chapter corresponds to a review article that was published in 2015 in Biotechnology journal, which described how the yeast Saccharomyces cerevisiae happened to be a good model to study the different steps in the Epstein-Barr virus life cycle.

Results

Chapter 1:

Identification of NCL as the first cellular factor involved in the GAr domain regulation of translation

• Graphical abstract:

• Manuscript presentation:

During this thesis, we studied the mechanism of the GAr domain-based inhibition of translation in order to identify cellular factors that are able to enhance or inhibit this translation regulation. Thanks to the genetic screening we performed in the yeast model that recapitulates the GAr domain-based regulation of translation, we were able to identify several candidate genes which overexpression alters the GAr-dependent translation inhibition. We focus our interest on NSR1 and its human homolog Nucleolin (NCL). So why did we get interested in NCL? First, it happens that NCL is a well characterized DNA/RNA binding protein which has been implicated in many cellular functions such as ribosome biogenesis, rRNA transcription, cell cycle control, apoptosis, transcription and translation regulation 120 . Second, many evidences showed that NCL has the ability to bind to G4 structures present on promoters (like c-Myc) and through this interaction NCL can modify genes transcription rate 123, 144 . Similarly, NCL can bind to G4 on the 5´-UTR or 3´-UTR of mRNAs and affect translation or mRNA stability. Moreover NCL has recently been shown to interact with the N-terminal domain of EBNA1 protein and enhance EBNA1-mediated OriP transcription.

The fact that there was already a demonstration that NCL can affect one of the two functions of EBV GMP, that the GAr domain mRNA is able to form G4 motif, and that NCL is a G4 binding protein indicate that NCL constitutes an excellent candidate and led us to focus primarily on this cellular protein and to study its effect on the GAr domain inhibition of translation.

In our first manuscript we demonstrated that NCL overexpression decreases GAr-OVA and endogenous EBNA1 protein level, and inversely that NCL downregulation enhances GAr-OVA and EBNA1 translation. Increasing GAr-OVA protein levels consequently enhances the presentation of OVA-derived antigenic peptides at the surface of APC and also the recognition by T CD8 + cells directed against OVA. Furthermore, we showed for the first time that NCL interacts with GAr mRNA through a direct interaction with the G4 motifs present inside the GAr-encoding sequence, and also that this interaction takes place in the nucleus. Finally, we demonstrated that targeting this interaction, for example by using small G4 interacting compounds like PhenDC3, interferes with the GAr domain translation inhibition thus demonstrating that NCL-EBNA1 mRNA interaction represents a novel and relevant therapeutic target to treat EBV-associated cancers.

These data led to the redaction of a publication that has been submitted to Nature Communications and which manuscript is placed below. The Epstein-Barr virus (EBV) is the first oncogenic virus discovered in human [1][2][3] and has been linked to various cancers that include Burkitt and Hodgkin lymphomas and 10% of gastric cancers. Another example is the nasopharyngeal carcinoma which is particularly frequent among men in China and Tunisia. Like all the gammaherpesviruses, EBV evades the host immune system but has an Achilles heel: its genome maintenance protein (GMP) EBNA1 4,5 . Indeed, EBNA1 is essential for EBV genome replication and maintenance and as such expressed in all EBV-infected cells. On the other hand, EBNA1 is highly antigenic and CD8 + T cells directed towards EBNA1

epitopes exist in all infected individuals. Hence, EBV has evolved a mechanism to limit EBNA1

production to the minimal level required for the viral genome replication and, at the same time, to minimize the production of EBNA1-derived antigenic peptides presented to the cytotoxic T cells through the MHC class I pathway 4,6 . The central glycine-alanine repeat (GAr) of EBNA1 plays a critical role in this mechanism of immune evasion as it is able to self-inhibit the translation of its own mRNA in cis 7,8 . The high level of EBNA1 protein and the efficient T cell response following the infection by an EBV strain encoding a truncated version of EBNA1 in which GAr has been deleted (EBNA1 GAr) demonstrates the critical role of GAr in EBNA1 immune evasion [7][8][9][10] . Of note, a polymorphism in the length of GAr exists and, importantly, the effect of GAr is length-dependent as a longer domain displays a stronger inhibitory effect on both mRNA translation and antigen presentation 11 .

The GAr-encoding mRNA sequence is GC rich and forms predicted G-quadruplex (G4) structures that have been implicated in the regulation of EBNA1 synthesis in vitro 12 . G4 are particular secondary structures of nucleic acid formed by the stacking of G-quartets which correspond to a planar arrangement of 4 guanines connected by Hoogsteen hydrogen bonds. G4 structures within G-rich DNA or RNA sequences have been implicated in gene regulation where they can affect transcription, alternative splicing and translation [13][14][15][16][17] . G4 modes of action are still relatively unknown but cellular factors that can interact with these structures are emerging [18][19][20] .

Nucleolin (NCL) is a multifunctional DNA/RNA-binding protein widely conserved among eukaryotes. It is involved in RNA metabolism, in particular in rRNA maturation 21 . NCL binds to Grich sequences in coding and non-coding regions of various mRNA, many of which encode cancer-related proteins, and enhance their translation 22 . In addition, NCL binds to some G4 structures within DNAs and RNAs. For example, it has been recently shown that NCL binds to and stabilizes G4 structures formed within the LTR promoter of HIV, thereby silencing the provirus transcription 23 . NCL also affects the transcription of c-MYC by binding to and stabilizing G4 present in the promoter of this oncogene and that negatively regulate its activity 17,24 .

GAr-based EBNA1 immune evasion has been considered a relevant therapeutic target to treat EBV-related cancers as most tumor cells from EBV-related cancers are infected by EBV whereas, in healthy individuals, the latent infection by EBV is primarily restricted to a specific small pool of memory B cells. Hence, overcoming GAr-based self-inhibition of EBNA1 translation should unveil EBV-carrying tumor cells to cytotoxic T cells without having significant effect on the vast majority of healthy host cells. As the mechanisms of GAr-mediated mRNA translation suppression in cis are not known, nor the cellular factors involved, we developed a yeast-based (Saccharomyces cerevisiae) assay that recapitulates all the aspects of the GAr-based inhibition of translation, including the GAr-length dependency 25,26 . This assay was successfully used to identify small molecular weight compounds that can stimulate EBNA1 expression both in yeast and mammalian cells and that relieve GAr-based limitation of antigen presentation 26 .

Based on the same yeast assay, here we have performed a genetic screen to identify host cell genes involved in the GAr-mediated inhibition of translation. This way, we isolated the yeast NSR1 gene which encodes the orthologue of human NCL and showed that NCL is critically involved in GAr-based limitation of translation and antigen presentation. We also found that NCL directly interacts in the nucleus with G4 formed in the GAr-encoding sequence of EBNA1 mRNA. Finally, we show that this interaction is druggable and that the G4 ligand PhenDC3

prevents NCL from binding to G4 formed in the GAr mRNA sequence and stimulates GAr-limited translation and antigen presentation.

Hence, NCL represents the first host cell factor critically involved in EBNA1 immune evasion and the NCL-EBNA1 mRNA interaction appears to be a relevant therapeutic target to treat EBVrelated cancers. Importantly, the results also show that G4 structures act as druggable protein binding modules.

RESULTS

Yeast nucleolin Nsr1p is required for GAr-based inhibition of protein expression in yeast and human nucleolin NCL can functionally replace Nsr1p

The yeast assay used in this genetic screen 26 is based on a fusion between the yeast Ade2p reporter protein and a GAr domain of 43 amino acid (43GAr). Because GAr is able to self-inhibit the translation of its own mRNA in yeast, this leads to a reduction in Ade2p level. This can easily be monitored in yeast as cells which express Ade2p at a functional level form white colonies, whereas cells that do not express Ade2p readily form red colonies and any intermediate level of

Ade2p leads to the formation of pink colonies whose intensity of coloration is inversely proportional to the level of Ade2p expressed. Hence, a yeast strain expressing the 43GAr-ADE2 construct from the constitutive ADH promoter forms pink colonies, whereas a control strain expressing ADE2 from the same promoter forms white colonies (Fig. 1a). We used the 43GAr-ADE2 strain to identify yeast genes whose overexpression leads to a redder phenotype meaning that they potentially exacerbate GAr-based inhibition of translation. For this purpose we transformed the 43GAr-ADE2 yeast strain by a yeast genomic DNA library consisting of small genomic fragments (~4kb) cloned into a yeast 2 multicopy plasmid which is present at ~50 to 100 copies per yeast cell, hence potentially allowing to assess the effect of overexpressing every yeast gene on GAr-based inhibition of translation. This way, we isolated two independent clones bearing overlapping genomic fragments that, among a few other genes, contained the yeast NSR1 gene. We then subcloned NSR1 gene alone under the control of the strong constitutive GPD promoter in a low copy number vector (CEN) and confirmed its ability, when overexpressed, to both confer a redder phenotype and exacerbate the ability of GAr to decrease 43GAr-Ade2p protein expression whereas having no significant effect on Ade2p protein in the control strain (Fig. 1b and Supplementary Fig. 1a) nor on 43GAr-ADE2 or ADE2 mRNA levels (Supplementary Fig. 1b).

We then determined the effect of NSR1 downregulation on GAr-based inhibition of protein expression. As NSR1 is not essential in yeast, we deleted this gene in both 43GAr-ADE2 and ADE2

strains and observed that its absence completely abolished the GAr inhibitory effect on 43GAr-Ade2p expression (first two lanes of the western blot in Fig. 1c). This effect was GAr-dependent as NSR1 deletion had no effect on Ade2p protein level in the control strain (last two lanes of the western blot in Fig. 1c). As controls, we checked that NSR1 deletion had no significant effect on 43GAr-ADE2 and ADE2 mRNA levels (Supplementary Fig. 1c).

Next, we assessed the potential ability of NCL, the gene encoding the human nucleolin, to functionally complement the deletion of NSR1 in yeast and found that the expression of NCL led to a decrease in 43GAr-Ade2p level whereas having no effect on Ade2p level (Fig. 1d). Of note, the human NCL was as efficient as the yeast Nsr1p (Supplementary Fig. 1d).

Taken together, these results show that Nsr1p, the yeast orthologue of nucleolin, is critically involved in the GAr-based inhibition of protein expression in yeast. As NCL, the human nucleolin, is able to fully complement the deletion of the yeast NSR1 gene, this suggests that NCL represents a host cell factor important for the GAr-mediated self-limitation of EBNA1 expression in EBV-infected human cells.

NCL also controls GAr-dependent EBNA1 expression in EBV-infected cells

We next assessed the role of human nucleolin (NCL) on GAr-based self-inhibition of translation in human cells. For this purpose, we first overexpressed HA-tagged NCL (HA-NCL) in three EBVinfected B cell lines: Mutu-1, B95.8 and Raji. As shown in Figure 2a, overexpression of HA-NCL led to a significant decrease in EBNA1 endogenous level in all these three cell lines, as compared to actin. Similar to yeast, this effect is GAr-dependent as overexpression of HA-NCL also decreased the level of transfected 235GAr-OVA (ovalbumin), the fusion protein that is used to assess the effect of GAr on MHC class I-restricted antigen presentation (see below), whereas having no significant effect on OVA alone, the control protein (Supplementary Fig. 2a).

We then determined the effect of downregulating endogenous NCL on GAr-dependent suppression of protein expression in H1299 cells using siRNA. As observed by others 27 , we were not able to knockdown more than ~40-50% the expression of NCL, probably because it is an essential gene in mammalian cells. However, this partial downregulation led to a significant increase in both EBNA1 (Fig. 2b) and 235GAr-OVA (Fig. 2c) protein levels, whereas having no effect on EBNA1 GAr or on OVA alone. Quantifications of three independent experiments are shown in Supplementary Figure 2b andc. Next we performed a metabolic 35 S methionine pulselabeling experiment, and we observed an increase in newly synthesized 235GAr-OVA following NCL down-regulation (Fig. 2d, upper panel). In contrast, no increase in newly synthesized OVA was observed (Fig. 2d, lower panel). We did not observe any significant effect on the levels of GAr-carrying mRNAs (Supplementary Fig. 2d and 2e respectively). Altogether, these results demonstrate that NCL downregulation does interfere with GAr-based suppression of translation.

These findings confirmed that, as in yeast, NCL represents a host cell factor critically involved in the GAr-dependent suppression of EBNA1 synthesis, a mechanism at the basis of EBV immune evasion in latently infected cells.

NCL downregulation overrides GAr-restricted antigen presentation

We next tested if downregulating NCL has an effect on GAr-restricted MHC class I antigen presentation. Indeed, as NCL downregulation led to a GAr-dependent increase in protein expression, it was also expected to stimulate antigen presentation. For this purpose, we determined the effect of siRNA-mediated NCL knockdown on the GAr-restricted presentation of the ovalbumin-derived SIINFEKL antigenic peptide (OVA 257-264 ) in complex with the murine k b MHC class I receptor using a specific monoclonal antibody. FACS analysis revealed that NCL knockdown significantly increased (+ 42.1% ± 1.37, p=0.0005) the formation of this complex in 235GAr-OVA-expressing cells (Fig. 3a). In contrast, NCL knockdown only had a modest effect on OVA-expressing cells (+ 19.7% ± 6.11, p=0.043; Fig. 3b). In all cases, the efficiency of siRNAmediated NCL downregulation and its effect on 235GAr-OVA or OVA expression were determined (Supplementary Fig. 3a).

We then tested if the observed increase in antigen presentation following NCL downregulation does have an effect on T cell activation. For this purpose, we determined the proliferation of whereas it had no effect in OVA-expressing H1299 cells (Fig. 3d, right panels). The efficiency of siRNA-mediated NCL downregulation and its effect on 235GAr-OVA or OVA expression are shown in Supplementary Fig. 3b.

NCL directly interacts in the nucleus with G4 present in GAr-encoding mRNA sequence

NCL has been reported to bind to some G-quadruplexes (G4) formed in both DNA 24 and RNA sequences 28 . G4 are composed and stabilized by the stacking of guanine tetrads which are assembled in a planar arrangement by Hoogsteen hydrogen bonding (Fig. 4a) and have been involved in the regulation of gene expression, DNA replication and telomere maintenance (for review 20 ). The G-rich sequence of GAr-encoding mRNA contains a cluster of 13 predicted G4 29 .

Hence, we determined the ability of NCL to bind to these structures. For this purpose, we adapted a pulldown assay recently developed to identify RNA G4 binding proteins 28 to an 18 ntlong oligonucleotide containing the most probable G4 that can form in the GAr-encoding mRNA sequence. Briefly, this oligonucleotide (GQ) was linked to biotin and pulldown experiments using streptavidin-conjugated sepharose beads were performed. As a negative control, we used an oligonucleotide (GM) with a similar sequence except that the four guanines forming the G4 were replaced by adenines or uridines in order to completely abolish the G4 structure, as predicted using the GQRS-H predictor software 30 . As a positive control, we used ARPC2 30 nt-long oligonucleotide which corresponds to a G4 found in the ARPC2 mRNA and that has been shown to bind NCL 28 . As shown in Figure 4b, NCL was precipitated from H1299 cell extracts when using GQ or ARPC2 oligonucleotides, but not when using GM or empty beads showing that NCL binds to G4 formed in the GAr mRNA sequence.

Next we performed the same pulldown experiment using recombinant NCL instead of cell lysate.

Similar results were obtained (Fig. 4c) showing that NCL directly binds GAr most probable G4.

Finally, we wanted to check if, and where, the NCL-GAr G4 interaction occurs in cellulo. Hence, we performed a proximity ligation assay (PLA) to assess if NCL associates with EBNA1 mRNA in H1299 cells expressing EBNA1. We observed nuclear PLA dots in cells expressing the full-length EBNA1 mRNA (Fig. 4d). In contrast, no dots were detected in cells expressing EBNA1 GAr as well as in the various controls (Fig. 4d).

Taken together, these results indicate that NCL directly interacts with the G4 of the EBNA1 mRNA in the nuclear compartment.

PhenDC3 prevents NCL-EBNA1 mRNA interaction and GAr-based inhibition of protein expression

Next, we tested the effect of various G4 ligands on GAr-based inhibition of protein expression.

Among the reported G4 ligands, pyridostatin (PDS, molecular structure depicted in Fig. 5a) and PhenDC3 (molecular structure depicted in Fig. 5b) [31][32][33] are the best benchmark probes compatible with cellular assays. Indeed, PDS and PhenDC3 at micromolar concentrations have been shown to efficiently target various G4 in cell-based experiments 34 . As, at the same range of concentrations (1-5 M), PDS has been shown to exacerbate GAr-based inhibition of protein expression in an in vitro coupled transcription-translation system 29 , we first tested the effect of this molecule on the level of 235GAr-OVA in H1299 cells. However, at the same concentration (5 M), no clear effect on 235GAr-OVA or OVA expression was observed (Fig. 5a) suggesting that PDS may not be able to interfere with GAr-mediated inhibition of EBNA1 expression in a cellular context. We then tested PhenDC3 at the same concentration (5 M) and found that it led to a significant increase in the steady-state level of 235GAr-OVA in H1299 cells (Fig. 5b, left panel).

This effect is GAr-dependent since PhenDC3 had no significant effect on OVA expression (Fig. 5b, right panel) and is not due to an effect on the level of the corresponding RNA (Fig. 5c). Hence, one possibility is that PhenDC3 prevents the binding of NCL on EBNA1 mRNA G4. To test this hypothesis, we performed the same G4 oligonucleotide pulldown assay as in Figure 4b in the presence or absence of 10 M PhenDC3 and we observed that PhenDC3 does prevent the binding of NCL on GAr G4 (Fig. 5d), readily explaining its effect on 235GAr-OVA expression.

Next, we tested PhenDC3 effect on EBNA1 expression and found that it also increased the endogenous EBNA1 level in Mutu-1 (EBV-infected B-cells; Fig. 5e left panel) and NPC-6661 (EBVinfected cells derived from a nasopharyngeal carcinoma 35 ; Fig. 5e right panel) cells. Importantly, PhenDC3 had no effect on EBNA1 mRNA level in Mutu-1 cells (Supplementary Fig. 3c).

To conclude, the PhenDC3 G4 ligand prevents the binding of NCL on GAr's G4 and, at the same time, leads to an increase in EBNA1 and 235GAr-OVA expression, thereby supporting the crucial role of NCL in GAr-based self-inhibition of translation by binding to G4 formed in the EBNA1 mRNA. Importantly, these results also indicate that NCL-EBNA1 mRNA interaction is druggable.

PhenDC3 activates GAr-limited antigen presentation

Finally, to assess the ability of PhenDC3 to interfere with GAr-based immune evasion, we performed the same OT1 T cell proliferation assay as in Figure 3c & d in the presence or absence of 5 M PhenDC3. As shown in Figure 6a, PhenDC3 significantly increased (two-fold change) the proliferation of OT1 T cells added to 235GAr-OVA-expressing H1299 cells whereas it had no effect on OT1 cells added to OVA-expressing cells. A western blot analysis confirmed that PhenDC3 at 5 M increases the level of 235GAr-OVA whereas it has no effect on OVA (Fig. 6b).

DISCUSSION

In this study we have identified nucleolin as the first host cell factor critically involved in GArbased EBNA1 immune evasion via its ability to bind G-quadruplexes formed in the GAr-encoding sequence of the EBNA1 mRNA. First, thanks to a genetic screen performed in the yeast model that recapitulates all the aspects of GAr-based self-inhibition of translation 26 , we isolated the yeast nucleolin Nsr1p as a critical host cell factor involved in GAr-based inhibition of protein expression in yeast. Indeed, the overexpression of Nsr1p exacerbates the GAr effect whereas the deletion of NSR1 gene fully abrogates GAr ability to self-inhibit translation. Second, as the human NCL gene, which encodes human nucleolin, is able to fully complement the effect of NSR1 deletion on GAr in yeast, we tested the role of NCL in GAr-based inhibition of protein expression in human cells and found that its overexpression and downregulation had the same effect on GAr than in yeast cells. Then we checked that, in line with its ability to interfere with GAr-mediated translation inhibition, NCL downregulation also increases antigen presentation and T cell proliferation. We also showed that NCL directly interacts with G4 present in GArencoding sequence of the EBNA1 mRNA in vitro and observed this interaction in cellulo in the nucleus. This suggests that the binding of NCL on EBNA1 mRNA G-quadruplexes is per se important for GAr-based translation inhibition and thereby for EBNA1 immune evasion. In line with this model, we showed that the G4 ligand PhenDC3 both prevents the binding of NCL on G4 of the EBNA1 mRNA, and increases EBNA1 expression and GAr-dependent antigen presentation.

This indicates that the interaction between NCL and G4 of the EBNA1 mRNA is a relevant and druggable therapeutic target to treat EBV-related cancers. Interestingly the G4 ligand pyridostatin (PDS) had no effect on EBNA1 expression in cellulo indicating that only some G4 ligands are able to interfere with NCL-EBNA1 mRNA interaction, which can be attributed to offtarget binding and/or differences in pharmacological properties (cell penetration, intracellular distribution etc.). This also points out two different possible mechanisms of action for G4 ligands whose binding on G4 may either stabilize them or prevent the binding of cellular partners, or both. In line with these hypotheses, we tested several PhenDC3 close chemical derivatives and observed that some, but not all, were active on EBNA1 expression (data not shown). Of note, PDS has been found to suppress EBNA1 expression in an in vitro coupled transcription-translation assay 29 but it is not known, in this experiment, if this effect is related to a change in EBNA1 mRNA level or to G4 stabilization that may exacerbate the GAr-dependent translation inhibition.

What could be NCL mechanism of action in GAr-based self-inhibition of translation and antigen presentation? EBNA1 G4 may constitute a recognition signal for NCL that is, itself, directly or indirectly, responsible for translation inhibition by interfering with either translation initiation and/or elongation machinery. Alternatively, NCL could stabilize G4 that, themselves, may inhibit the ribosome progression. Of note, either of these two possible mechanisms would explain why EBNA1 mRNA is translated mainly in mitosis, at a time when the nuclear envelope has been disaggregated 4,25 . Finally, NCL could act by trapping EBNA1 mRNA in the nucleus given that NCL is a nuclear protein, thereby preventing its efficient translation by the ribosome. Importantly, these mechanisms are not mutually exclusive and a model in which NCL would inhibit translation by preventing ribosome progression during mitosis and by trapping EBNA1 mRNA in the nucleus during the other phases of the cell cycle can be envisioned. In either case, it is unlikely that the virus has developed a novel mechanism to exploit nucleolin for controlling gene expression. Rather, it is likely that this reflects a more general evolutionary conserved cellular pathway. The fact that NCL effect on GAr-based limitation of protein expression is also operant in yeast strengthens this hypothesis as yeast has no common evolutionary history with EBV.

Hence, PhenDC3 and more generally G4 ligands represent chemical probes that will greatly help to characterize this pathway and to identify its physiological mRNA targets. To finish, as other oncogenic gammaherpesviruses like the Kaposi sarcoma-associated herpesvirus have evolved similar strategy of G4 clustering in the coding regions of their GMP to evade the immune system, the results presented here may have applications for other gammaherpesvirusesrelated diseases 29,36 .

METHODS

Yeast strains and culture media

All the yeast strains used in this study are derived from the W303 WT strain 37 : MATa, leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15. The ade2 strain genotype is: MATa, leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1::his5 S. pombe. Yeast cells were grown and used as previously described 38

Yeast-based genetic screen for isolation of enhancers of GAr-based translation inhibition

A yeast genomic DNA library (a kind gift by F. Lacroute) constructed by inserting ~4 kb genomic DNA fragments (obtained by Sau3A partial digestion) at the unique BamHI site in the replicative 2 multicopy pFL44L vector containing URA3-marker, was transformed into 43GAr-Ade2p pink yeast strain using standard lithium acetate procedures 39 . This multicopy plasmid is present at ~ 50-100 copies per yeast cell. Transformants were selected on uracil-free minimal solid medium and a positive selection was carried out based on the redder color phenotype. Out of ~20,000 transformants growing on uracil-free medium, 39 gave a redder phenotype. Plasmids originated form the pFL44-based library were extracted from these 39 redder transformants, purified and amplified in E. coli and then re-transformed into 43GAr-Ade2p yeast strain for confirmation of the redder phenotype. The extremities of the confirmed clones were sequenced using the following primers: F-5' GTGCTGCAAGGCGATTAAGT 3' and R-5'TGTGGAATTGTGAGCGGATA 3'.

Two confirmed clones contained overlapping genomic fragments containing the yeast NSR1 gene.

Plasmid constructions

All vectors were constructed using standard cloning procedures. T4 DNA ligase and restriction enzymes were purchased from New England Biolabs. Plasmid maintenance was carried out in TOP10 E.coli strain. The p416 (GPD) containing NSR1 gene was constructed as follows: NSR1

coding sequence was amplified from genomic DNA of the S. cerevisiae W303 WT strain using the following primers: NSR1-F 5' CGCGGATCCATGGCTAAGACTACTAAAGTAAAAGGTAAC 3' and NSR1-R 5' CCGCTCGAGCGGTTAATCAAATGTTTTCTTTGAACCAG 3'. The corresponding PCR fragment was cloned into BamHI and XhoI cloning sites of p416 (GPD) centromeric vector. In order to introduce a HA tag in frame with human NCL, its coding sequence was PCR-amplified from cDNA extracted from HEK293T cells using HA-NCL F-5' CGCGGATCCATGTACCCATACGATGTTCCAGATTACGCTGTGAAGCTCGCGAAGGCAG 3' and NCL R-5' CCGCTCGAGCGGCTATTCAAACTTCGTCTTCTTTCC 3' primers and cloned into pCDNA3 vector (Invitrogen) using BamHI and XhoI cloning sites. HA-NCL was then sub-cloned into the S.cerevisiae vector p414 (GPD). All generated constructs were amplified in the TOP10 E.coli strain, and sequenced by the Sanger method.

Generation of nsr1 yeast strains

NSR1 gene deletion was carried out by replacement with kanMX6 cassette amplified from PFA6a-kanMX6 vector 40 , using the following primers: F-5' ACCAATTTCGGATCACTCAACCCAGGCAGGATAAAATAAGCGGATCCCCGGGTTAATTAA 3' and R-5' AAGAGAAAAAATTGAAATTGAAATTCATTTCATTTTCTCAGAATCCGAGCTCGTTTAAAC 3'. Then the PCR fragment was transformed into W303 ade2 , 43GAr-ADE2 and W303 ade2 , ADE2 yeast strains using standard lithium acetate procedures 39 . The transformed cells were spread on YPD + 100 µg/mL kanamycin plates which were then incubated 5 days at 29°C, after which the plates were replicated on fresh YPD + 100 µg/mL kanamycin plates, and the deletion of NSR1 gene in kanamycin-resistant colonies was checked by PCR, using the following primers: nsr1

Fbis-5' GTACTTAAGTGTAGCTGTTGC 3' and nsr1 Rbis-5' TAGAGATGGTGAATGAAAGG 3'.

Yeast protein extracts

5 mL of 0,8-1,0 OD 600nm exponentially growing cells were harvested and cell pellets were resuspended into 300 µL of lysis buffer (25 mM Tris-HCl pH 6.8; 10% glycerol; 5%mercaptoethanol; 5% SDS; 8 M Urea; 0.02 % Bromophenol Blue).

Mammalian cells protein extracts

Whole cells were harvested 48 hours post-transfection and lysed in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Igepal containing protease inhibitors (Roche, Germany). Samples were centrifuged at 16,000 g during 20 min at 4°C and protein concentrations were measured using a Bradford assay.

Western blotting

Equal protein quantities and volumes of all samples were loaded onto 10% NuPAGE® Bis-Tris gels (Invitrogen), and transferred onto 0.45 m nitrocellulose membranes (GE Healthcare).

Membranes were blocked during 1 hour at room temperature in PBS 1X containing 0.1% Igepal and 3% BSA.

Membranes were analyzed using the following antibodies: anti-HA serum (1:2500); anti-Nsr1p mouse monoclonal antibody (Abcam, 1:5000), anti-NCL rabbit polyclonal antibody (Abcam, 1:5000), anti-GAPDH (Sigma, 1:5000), anti-EBNA1 mouse monoclonal antibody (OT1X, 1:2000), anti-OVA rabbit polyclonal antibody (Sigma, 1:2500), anti-actin (Sigma, 1/5000). The membranes were then washed with fresh PBS 1X + 0.1% Igepal and incubated for 45 min with swine antirabbit or goat anti-mouse secondary antibodies (Dako) conjugated to horseradish peroxidase at a 1:3000 dilution, and analyzed by enhanced chemiluminescence (ECL, GE Healthcare) using a

Vilbert-Lourmat Photodocumentation Chemistart 5000 imager. All the experiments were repeated at least three times. Relative protein levels for each sample were normalized to GAPDH or Actin protein levels as indicated, using Fusion-Capt Advance software.

Cell culture, Transfection. according to the manufacturer's protocol or electroporation using Gene PulserXL system (Biorad).

H1299

RNA extraction and quantitative real-time PCR

Total yeast, H1299 and Mutu-1 cellular RNA was extracted using RNAeasy and RNAase-free, DNase kits (Qiagen). cDNA synthesis was carried out using 1 µg of DNA-free RNA using M-MLV reverse transcriptase (Invitrogen) and Oligo-dT primer. Triplicated cDNA samples were analysized by quantitative PCR using PERFECTA SYBR fastmix (Quanta Bioscience). The relative abundance of target mRNA was normalized using Actin as an endogenous control.

Quantification of gene expression was determined using the -2 Ct method. The primers used for

RNA pull-down experiments

For the preparation of whole cell extracts, confluent H1299 cells were harvested after trypsin treatment and washed twice with 1X PBS (Gibco). Cells were suspended in 500 µL of lysis buffer (20 mM Tris-HCl pH 7.5; 200 mM NaCl and 0.1 % Igepal) containing 1X protease inhibitor cocktail (Roche). Cell lysis was performed by 5 series of vortex followed by 10 min incubation on ice, and 3 series of 3 sec sonication at 20% amplitude. After lysis cells were centrifuged at 4°C for 15 min at 16,000g, and the supernatant was quantified by Bradford. The whole cell extracts or recombinant GST-NCL (Abnova) were used for pull-down assays with the following G-quadruplex forming oligonucleotides: GQ-5'-GGGGCAGGAGCAGGAGGA-3'Biotin TEG, ARPC2-5' AGCCGGGGGCUGGGCGGGGACCGGGCUUGU-3'Biotin TEG. The negative control for EBNA1 Gquadruplex was the GM-5' GAGGCAGUAGCAGUAGAA-3'Biotin TEG oligonucleotide which, according to the GQRS-H predictor software 30 , is unable to form G4 structures. To avoid unspecific binding, high-affinity streptavidin sepharose beads (GE Healthcare) were incubated in 1 mL blocking buffer containing 10 mM Tris-HCl pH 7.5; 100 mM KCl; 0.1 mM EDTA; 1 mM DTT; 0.01% Triton X-100; 0.1% BSA; 0.02% S. cerevisiae tRNAs (Sigma), for 1 hour at 4°C on a rotating wheel. 10 pg of each folded biotinylated RNA oligos were incubated with 50 µL of solution containing the streptaviding sepharose beads for 90 min at 4°C on a rotating wheel. 500 µg of cell extract or 200 ng of recombinant GST-NCL were incubated with the RNA oligonucleotides bound to the streptavidin beads during 90 min at room temperature. Beads were washed with increasing KCl concentration (200-800 mM). Protein still bound to beads after the washes were eluted using 2X SDS loading buffer and analyzed by western blotting against NCL, as previously described.

Proximity Ligation Assay (PLA)

Cells were fixed with 4% paraformaldehyde in PBS 1X for 20 min and permeabilized with 0.4% Triton X-100, 0.05% CHAPS for 5 minutes at room temperature. 50 ng of EBNA1-digoxigenin DNA probe (5' CTTTCCAAACCACCCTCCTTTTTTGCGCCTGCCTCCATCAAAAA 3') were denaturated 5 minutes at 80 °C and the hybridization reaction was carried out overnight at 37 °C. A blocking solution of 3% BSA 0.1% saponine in 1X PBS was added for 30 min followed by 2 hours incubation with the primary antibodies (anti-digoxigenin 1/200 -Sigma-and anti-NCL 1/1000 -Abcam-) diluted in 0,3% BSA, 0.1% saponine. The proximity ligation assay (PLA) was carried out using the Duolink PLA in situ kit (Sigma) following the manufacturer's protocol.

35 S methionine pulse-labeling 8 X 10 5 cells were transiently transfected with 4 µg of 235GAr-OVA or OVA vectors and, 8 hours later, NCL silencing was performed using 40 nM of NCL siRNA or control siRNA (as previously described). 40 hours after the transfection cells were incubated 30 min in a methionine-free medium. After incubation, 25 µM of MG132 proteasome inhibitor was added to the medium and cells were incubated for 45 min. Cells were then cultured in a medium containing 0.15 mCi/mL 35 S-methionine (Perkin Elmer, Boston, USA) for 1 hour and harvested. Cell pellets were suspended in 20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Igepal and treated as described above.

Lysates were pre-cleared with 1 µg normal rabbit serum (Dako) bound to protein G-Sepharose magnetic beads (GE Healthcare) for 30 min at 4°C and further immunoprecipitated with 1 g of anti-OVA polyclonal antibody (Sigma) or IgG-rabbit (Dako), pre-bound to protein G-Sepharose magnetic beads overnight at 4°C. Beads were then washed and proteins eluted using 2X SDS loading buffer. Immunoprecipitates were analyzed by SDS-PAGE using 10% precast NUPAGE gels (Invitrogen). The amount of radiolabeled proteins was visualized using a Storm Phosphorimager (GE Healthcare).

T cell proliferation assay

Naive OVA 257-264 specific CD8+ T cells were isolated by negative selection from peripheral and mesenteric lymph-nodes of 12-week-old female OT1 mice using the CD8 + T cell isolation kit (Miltenyi Biotec, Germany). Afterwards, CD8 + T cells were stained with CellTraceTM Violet (Thermo Fisher Scientific, USA) according to the manufacturer's protocol and mixed with H1299 cells cotransfected with mouse k b expression vector and OVA or GAr-OVA constructs. For all the assays, 10 5 H1299 cells were harvested 48h after transfection and co-incubated with 4 x 10 5

stained T cells at 37°C in humidified air/CO 2 atmosphere in 1 ml of RPMI medium containing 10% FBS, 4 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 5 mM HEPES and 0.05 mM 2-mercaptoethanol (Sigma-Aldrich). After 3 days, cells were harvested, stained with hamster anti-mouse CD3-APC (Miltenyi Biotec) and fixable viability dye eFluor® 780 (eBioscience, USA) and analyzed by FACS on a CANTO II flow cytometer (BD Biosciences, USA). Cells were gated for live CD3 + cells (10.000 events collected) and data were analyzed using BD FACSDiva software version 8.0.1. The percentage of proliferating T cells was considered for statistical analysis. GAPDH was used as a loading control. The 43GAr-Ade2p/GAPDH or Ade2p/GAPDH ratio are indicated below the gels.

1

INTRODUCTION

The Epstein-Barr virus (EBV) is the first oncogenic virus described in human. EBV is worldwide spread causing a latent infection of B cells which is often asymptomatic. Nevertheless, EBV infection has been linked to several types of cancers like Burkitt and Hodgkin lymphomas, nasopharyngeal carcinoma and 10% of gastric cancers 26 . EBV´s EBNA1 protein is the only protein expressed in all EBVinfected cells as it is essential for viral replication and maintenance. EBNA1 is also highly antigenic and T cells against EBNA1-derived antigenic peptides exist in all EBV-infected individuals. Therefore, EBV has evolved a strategy in order to tightly regulate EBNA1 protein level to ensure genome replication and maintenance and at the same time avoid T CD8 + recognition of EBV-infected cells.

EBNA1-based mechanism of immune evasion is based on its central glycine-alanine rich domain (GAr)

which is able to self-limit the synthesis of its own mRNA in cis, and thus the production of EBNA1derived antigenic peptides [START_REF] Tellam | Messenger RNA sequence rather than protein sequence determines the level of self-synthesis and antigen presentation of the EBV-encoded antigen, EBNA1[END_REF][START_REF] Tellam | Targeting of EBNA1 for rapid intracellular degradation overrides the inhibitory effects of the Gly-Ala repeat domain and restores CD8+ T cell recognition[END_REF]143 . The mechanism involved in this GAr-based translation inhibition is still unknown and highly debated 5,[START_REF] Cardinaud | The synthesis of truncated polypeptides for immune surveillance and viral evasion[END_REF] . Recent evidence has shown that the GAr-encoding mRNA form particular secondary structures known as G-quadruplexes (G4) which seem to be responsible for GAr-mediated inhibition of translation and antigen presentation [START_REF] Tellam | Regulation of protein translation through mRNA structure influences MHC class I loading and T cell recognition[END_REF] . Our group recently identified Nucleolin (NCL) as the first cellular factor critically involved in both GAr-based inhibition of translation and antigen presentation (Lista et al submitted, see chapter 1). We demonstrated that NCL directly binds to G4 formed by GAr-encoding mRNA and that this interaction takes place in the nucleus.

Furthermore, impairing NCL binding to GAr mRNA by using PhenDC3 G4 ligand abolishes GAr-based inhibition of translation and antigenic presentation. Nevertheless, we still don t know how this interaction inhibits mRNA translation (Lista et al submitted, see chapter 1). Since NCL-GAr mRNA interaction takes place in the nucleus, we can hypothesize three mechanisms: first, GAr G4s may act as a recognition signal for NCL binding which would repress translation; second, NCL could trap GAr RNA in the nucleus which would explain its low translation level; third, a combination of the two hypotheses described above. Here we showed that loosing NCL-GAr RNA interaction completely abolishes GAr-based translation inhibition and also antigen presentation. Moreover, our data show that GAr-based translation inhibition and antigen presentation are effective as long as GAr RNA and NCL are allowed to interact, i.e: as long as NCL and GAr RNA are localized in the same cellular compartment.

Hence, the nuclear localization of the interaction between NCL and GAr RNA seems to be rather due to the cellular localization of NCL in the cell.

RNA extraction, RT-PCR and RT-qPCR

1, 5 x 10 5 H1299 cells were plated in six-well plates and transfected with the indicated constructs. At 48 h post-transfection, cells were washed with cold PBS 1X and RNA was purified using the RNeasy Mini Kit and on-col mn DNa e ea men Qiagen follo ing he man fac e p o ocol Nuclear/cytoplasmic RNA extractions were carried out according to Apcher et al 144 employing transfected H1299 cells. RT was carried out using M-MLV reverse transcriptase and random hexamers (Invitrogen). q-PCR was performed using a set of primers targeting OVA or globin (see Supplementary

Table 2) and Phusion high-fidelity PCR master mix (Thermo Scientific). RT-qPCR was performed as previously described 144 using specific primers for each gene fragment of interest (see Supplementary Table 2).

Western blot analysis

Whole cell lysates were prepared 48 hours post-transfection and protein concentration was measured using a Bradford assay. Samples were electrophoretically separated in NuPAGE® 10% Bis-Tris gels (Invitrogen), transferred onto 0.45 m nitrocellulose membranes (GE) and bloted under standard conditions using the following antibodies: mouse monoclonal anti-globin antibody (2H3, Sigma), rabbit polyclonal anti-OVA antibody (C6534 Sigma), mouse monoclonal anti-actin (AC-15 Sigma), mouse polyclonal anti-HA antibody (a kind gift from Borek Vojtesek, Masaryk Memorial Cancer Institute, Brno, Czech Republic) and chicken polyclonal anti-HIV1 Rev antibodies (ab36623, Abcam). Anti-mouse (Dako), anti-rabbit (Dako) or anti-chicken (Sigma) secondary antibodies conjugated to horseradish peroxidase were used according to manufacturer s recommendation to generate immunocomplexes revealed with enhanced chemiluminescence (Thermo Scientific).

Membranes were scanned in a MyECL imager (Thermo Scientific) and signal intensity was determined using My Image software (Thermo Scientific).

Protein immuno-detection and

Antigen presentation assays

Naive OVA257-264 specific CD8 + T cells were isolated by negative selection from peripheral and mesenteric lymph-nodes of 12-weeks-old female OT-1 mice using a CD8 + T cell isolation kit (Miltenyi Biotec, Germany). Afterwards, CD8 + T cells were stained with CellTrace TM Violet (Thermo Fisher Scientific, USA according to the manufacturer s protocol and mixed with H cells co-transfected with mouse Kb expression vector and the indicated constructs. For all the assays, 10

RESULTS and DISCUSION

Interaction of Nucleolin with GAr-carrying RNAs only takes place in the nucleus

We recently demonstrated that GAr mRNA-Nucleolin (NCL) interaction takes place in the nucleus and that this interaction is essential to ensure GAr-based translation inhibition (Lista et al, submitted).

We first wondered if EBNA1 mRNA translation inhibition may be due to its trapping by NCL in the nucleus, thus avoiding EBNA1 mRNA cytoplasmic transport and translation. For this purpose, we took advantage of the fact that pre-RNAs (intron-containing RNA) that need to be spliced are taken over by the splicing machinery which rapidly mediates the transport of spliced-mRNAs into the cytoplasm for their subsequent translation. For this reason, we created several constructs containing the GArencoding sequence in fusion with globin or ovalbumin cDNA (GGC and GOC) or complete gene sequence (GGG and GOG), as well as the GAr-free corresponding controls (GC,GG,OC,OG) (Figure 1A). Each construct contains the ovalbumin (OVA) SL8 antigenic peptide SIINFKEL (OVA257-264) sequence in order to measure the antigenic presentation of the peptides derived from all construct used. By fluorescence in situ hybridization (FISH) and subcellular fractionation, we showed that, as expected, both GAr-free RNAs (GG and OG) and GAr-carrying RNAs (GGG and GOG) from the gene constructs are localized in the cytoplasm (Figure 1B-C). However, GAr-carrying RNAs from cDNA 6 constructs (GGC and GOC) are localized in the nucleus (Figure 1B-E), contrary to GAr-free RNAs from cDNA constructs (GC and OC). We then checked the ability of GAr-carrying RNAs localized in the nucleus or the cytoplasm to interact with NCL by proximity ligation assay (PLA) and RNA coimmunoprecipitation. We demonstrated that only GAr RNAs localized in the nucleus are able to specifically interact with NCL whereas no interaction was detected between NCL and GAr cytoplasmic RNAs (Figure 2A-B). To support our findings, we treated cells expressing GAr cytoplasmic RNAs with the splicing inhibitor isoginkgetin. As expected, this treatment inhibited the cytoplasmic transport of GAr RNAs from gene constructs (GGG) which were therefore located in the nucleus. Using PLA, we demonstrated that these GAr RNAs from gene constructs (GGG) which are held back in the nucleus, are able to establish an interaction with NCL (Figure 2C). Taken together our results showed that in order to be able to interact with NCL, GAr-carrying RNAs need to be located in the nucleus, which is in good agreement with the location of NCL in the cell.

GAr-based inhibition of translation and antigen presentation is solely efficient from nuclear RNAs.

After having demonstrated that only GAr RNAs present in the nucleus are able to interact with NCL, we tested the translation efficiency from nuclear and cytoplasmic GAr-carrying RNAs. For this purpose, H1299 cells were transfected with the globin (Figure 3A) and OVA (Figure 3D) constructs, and their expression levels were analyzed by western blot. We showed that GAr is solely able to inhibit the expression of nuclear RNAs (GGC, GOC) whereas its effect is completely abolished when the RNAs are located in the cytoplasm (GGG, GOG; Figure 3A andD (GG, GC, OG and OC). These results showed that NCL interaction is essential to ensure the GAr-based inhibition of translation and antigenic presentation.

Export of nuclear GAr RNAs to the cytoplasm abolishes GAr RNA-NCL interaction and suppresses the GAr-based inhibition of translation and antigen presentation.

We next wondered if the relocation of nuclear GAr RNAs in the cytoplasm may affect its interaction with NCL, GAr-fused protein expression and antigenic presentation.

In order to modify the localization of nuclear GAr RNAs we inserted the RRE recognition sequence for HIV-1 REV protein in the globin cDNA constructs (Figure 4A-B). During HIV-1 infection some unspliced viral mRNAs coding for structural proteins need to be transported to the cytoplasm to be rapidly expressed. To do so, the REV viral protein is expressed in the early stage of HIV-1 infection and promotes the cytoplasmic transport of RRE-mRNAs. By using this REV-based system we showed that nuclear GAr-globin RNAs (GGC) are efficiently exported to the cytoplasm by REV (Figure 4B). The interaction between GAr RNA and NCL, that is taking place in the nucleus (Figure 4C), is completely abolished after RNA transport to the cytoplasm (Figure 4D). Furthermore, the cytoplasmic transport of GAr RNA from cDNA construct (GGC) enhances GAr globin expression (Figure 4E) and also antigen presentation (Figure 4G). Altogether these data, demonstrate that NCL co-localizing with GAr RNA in the nucleus is crucial to ensure their interaction and a further GAr-based translation inhibition effect.

NCL-mediated GAr inhibition of translation is also efficient in the cytoplasm.

We finally sought to determine whether the nuclear location of NCL-GAr RNA interaction is essential to ensure the GAr inhibition of translation effect. For that purpose, we constructed a cytoplasmic NCL mutant by modifying its nuclear localization signal NCLΔNLS, Figure 5A). We first showed by PLA that changing NCL location from the nucleus to the cytoplasm restores its interaction with cytoplasmic GAr RNAs GGG (Figure 5B-C). These data were confirmed by using the REV-based relocation of GAr RNAs from cDNA constructs in the cytoplasm: NCLΔNLS interacts with cytoplasmic GAr RNAs from cDNA constructs GGC (Figure 5F-G). These data proves that NCL-GAr RNA interaction can take place outside of the nucleus. Moreover, we observed that the cytoplasmic interaction of NCL with GAr RNA restores the GAr-based translation inhibition (Figure 5D-E), demonstrating that to ensure EBNA1 translation inhibition, the interaction of GAr-containing RNA with NCL is essential no matter where it takes place in the cells (Figure 5H). Nevertheless, our results do not allow to 8 determine if NCL inhibits translation of EBNA1 mRNA by a direct binding or if EBNA1 mRNA trapping in the nucleus by itself is sufficient enough for the inhibition of EBNA1 translation and antigenic presentation. However, the fact that GAr mRNA-NCL cytoplasmic interaction also leads to translation inhibition indicates that NCL can inhibits protein translation without RNA nuclear trapping.

Moreover, we can hypothesize that NCL interaction with GAr RNA may block the binding of the ribosomal subunit 40S, the ribosomal assembly of the subunit 60S or the ribosomal progression.

Another possibility is that GAr polypeptide may also be involved in the translation inhibition by blocking ribosomal read-through.

Taken together our results demonstrated that in order to ensure EBNA1 GAr-based translation inhibition, NCL is recruited to the EBNA1 mRNA through its interaction with GAr G4s motif which inhibits EBNA1 translation and thus antigen presentation. (B) Cells transfected with indicated constructs were subjected to RNA co-immunoprecipitation (RNA-coIP) using antibodies against nucleolin. NCL and IgG bound RNAs were analyzed by RT-qPCR against globin. This confirms the interaction of NCL with GAr-globin RNA from cDNA construct. Graphs represent the mean of three independent experiments. **p<0.01, ns: not significant.

(C) Cells transfected with GAr-globin gene construct (GGG) were treated with 30 µM of the spliceosome inhibitor isoginkgetin and subjected to proximity ligation assays (PLA) between NCL and the GGG RNA. Isoginkgetin treatment induces the retention of GGG RNA in the nucleus and the interaction between NCL and GGG RNA.

(D) Cells transfected with GAr-globin gene (GGG) and treated with 30 µM of isoginkgetin were analyzed by RT-qPCR using primers located in introns, revealing that isoginkgetin treatment induces an accumulation of unspliced GGG RNA. Yeast as a tool to decipher some of the steps of EBV life cycle

• Manuscript presentation:

During the past 30 years the yeast Saccharomyces cerevisiae happens to have been an excellent tool to study some of the steps of the Epstein-Barr virus life cycle. From the beginning of the eighties yeast has been used in order to identify the autonomous replicative sequences (ARS) of EBV genome, and also to study EBV lytic cycle. Later on, yeast has contributed to the study of EBNA1 function in the maintenance of the viral episome. Moreover, thanks to the yeast two-hybrid assay the cellular proteins that collaborate with EBNA1 in order to ensure the episome maintenance were identified.

Finally, yeast served to explore EBNA1 GAr-based mechanism of immune evasion. These topics were the subject of a review that we recently published in Biotechnology Journal which manuscript is presented below.

Introduction

The Epstein-Barr virus (EBV) is the first oncogenic virus described in humans [1-4] and EBV is still one of the most effective means for transforming and immortalizing human B cells. Over 90% of the population is infected by this ubiquitous gammaherpesvirus. Primary infection with EBV is normally asymptomatic but, in teenagers and adults, it can be responsible for a self-limiting lymphoproliferative disease termed infectious mononucleosis. In most individuals this virus persists as a lifelong latent asymptomatic infection but it can be responsible for severe lymphoproliferative disorders in patients suffering from some forms of immune suppression (e.g. grafted patients or HIV-infected people). EBV has also been linked to certain types of cancers ( [4, 5], for a comprehensive list see [6]), the best known being the endemic form of the Burkitt's lymphoma to which the malaria infection has

Review

The long-lasting love affair between the budding yeast Saccharomyces cerevisiae and the Epstein-Barr virus María José Lista The Epstein-Barr gammaherpesvirus (EBV) is the first oncogenic virus discovered in human. Indeed, EBV has been known for more than 50 years to be tightly associated with certain human cancers. As such, EBV has been the subject of extensive studies aiming at deciphering various aspects of its biological cycle, ranging from the regulation of its genome replication and maintenance to the induction of its lytic cycle, including the mechanisms that allow its immune evasion or that are related to its tumorogenicity. For more than 30 years the budding yeast Saccharomyces cerevisiae has fruitfully contributed to a number of these studies. The aim of this article is to review the various aspects of EBV biology for which yeast has been instrumental, and to propose new possible applications for these yeast-based assays, as well as the creation of further yeast models dedicated to EBV. This review article illustrates the tremendous potential of S. cerevisiae in integrated chemobiological approaches for the biomedical research.

been proposed as a co-factor [3]. Other examples are the nasopharyngeal carcinoma, which is particularly frequent in Chinese and Tunisian men [7], the Hodgkin's lymphoma and 10% of the gastric cancers. The link between EBV and human diseases has been known for 50 years and as such has been the subject of extensive studies aiming at deciphering its life cycle and its role in tumorogenicity (Fig. 1). Among these studies, the budding yeast Saccharomyces cerevisiae has been fruitfully used for more than 30 years, both as a model and a tool, to unravel many of the different aspects of EBV mysteries. The aim of this article is to review these uses of S. cerevisiae and to propose new possible applications for these yeast-based assays, as well as the creation of further yeast models dedicated to EBV. Overview of the EBV life cycle. Following a primary infection of naive B-cell by EBV, a latent infection settles down in most cases (A). In this situation the virus has to replicate and maintain its genome (B) thanks to its genome maintenance protein (GMP) EBNA1 which is highly antigenic. EBNA1 is thus the Achilles heel of EBV and the virus evolved a mechanism to tightly control its level, thereby allowing the immune evasion of EBV-infected cells and, at the same time, the replication and maintenance of the viral genome (C). In a few cases, the virus is reactivated (D) which leads to the activation of the lytic cycle by ZEBRA1 and thus to the lysis of the infected B cells, thereby releasing many viral particles that can infect some more naive B-cells. Because of its oncogenic activity, EBV can also, in a few cases, promote tumors (E), in particular some lymphomas like the Burkitt's and Hodgkin's lymphomas. The budding yeast Saccharomyces cerevisiae has been successfully used to study all these aspects of EBV biology.

2 Using yeast to explore EBV genome replication and maintenance

EBV genome replication

Regarding EBV, yeast has been first used from the beginning of the eighties to identify autonomous replicating sequences (ARS) in the EBV genome [8]. Indeed, the EBV genome is an extrachromosomal double-stranded circular DNA episome which uses the DNA polymerase of the host cell to replicate during latent infection in which this episomal element has to persist. Hence, this plasmidic DNA necessarily contains its own autonomous origin of replication that has been intensively searched for years without any success [9, 10]. Then, at the beginning of the eighties, the group of J. S. Pagano exploited a recently described yeast-based system [11-14] to detect ARS in the EBV episome [8]. This system (described in Fig. 2A) is based on a plasmid which contains a selective marker (usually the yeast URA3 gene) which allows its positive selection in yeast. Importantly, this plasmid does not contain any yeast origin of replication and thus cannot confer to a ura3-auxotroph yeast strain the ability to grow in a medium deprived of uracil, unless an exogenous autonomous origin of replication is incorporated in the plasmid. This system has thus been used to screen eukaryotic or viral DNA fragments for their ability to self-replicate and therefore has allowed ARS identification in the EBV genome (Fig. 2A). Of note, none of the identified ARS correspond to oriP, the genetically-defined EBV latent EBNA1-dependent origin of replication which was defined shortly after [15] (EBNA1 is an EBV-encoded protein presented in the next section). They rather overlap with EBNA1-independent sites of replication identified later on by the team of C. L. Schildkraut [16, 17].

EBV genome maintenance

In addition to its need to replicate, the viral genome has to remain within the infected cells, which is particularly important in the case of EBV as it latently infects only a few resting B-lymphocytes which then may become highly dividing cells. The EBV genome is maintained in latently infected human cells as low copy number, doublestranded circular DNA episomes [18] which replicate once per cell cycle and are efficiently partitioned during cell division, resulting in a constant copy number per cell. Hence EBV episomes behave like minichromosomes, which implies that they contain sequences allowing their direct or indirect anchoring to the mitotic spindle or host cell chromosomes. Upon primary B-cell infection, EBV directly enters latent infection which is characterized by a reduced viral gene expression limited to a small subset of proteins termed latent antigens. These latent proteins include at least the genome maintenance protein (GMP), expression of which is crucial for the maintenance of the viral genome in latently infected cells [19]. The GMP of EBV is the EBNA1 protein which is also essential for the efficient replication of the EBV genome. Therefore, both the replication and maintenance of EBV episomes critically depend on the virally-encoded EBNA1 protein which is expressed as such in all EBV-infected cells. In addition, EBNA1 is highly antigenic and CD8 + T cells directed towards EBNA1 epitopes exist in all the infected individuals, including individuals with cancers. For these reasons, EBNA1 is widely recognized as the Achilles heel of EBV towards the immune system (see the last section dedicated to the EBNA1-based EBV immune evasion) [19]. Hence, EBNA1 is the most studied EBV-encoded protein and, at the end of the nineties, the team of L.

Frappier used yeast as a tool to identify in a two-hybrid screen the human proteins that interact with EBNA1. EBNA1 was used as a bait and EBP2 (EBNA1 binding protein 2) was shown to specifically interact with EBNA1 [20]. EBP2 was also shown to bind to DNA-bound EBNA1 in a one-hybrid system and the EBP2-EBNA1 interaction was confirmed by co-immunoprecipitation. Thanks to the yeast two-hybrid system, the EBNA1 region that binds to EBP2 has been mapped to the small central glycine-arginine (GR) rich domain. The deletion of this GR domain in EBNA1 prevents EBP2 interaction and consequently the ability of EBNA1 to support the long-term maintenance of a plasmid containing the EBV origin (OriP) in human cells.

Later on, a minor contribution of the small N-terminal GR of EBNA1 was also reported using a similar two-hybrid mapping [21]. More recently, the yeast two hybrid system was used to establish at the genome scale the interaction maps between EBV proteins and between EBV and human proteins [22].

Function of EBP2 in EBV genome maintenance

EBP2 is a 35 kDa protein conserved in eukaryotes, which led the teams of M. A. McAlear and L. Frappier to study the function of its ortholog in the budding yeast [23]. The yeast EBP2 gene has been shown to encode an essential protein that localizes to the nucleolus and yeast cells expressing temperature-sensitive mutants of EBP2 become depleted of ribosome and cease to divide after several generations at the restrictive temperature.

A careful analysis of these mutants suggested that the EBP2 protein is involved in the processing of the ribosomal RNAs (rRNAs), thereby giving some clues about its function. Since then, the human EBP2 protein (hEBP2) has been shown to be also a nucleolar protein [24], thus probably fulfilling the same cellular function as the yeast protein. Of note is that EBNA1 was very recently shown to interact with nucleolin, another nucleolar protein essential for rRNA maturation. Nucleolin is conserved from yeast to human and its interaction with EBNA1 is critical for the genome maintenance function of the latter [25]. hEBP2 was shown by the group of L. Frappier to bind to mitotic chromosomes following the nucleolus disassembly observed in mitosis [26]. In the same study, an RNA interference (RNAi) approach showed that hEBP2 is essential for the human cell proliferation and for the binding of EBNA1 and EBV-based plasmids to mitotic chromosomes. Furthermore, hEBP2 undergoes the same cell cycle-regulated association with the mitotic chromatin in yeast and hEBP2 attachment to mitotic chromosomes is dependent on the yeast Aurora B kinase ortholog Ipl1, suggesting that the key role of hEBP2/EBNA1 complex in the EBV genome maintenance is regulated by Aurora B kinase activity. This notion is supported both by RNAi silencing of Ipl1 homolog in human cells and by using ZM447439, a small molecule that specifically inhibits Aurora B [26]. Hence, the functional link between hEBP2 and EBNA1 highlighted by the interplay of the yeast-and human cell-based approaches nicely illustrates the two types of yeast utilization for biomedical studies: first as a tool to isolate hEBP2 in a two-hybrid screen, and then as a model system to decipher hEBP2 biological function and the nature of its role in the EBV genome maintenance.

Reconstitution of EBV-based plasmid partitioning in yeast

Following the discovery of hEBP2 crucial role in the EBV genome maintenance, the group of L. Frappier successfully reconstituted EBV-based plasmid partitioning in yeast [27]. This system (described in Fig. 2B) is based on a plasmid that contains a yeast replication origin (ARS) and the TRP1 gene as a selective marker but that lacks a yeast segregation element (centromeric sequence or CEN), thereby being unable to stably segregate in yeast.

The insertion of the FR segregation sequence of EBV in this plasmid allows its stable segregation in yeast only if EBNA1 and hEBP2 are co-expressed in the same yeast cells (Fig. 2B, lower panel), demonstrating that the EBV segregation system can be reconstituted in yeast. Using The yeast assay that allowed the identification of ARS in EBV episome. The EBV episome was first digested and the resulting fragments were cloned into a plasmid that contained the yeast URA3 gene which allows an ura3yeast strain to grow on a minimal medium deprived of uracil (Step 1). This plasmid lacks of a eukaryotic origin of replication and is thereby rapidly lost by dividing yeast cells making them unable to grow on minimal medium not supplemented with uracil. The resulting library of vectors was then used to transform a recipient ura3yeast strain and the transformed cells were spread on a minimal medium not supplemented with uracil (Step 2). The colonies which appeared on this selective medium should, in principle, have been transformed by a vector that contains a fragment of EBV episome that acts like a eukaryotic origin of replication. The plasmids from these colonies were extracted and the activity of the EBV ARS was confirmed by the retransformation of the same yeast strain, thereby allowing the ARS characterization (Step 3). (B) Yeast model for the maintenance of EBV episome. The rationale is similar to the one of the assay presented in (A), except that the designed plasmid contains the TRP1 selection marker as well as its own origin of replication and yeast centromeric sequences (Yeast CEN, left) or the EBV FR sequence (right) which is known to be important for the maintenance of EBV episome, presumably by anchoring it to the host cell chromosomes via EBV-encoded EBNA1 and the human host cell-encoded hEBP2 proteins (Step 1). These plasmids were introduced in a recipient trp1 -yeast strain that expresses, or not, EBNA1 and/or hEBP2 (Step 2). Whereas the yeast vector that contains a yeast CEN sequence stably propagates irrespectively of the presence or absence of EBNA1 and/or hEBP2, the yeast vector that contains the EBV FR sequence is stably maintained only in yeast cells that expressed both EBNA1 and hEBP2 (Step 3). Hence, the crucial role of these two proteins in the EBV genome maintenance was recapitulated in yeast.

this system, the role of the EBNA1/hEBP2 complex in the EBV genome maintenance was carefully investigated [28]. hEBP2 was found to attach to yeast mitotic chromosomes in a cell cycle-dependent manner, thus allowing the binding of the EBV episome/EBNA1 complex to the mitotic chromosome (see Fig. 3 for an integrated model). This elegant yeast-based system was thus definitively validated. Thanks to the tremendous power of yeast genetics it may further be used to decipher the cell-cycle regulated binding of EBV episomes to host cell chromosomes, a mechanism which allows their maintenance at a constant copy number in dividing cells. In this line, this yeast model has been used to highlight the importance of the phosphorylation of the four serines within the central GR rich domain of EBNA1 (the domain which interacts with hEBP2) in the regulation of the EBNA1-based segregation of EBV-derived plasmids [29]. The role of these serine phosphorylations was then validated in human cells.

Isolation of other host cell factors important for EBV genome segregation

Finally, the EBV-based plasmid partitioning assay also allowed the group of L. Frappier to test the ability of other host proteins, Brd2, Brd4 (which are members of the bromodomain and extra terminal domain-(BET-)family) and MeCP2 that are implicated in the genome segregation of papillomavirus and Kaposi's sarcoma-associated herpesvirus, to support EBNA1-mediated segregation of EBV-based plasmids [30]. They found that Brd4 enabled EBNA1-based segregation of the EBV genome, while Brd2 and MeCP2 had a general stimulatory effect on the plasmid maintenance. In the same study, this effect of Brd4 was subsequently validated in human cells. Of note, in the human system the significance of this interaction appeared to be rather in transcriptional activation as opposed to EBV genome segregation. Nonetheless, it was the yeast assay that identified the Brd4-EBNA1 interaction.

Using yeast to analyze the EBV lytic cycle

As stated above, upon primary B-cell infection, EBV directly enters the latent phase. The switch from the latent phase to the lytic cycle (Fig. 1D) has been identified as a potential therapeutic target, since favoring this switch should lead to the specific lysis of EBV-carrying tumor cells (reviewed in [6]). ZEBRA is an EBV-encoded transcription factor that first drives the expression of Rta (another EBV-encoded transcription factor) and then, together with Rta, controls the expression of several early EBV lytic genes. ZEBRA is thus at the top of a cascade of events leading to the expression of EBV lytic cycle genes (Fig. 1D). ZEBRA activates the transcription by binding to ZEBRA response elements (ZREs) found in the promoters of its EBV lytic cycle target genes. ZEBRA transcriptional activity has been recapitulated in S. cerevisiae by the group of G. Miller which expressed ZEBRA in yeast cells that contain a reporter gene (LACZ) under the control of several ZREs [31]. This system allowed the identification of the regions of ZEBRA that play a role in the transcriptional activation of its target genes. The group of G. Miller then further showed that two natural EBV promoters are activated by ZEBRA in yeast: Zp, the promoter that regulates the expression of ZEBRA itself and EAp, the promoter controlling the expression of BMRF1, the EBV DNA polymerase processivity factor. Importantly, as yeast is a EBV has evolved a mechanism to express EBNA1 at a functional but minimal level to allow the maintenance of its episome and, at the same time, to evade the immune system.

naive system regarding EBV, these results indicate that neither mammalian-specific nor virally-encoded cofactors are mandatory for ZEBRA-based activation of the transcription from these two promoters [32].

4 Using yeast to study the oncogenic activity of EBV

CDK-like activity of the CHPK family of herpesviruses kinases

Altogether, the genomes of the eight known human herpesviruses, including EBV, encode at least sixteen protein kinases that are grouped in three distinct families. One of these families, the CHPK (for conserved herpesvirus-encoded protein kinase) family, comprises eight members, one from each herpesvirus. Several of these CHPKs have been shown to display a CDK-like activity by inducing the phosphorylation of the retinoblastoma (Rb) tumor suppressor protein and of the lamin A/C, two events normally catalyzed by human CDKs, thereby promoting the entry in the cell cycle and mitosis of their host cells [33, 34]. This ability most certainly accounts for the ability of the herpesviruses to promote tumor formation. UL97, the CHPK of the human cytomegalovirus (HCMV) was hence first shown to phosphorylate and thus inactivate the Rb tumor suppressor protein [35]. In this study, the budding yeast was used to demonstrate that UL97 possesses a CDK-like function. Indeed, UL97 is able to complement a defect in the function of Cdk1/Cdc28 at the G1/S transition in yeast. The essential Cdk1/Cdc28 protein is the main CDK that controls the progression through all the phases of the yeast cell cycle and various conditional (thermosensitive) mutant alleles of the CDK1/ CDC28 genes, that block yeast cells at specific stages of the cell cycle when they are grown at the restrictive temperature (36°C), have been isolated. In this study, a haploid yeast strain which contains the cdc28-13 allele that blocks cells at the G1/S transition when grown at the restrictive temperature was used and the budding index, which reflects the ability of yeast cells to resume their cell cycle, was estimated. As expected, the expression of the human CDK1 gene from a plasmid led to a high budding index (around 70%), as compared to the low budding index (around 40%) using an empty vector. The expression of UL97 led to a quite high budding index (around 60%) whereas a catalytically inactive form of UL97 led to the same budding index as the empty vectors (around 40%).

Taken together these results show that UL97 possesses a CDK-like activity which allows yeast cells deprived of a functional Cdk1/Cdc28 to resume the cell cycle, almost as efficiently as the human CDK1, which is long known to complement yeast cdk1/cdc28 mutants [36].

Systematic analysis of the eight CHPK in yeast

In a recent study [37], the eight CHPKs have systematically been tested for their ability to complement the yeast cdc28-13 mutant at the restrictive temperature.

The EBV-encoded CDK-like proline-dependent serine/ threonine kinase BGLF4 is the EBV representative of the CHPK family. BGLF4 was shown to display the highest CDK-like activity among all the CHPKs. UL97 from HCMV, and U69 from both human herpesviruses 6 and 7 (HHV-6 and HHV-7) also displayed a CDK-like activity, although to a lower extent than BGLF4, whereas other CHPKs, including UL13 from both herpes simplex viruses types I and II (HSV-1 and HSV-2), ORF47 from varicella zoster virus (VZV) and ORF36 from the Kaposi's sarcoma associated herpesvirus (KHSV) had no significant activity. Thus, according to this yeast-based assay, CDK-like activity seems to be absent in alphaherpesviruses (HSV-1, HSV-2 & VZV). Interestingly, these results fully corroborated the observations that Rb is not well phosphorylated in the cells infected by alphaherpesviruses [38], but is properly phosphorylated in the cells infected by EBV and HCMV [39, 40]. However, a more recent study did not reproduce this result [41]. In this paper, the same cdc28-13-based assay was used except that the yeast cells were synchronized in early S-phase using hydroxyurea prior to the shift at the restrictive temperature after which the budding index was estimated. This additional synchronization step may explain the discrepancies observed between these two studies. Furthermore, another mutated allele of the CDC28 gene, cdc28-4 which leads to an arrest at the G2/M transition when yeast cells are shifted at the restrictive temperature, was used to determine the ability of the various genes (human CDK1, BGLF4 and K102I, a catalytically inactive version of BGLF4) to complement the growth defect of a cdc28-4 strain at the restrictive temperature. Only the human CDK1 was able to complement the growth defect of the cdc28-4 strain. This test being much more stringent and based on a mutation that affects the G2/M transition, it may account for this additional discrepancy. It would clearly be interesting to fully exploit the large collection of cdk1/cdc28 mutants to unravel the CDK-like activity of BGLF4, for example by systematically determining its ability to rescue, or not, the growth defects of these various mutant strains (see the perspective section). Finally, yeast two-hybrid approaches have allowed the identification of xeroderma pigmentosum C (XPC) [42] and a splicing variant of interferon regulatory factor 3 (IRF3) [43] as host cell interactants of BGLF4.

5 Using yeast to investigate the immune evasion of EBV 5.1 EBV strategy to evade the immune system is based on control of EBNA1 level

As stated above, most of EBV strategy to evade the immune system is based on EBNA1, its GMP. Indeed, due to its crucial role in the genome replication and maintenance, EBNA1, which is highly antigenic, has to be expressed in every EBV-infected cell. In addition, all infected individuals, including those having a cancer linked to EBV, have cytotoxic CD8 + T cells directed towards EBNA1 epitopes. However, their immune system fails to detect and destroy EBV-infected cells. This is due to EBV's strategy to evade the immune system which is based on a tight control of the expression of EBNA1. EBNA1 is thus expressed at a level low enough to avoid the immune detection, but sufficient to ensure its crucial GMP function. For this reason, various teams have considered the mechanism that tightly controls the level of EBNA1, and thus responsible for its stealthiness, as a pertinent therapeutic target for EBV-associated cancers [6]. Indeed, whereas in healthy individuals the latent infection by EBV is primarily restricted to a specific pool of memory B-cells and the viral replication is spontaneously activated in only a small percentage of these cells, most tumor cells from EBV-related cancers are infected by EBV and express EBNA1 at a functional but minimal level that allows the cancer cells to evade the immune system whilst retaining the EBV episome. Therefore, overcoming the EBNA1-based mechanism of EBV stealthiness should unveil EBV-carrying tumor cells as targets for the immune system without having any significant effect on the vast majority of healthy host cells.

Mechanism that tightly controls EBNA1 expression

The mechanism that tightly controls EBNA1 expression has been thoroughly studied. These studies started twenty years ago when the central glycine alanine rich (GAr) domain of EBNA1 was shown to be pivotal for the ability of EBNA1 to evade the immune system [44]. EBV's GAr domain consists of single alanines separated by one, two or three glycines. The deletion of this GAr domain significantly increased the presentation of EBNA1-derived antigenic peptides by the major histocompatibility complex (MHC) class I pathway. This result was then rapidly attributed to the ability of the GAr domain to inhibit the protein degradation by the 26S proteasome [45]. Indeed, when fused to a number of known substrates of the 26S proteasome, like IkB [45] or p53 [46], the GAr domain clearly inhibited their degradation in a length-dependent manner (the longer the GAr domain, the stronger the inhibition of the proteosomal degradation is). These results were fully consistent with the current dogma that prevailed at that time according to which the main source of antigenic peptides comes from the proteosomal degradation of full length proteins. In line with this model, the budding yeast S. cerevisiae was also used to confirm that the GAr domain possesses a length-dependent ability to prevent the proteosomal degradation [47]. In this study, destabilized versions of the green fluorescent protein (GFP), harboring N-end rule or ubiquitin fusion degradation signals, were fused to GAr domains of various lengths and expressed in yeast. The length-dependent effect of the GAr domain on the protein stability was thus confirmed, suggesting either that the GAr domain possesses an intrinsic ability to inhibit the 26S proteasome, or that this effect is mediated by host cell factor(s) which is/are conserved in yeast. Of note, one curious result in this article was that, despite the GAr length-dependent stabilization of the unstable version of the GFP by the GAr domain, the steady-state level of the various GAr-GFP fusions was inversely correlated to their degree of stabilization: the longer the GAr domain, the more the reporter protein was stable but the smaller its steady state level, which appears rather counterintuitive. These discrepancies were also observed at the same time for EBNA1 as the deletion of the GAr domain from EBNA1 (EBNA1 GAr) increased its level of expression instead of decreased it, as one could expect as a consequence of the deletion of a stabilization signal [48]. Finally, the model stating that the inhibition of the proteosomal degradation of EBNA1 by its GAr domain accounts for the ability of the latter to inhibit the presentation of antigenic peptides derived from EBNA1 was further challenged when it was observed that, with or without its GAr domain, EBNA1 is a very stable protein [48, 49]. Hence, even if the GAr domain is able to inhibit the proteasomal degradation of unstable proteins to which it is fused, this effect in not operant in EBNA1.

In the study by Yin et al., the use of in vitro expression systems and cell-based metabolic labeling led to the demonstration that the increased EBNA1 level observed following the deletion of the GAr domain was due to an increase in the rate of EBNA1 synthesis [48]. This striking result was the starting point of other studies that demonstrated that the GAr domain is in fact an effective inhibitor in cis of the translation of any messenger RNA (mRNA) to which it is fused, without affecting the translation in trans of other mRNAs [49-53]. These results were fully in line with the DRiPs model that was at that time recently proposed by Yewdell [54, 55] and that challenged the entire concept of full length proteins being processed by the 26S proteasome to supply the MHC class I pathway with antigenic peptides. According to this model, antigenic peptides would come mainly from the DRiPs (for "defective ribosomal products"), an alternative source of peptide material that would correspond to byproducts of translation. Hence, most of the antigenic peptides would be generated during translation of mRNAs (especially during the initiation phase). This is fully consistent with several observations among which the very rapid kinetics of the presentation of antigenic peptides by the MHC class I pathway after cell infection (a few minutes) which is not compatible with the synthesis of full length proteins followed by their degradation. In addition, inhibiting the transcription of the gene encoding the antigenic protein prevents the presentation of antigenic peptides derived from this protein during a time frame when the levels of the protein remain high [56]. Altogether, these data demonstrate that neosynthesis is required for the presentation of T cell epitopes from a long-lived viral protein.

A yeast model for EBV immune evasion

Of note, the discrepancies in the steady-state level of the various GAr-GFP fusions observed in yeast were thereby explained if, in yeast cells as in human cells, the GAr domain inhibits in cis the translation of its own mRNA in a GAr length-dependent manner. Based on this assumption, we have recently developed a yeast-based assay for monitoring the GAr domain ability to inhibit the translation of its own mRNA in cis [57]. In this model (described in Fig. 4), the yeast Ade2p protein was used as a reporter protein as: (i) it is a stable protein (like EBNA1); and (ii) a convenient colorimetric system exists to easily determine its steady state level in living cells. Indeed, whereas an ade2 strain readily forms red colonies on rich YPD medium, cells that express Ade2p at a functional level form white colonies and any intermediate level of Ade2p leads to the formation of pink colonies whose intensity of coloration is inversely proportional to the level of Ade2p expressed. Hence, any subtle change in the level of Ade2p can easily be monitored. The ADE2 gene expressed from the constitutive ADH promoter, that allows its minimal expression to get white colonies, was fused to GAr domains of different lengths (21, 43 or 235 amino acids), or to the GAr domain of the Papio herpesvirus, which is known to have no effect on translation due to the insertion of one serine every seven residues of the repeat. As in human cells, the length-dependent ability of the GAr domain to inhibit the translation of its own mRNA in cis was demonstrated whereas the Papio GAr domain had no effect [57]. Thanks to the Ade2p reporter protein, the effect of GAr can easily be monitored (Fig. 4A) and served as the screening basis for identifying compounds able to interfere with its inhibitory effect on translation using a method previously described [58-60]. This way doxorubicin (DXR) was identified and shown to specifically counteract the inhibitory effect of GAr on the expression of GAr-Ade2p. This effect was GAr-dependent since DXR had no effect on the expression of Ade2p (Fig. 4B). This yeast assay was then definitively validated when DXR was not only shown to stimulate, in a GAr-dependent manner, EBNA1 expression in mammalian cells (Fig. 4C), but also to overcome the GAr-dependent restriction of MHC class I antigen presentation (Fig. 4D) [57]. In addition, as many chemical analogs of DXR are commercially available, a structure-activity relationship study was performed using both the yeast and T-cell assays. The good correlation between the results obtained using the two assays definitively validated the yeast model for EBNA1-based EBV's stealthiness. They also indicated that the effect of DXR on the GAr-mediated inhibition of translation is uncoupled from its already known genotoxic effect [57]. What's next?

The yeast assay could also be used now for various kinds of genetic screening aiming at identifying the conserved host cell factors involved in the GAr-mediated inhibition of translation and thus of antigen presentation. Of note, similarly to the drug screening, this assay should allow to identify both positive and negative regulators of the GAr effect that could then constitute attractive therapeutic targets for the treatment of cancers linked to EBV.

Concluding remarks

As with all long-lasting love affairs, there is a need to reinvent the story and thus rekindle the flame. This could be done for example by fully exploiting the yeast-based assays that already exist and model various aspect of EBV biology. Indeed, most of these assays could be used for drug screening whereas, for the time being, the yeast model for the EBNA1-based mechanism of EBV immune evasion is the only one that has been exploited this way. The possibilities were even less explored regarding genetic screenings that aim at identifying in a comprehensive manner the host cell factors necessary for the various aspects of the EBV biological cycle. In this view, exploiting the yeast models for the EBV genome maintenance and the immune evasion could certainly give some interesting clues about new mechanisms, and thus new potential therapeutic targets that are relevant for the treatment of EBV-related diseases, including cancers. In addition, regarding EBV oncogenicity, yeast strains in which the endogenous CDK1/CDC28 gene is replaced by BGLF4, the EBV member of the CHPK family, may be constructed, first to rigorously determine if BGLF4 is a bona fide CDK-like kinase and, second, if so, to identify candidate drugs able to inhibit this activity without any significant effect on a similar yeast strain in which the endogenous CDK1/CDC28 gene is replaced by the human CDK1. In this way specific inhibitors of BGLF4 might be identified.

Finally, one interesting aspect of the yeast models for EBV is that yeast is a "naive" eukaryote regarding EBV, considering that, contrary to human EBV host cells, no co-evolution between EBV and yeast has occurred. This obvious remark has an important consequence: even if some yeast factors could functionally substitute their human counterparts, they were for sure not evolutionary optimized, which means that their overexpression should The GAr domain of EBNA1 which is responsible for its self-limiting expression (thereby allowing the immune evasion of EBV) was fused to the yeast ADE2 reporter gene which encodes Ade2p, a very stable protein, like the EBNA1 protein. Full expression of Ade2p in yeast leads to the formation of white colonies, whereas, due to the inhibition of its translation in cis by the GAr domain, the GAr-ADE2 construct leads to the formation of pink colonies. This effect, which is GAr length-dependent as in human cells, demonstrates that the ability of the GAr domain to inhibit the translation of its own mRNA is also operant in yeast. This assay based on the pink phenotype of the GAr-Ade2p expressing yeast cells was used to perform drug screening aiming at isolating compounds able to interfere with the GAr effect on translation (A). Drugs that suppress the inhibitory effect of GAr on translation lead to the formation of whiter colonies that are easily monitored on Petri plates. The effect of these drugs on the GAr-dependent inhibition of the protein expression can then be evaluated both in yeast (B) and in human cells (C). Finally, their effect on the GAr-mediated restriction of the antigen presentation can also be evaluated using a T-cell assay (D). At each step it is important to check if the effects observed are GAr-dependent.

in principle enhance the efficiency of the considered EBVrelated process, which makes them easier to detect in global approaches than using approaches based on their inactivation which are more tedious, even in yeast [61].

In conclusion, many new exciting yeast-based adventures to decipher EBV biology are still to come, thus promising some rejuvenation of the long-lasting love story between the budding yeast and the Epstein-Barr virus. In addition the success of this love story may inspire similar yeast-based stories to study other gammaherpesviruses also involved in human diseases, in particular some cancers, like the Kaposi and papilloma viruses. Hence, despite the fact that it has been used for decades and that one may have initially considered it as an unlikely match, the yeast-EBV couple is still as glamorous as at its beginnings, and will certainly keep playing a key role in deciphering some of the EBV mysteries still to be discovered. to inhibit its own mRNA translation in cis, a process led by its central GAr domain, and in that way reduces the production of EBNA1-derived antigenic peptides and the further recognition of infected cells by EBNA1 specific T cells [START_REF] Yin | Self-inhibition of synthesis and antigen presentation by Epstein-Barr virus-encoded EBNA1[END_REF] . When I started this PhD, the mechanism involved in this process was undeciphered, and two hypotheses that are not mutually exclusive were ventured:

• The team of R. Fåhraeus showed evidence supporting a model by which the GAr nascent polypeptide would block the translation of the upcoming mRNA. They demonstrated in vitro that the polysomal profile of the EBNA1 mRNA mimics the one of EBNA1ΔGAr in which they have introduced a hairpin secondary mRNA structure in the 5´-UTR 5 , suggesting that GAr may block the translation initiation process. In line with this observation, the insertion of the c-MYC IRES into GAr-OVA mRNA completely abolishes the GAr inhibition of translation effect, suggesting that this mechanism depends of the CAP-dependent translation initiation.

• The team of J. Tellam showed that the GAr mRNA formes G4 structures that seem to be responsible for the ribosomes stalling and thus block the translation elongation process [START_REF] Murat | G-quadruplexes regulate Epstein-Barr virus-encoded nuclear antigen 1 mRNA translation[END_REF] . Furthermore, frameshifts of EBNA1 internal repeat altering protein sequence but not mRNA sequence were still able to inhibit their own mRNA translation and antigen presentation [START_REF] Tellam | Messenger RNA sequence rather than protein sequence determines the level of self-synthesis and antigen presentation of the EBV-encoded antigen, EBNA1[END_REF] . In addition, the treatment with small G4 ligands (for example PDS) that stabilize G4s was shown to decrease EBNA1 translation in an in vitro transcription-translation coupled system. Hence, this hypothesis is only based on in vitro experiments.

When I started my PhD, these two hypotheses had in common the fact that the cellular factors involved in this GAr-dependent regulation of translation were unknown.

In order to identify such cellular factors no matter the mechanism involved, we performed a genetic screening using the yeast-based assay that recapitulates GAr translation inhibition (Figure 13). This approach allowed us to identify the first cellular factor that is involved in GAr-based translation inhibition and immune evasion: Nucleolin (NCL).

Identification of NCL, the first cellular factor able to affect the GAr-based EBNA1 translation inhibition

We proved that NCL overexpression in EBV-infected cells enhances EBNA1-dependent regulation of translation and inversely that its downregulation by siRNA increases EBNA1 translation (Figure 2, chapter 1 of the results section). An increase in EBNA1 translation may constitute a therapeutic solution against EBV-associated cancers, since it also increased the antigenic peptides presented at the surface of APC and also specific T CD8 + proliferation (Figure 3, chapter 1 of the results section).

This means that virus-infected cells would be unveiled to T cells directed towards EBNA1 which exist in all EBV-infected individuals.

The relationship between NCL and EBV was showed for first time by the group of YL. Chen that proved that the RNA binding domains (RBDs) of NCL are able to interact with the N-terminal domain of the EBNA1 protein and that this interaction enhances EBNA1-mediated OriP transcription. These studies where made in EBV negative cells which were transiently transfected with flag-tagged EBNA1. Coimmunoprecipitation assays showed the association between NCL and EBNA1, and the authors concluded that NCL/EBNA1 association is not dependent on the presence of DNA or RNA. The authors showed that NCL overexpression in these cells significantly increased the ability of EBNA1 to activate OriP transcription. They also claimed that NCL overexpression does not affect EBNA1 protein levels 145 . However, the published results do not fit with their conclusions as their data clearly show that NCL overexpression decreases EBNA1 levels by 50%. Two years later the same authors demonstrated that another cellular factor, the ribosomal protein L4 (RPL4), is also implicated in EBNA1 OriP transcription activation 146 . Altogether our data and Chen's group data show that NCL is highly linked to at least two of EBNA1 functions: transcription enhancement and immune evasion. The identification of NCL as a GAr mRNA binding protein represents another argument in favor of the G4 hypothesis, but does not exclude a possible involvement of the GAr polypeptide.

We demonstrated that NCL-GAr RNA interaction takes place in the nucleus (Figure 4, chapter 1 of the results section). Nevertheless, we still don't know how this interaction occurs and what the exact mechanism is. Three different scenarios are possible: → first, the interaction between NCL and GAr mRNA may trap the GAr mRNA in the nucleus, thereby preventing its translation by the cytoplasmic ribosomes.

→ second, GAr G4 motifs may act as a recognition signal for NCL which interaction with G4 is able to directly affect translation initiation and/or elongation. → third, NCL may act as a stabilizer of the G4s motif which are themselves able to inhibit translation.

These hypotheses are equally possible and not mutually exclusive. No matter the mechanism, it is unlikely that the virus has developed a new bright mechanism involving NCL in order to control its own EBNA1 RNA translation. More probably, the virus is using a mechanism that already exists in the cells. Furthermore, the fact that GAr-based translation inhibition works in yeast reinforces the hypothesis of an evolutionary conserved mechanism.

How does NCL impairs the GAr-based EBNA1 translation inhibition?

To get deeper into GAr RNA-NCL interaction, and since this interaction takes place in the nucleus, we wondered what may happen if we mislocalize GAr RNA from the nucleus to the cytoplasm. Using proximity ligation assay (PLA) and RNA co-immunoprecipitation we demonstrated that the mislocalization of GAr RNA to the cytoplasm abolishes its interaction with NCL (Figure 2, chapter 2 of the results section).

Furthermore, the cytoplasmic localization of GAr RNA totally abolishes the GAr effect on the regulation of translation (Figure 3, chapter 2 of the results section). In line with this observation, we remarked that RNAs that are naturally targeted to the cytoplasm are not subject to GAr translation inhibition. However, the retention of these pre-RNA in the nucleus allows their interaction with NCL in the nucleus (Figure 4H, chapter 2 of the results section), as well as the decrease of the translation of these RNAs and the reduction of antigenic presentation.

These results indicate that NCL interaction is essential in order to ensure GAr-based translation inhibition, whereas it seems that the nuclear localization of GAr RNA is due to the fact that NCL is mostly present in the nucleolus and the nucleoplasm. In order to reinforce our hypothesis, we constructed a NCL mutant which nuclear localization signal (NLS) is mutated (NCL mutNLS), and that is consequently unable to return to the nucleus once synthesized (Figure 5A, chapter 2 of the results section). We demonstrated that the cytoplasmic NCL mutNLS is able to interact with GAr RNAs that have been relocalized in the cytoplasm (Figure 5C, chapter 2 of the results section), and that this interaction affects the GAr-based translation inhibition (Figure 5D-E, chapter 2 of the results section). We showed that NCL binding to GAr RNA is sufficient to ensure GAr-based inhibition of translation. Taken together the results obtained in the second part of this thesis showed that NCL interaction with GAr RNA is essential to ensure GAr-based translation inhibition.

The results presented in this second chapter showed for the first time that EBV escapes from the immune system recognition during the latent phase by taking advantage of a common cellular mRNA motif (G4) that recruits NCL thereby controlling the levels of EBNA1 expression. Nevertheless, we have not determined yet if GAr RNA retention in the nucleus by NCL is sufficient enough to inhibit translation or if its own inhibitory effect through G4 binding is also necessary. Moreover, we still don't know how NCL inhibits translation: does NCL binding to GAr RNA block the binding of the ribosomal subunit 40S, the ribosomal assembly of the 60S or the ribosome progression? Another possibility is that the GAr polypeptide could also be involved in this interaction and thus the complete mechanism of translation inhibition would implicate the triple interaction between GAr mRNA, GAr polypeptide and NCL.

To decipher if ribosome assembly takes place when NCL interacts with GAr RNA, we determined if cytoplasmic GAr RNAs are able to interact with the ribosomal protein L11 when a cytoplasmic version of NCL is expressed. Our preliminary results show that cytoplasmic GAr RNAs are able to interact with the ribosomal protein L11 that is associated to the large subunit of the ribosome. These data suggest that the ribosome is probably assembled on GAr mRNA but that translation initiation and/or elongation is blocked by NCL binding (Figure 14). Some more experiments will help confirming these results.

Another intriguing question that raised on concerns the timing of the interaction between NCL and GAr G4s. A regulation is most probably controlling this mechanism in order to permanently ensure a minimal level of EBNA1 which is essential for the episome replication and maintenance.

In normal conditions most of the available NCL is localized in the nucleolus where it accomplishes crucial functions among which ribosome biogenesis. During cell division, the nucleolus is disrupted and re-generated after mitosis, which frees all NCL that was trapped in the nucleolus. At this stage of cell division, EBNA1 needs to be produced to ensure the transmission of the viral genome to daughter cells by tethering the viral episome to the mitotic host chromosomes. However, the uncontrolled EBNA1 high rate of expression would lead to the increase of EBNA1-derived antigenic peptides and thus the recognition of infected cells by specific T CD8 + . For this reason, NCL-GAr RNA interaction should likely take place when EBNA1 translation rate is higher in order to avoid the recognition and elimination of EBV-infected cells. It would be interesting to challenge this hypothesis by performing a PLA assay between NCL and GAr RNA at different steps of the cell cycle of synchronized cells.

Since NCL is subjected to several post-translational modifications which may control its localization in the cell, another possibility is that a particular population of NCL (eg, phosphorylated NCL) which is localized in the nucleoplasm may be interacting with GAr G4 motifs. It would be interesting to try to identify this population of NCL by performing a G4 pull-down experiment followed by the detection of NCL with antibodies that can reveal NCL phosphorylation's or acetylation's. Proximity ligation assay carried out between GGG RNA and L11 combined with NCL∆NLS immunostaining showed that GAr-carrying RNAs present in the cytoplasm are able to interact with the ribosomal protein L11, and that this interaction co-localized with the location of cytoplasmic NCL∆NLS.

To conclude, we described a novel function of NCL in EBV latency during which the interaction of NCL with EBNA1 GAr mRNA is directly implicated in translation inhibition and hence responsible for the poor recognition of infected cells by T cells directed against EBNA1. These data suggest that targeting GAr mRNA-NCL interaction constitutes a novel therapeutic target to treat EBV-associated cancers.

In the light of our data showing the nuclear localization of EBNA1 mRNA and the cytoplasmic export driven by c-MYC IRES, another possible interpretation would be that c-MYC IRES-carrying mRNAs have no GAr-based translation inhibition because they are exported to the cytoplasm (see introduction section 2.3.1).

Targeting GAr mRNA-NCL interaction

In order to disrupt GAr mRNA-NCL interaction, both interactors can be targeted. Since NCL is essential and implicated in several cellular pathways, targeting NCL is probably not the best option. We observed that NCL overexpression is often toxic for cells, and inversely its knockdown is only about 40-50%, meaning that NCL cell levels should stay mostly stable for cell viability. On the other hand, targeting G4s motifs should be easier, but since these motifs are present all over the human genome there is a selectivity issue, as modifying G4 stability could also affect several steps in gene regulation.

-Targeting NCL:

Due to its numerous cellular functions, NCL has been associated to cancer development and progression and also to neurodegenerative disorders. During tumorigenesis NCL is involved in several steps: first, its overexpression causes an increase in rRNA synthesis and ribosome biogenesis in the nucleolus. Second, NCL is able to enhance oncogene expression which leads to anti-apoptotic and cell survival signals.

However, no NCL mutations or splicing variants have been identified yet. Nevertheless, NCL localization (Figure 15) is often modified and associated with several human pathologies as cancer.

It has been shown that in many types of human cancers (like gastric cancers, melanomas, glioblastomas), NCL is overexpressed and mislocalized to the nucleoplasm, cytoplasm or the cell surface 147 . For example, in gastric cancer, NCL is highly overexpressed and the accumulation of cytoplasmic NCL is associated with a poor prognosis for patients. When situated at the cell surface, NCL is able to interact with several cellular proteins that help with the tumorigenesis process. In B lymphocytes, NCL interacts with Fas and blocks the interaction with FasL, causing a decrease in apoptosis. Because of this, targeting NCL has been considered as a novel therapeutic target against cancer over the past few years. Nevertheless, NCL targeting is not an easy task. The G-rich aptamer AS1411 which is currently in phases I and II of clinical trials, is promising for therapy as it is able to target different The red dots represents NCL at its different locations. In the nucleolus NCL association with chromatin (rDNA) could regulate Pol I transcription. Also, NCL binding to pre-rRNA affects its maturation and its assembly with ribosomal proteins. Furthermore, the shuttling of NCL between the nucleus and the cytoplasm might participate in the transport of several ribosomal proteins. In the nucleoplasm, NCL has been associated to various mRNAs affecting their translation or stability. Finally, NCL has also been identified at the cell surface where it can affect cellular migration and adhesion, as well as viral infection.

NCL signaling pathways. The overexpression of the miRNAs miR-21, miR-103, miR-221 and miR-222 is often associated with aggressiveness in breast cancer. It has been shown that NCL is able to interact with the microprocessor complex and enhance the production and maturation of these miRs. The treatment with AS1411 inhibits the interaction of NCL with the microprocessor complex which indeed reduces the production of these miRs thus leading to the upregulation of genes involved in apoptosis 149 . AS1411 also blocks the interaction of cytoplasmic NCL with many other mRNAs like the antiapoptotic Bcl2 mRNA thus inducing apoptosis in cancer cells. Most interesting, AS1411 is able to specifically bind and trap cell surface NCL avoiding its interaction with key proteins. This is the case in HIV-1 infection where AS1411 treatment dramatically decreases HIV-1 attachment to the cell, which makes this G-rich aptamer a potential drug against HIV-1 infection 133 .

We performed a preliminary experiment treating 235GAr-OVA and OVA expressing cells with 10 µM of AS1411 in order to determine if it has any effect on 235GAr-OVA expression (data no shown). As expected, since GAr mRNA-NCL interaction occurs in the nucleus, AS1411 has no effect on trapping the NCL population present in the nucleus and thus AS1411 treatment has no effect on GAr-based translation inhibition.

-Targeting G4 structures:

In order to target G4 structures, two different ways have been ventured: first, the use of antisense oligonucleotides that may affect the stability of the structure; second, the use of small G4 ligands that would stabilize or destabilize the motif. Antisense oligonucleotides (ASO) targeting G4s have been successfully used by the group of J.P Perreault. For example, the histone variant H2AFY (which is often overexpressed in cancer) contains a G-rich tract in its 5´-UTR which should be able to form G4. However, the G4 motif cannot be formed because the G-rich tracts are already engaged in a Watson-Crick-based stable structure (Figure 16).

The group of JP. Perreault showed using cell-based assays that an ASO that is complementary to the G-tracts upstream region can promote the release of the G-tracts and thus allow their folding into a G4 motif 150 which will then inhibit the translation of H2AFY. Furthermore, the group of P. Murat also used an ASO to target the GAr domain G4s. They showed that it abolishes G4 formation and thus causes an increase in EBNA1 translation in vitro [START_REF] Murat | G-quadruplexes regulate Epstein-Barr virus-encoded nuclear antigen 1 mRNA translation[END_REF] . It will be interesting to test the ability of this ASO to disrupt NCL binding to EBNA1 RNA.

On the other hand, we also tested several small G4 ligands predicted to stabilize these structures and which should decrease EBNA1 translation. First, we tested pyridostatin (PDS) which has already been tested by the group of J. Tellam in vitro. They showed that PDS treatment specifically decreases EBNA1 translation by stabilizing GAr G4s motif [START_REF] Murat | G-quadruplexes regulate Epstein-Barr virus-encoded nuclear antigen 1 mRNA translation[END_REF] . Unfortunately, in cellulo we didn't see any effect showed that PhenDC3 competes with NCL for binding to GAr domain G4s (Figure 5C, chapter 1 of the results section). Furthermore, PhenDC3 treatment also increases T CD8 + proliferation (Figure 6, chapter 1 of the results section). These results constitute the proof of concept that NCL-GAr mRNA interaction is druggable and thus represents novel therapeutic target to treat EBV-associated cancers. In addition, we tested other G4 ligands (data no shown) and observed that some of them have a stabilizing effect (thus enhancing GAr-dependent translation inhibition effect), whereas some others display a similar destabilization effect as PhenDC3, and some others have no effect at all on GAr translation inhibition, showing that these ligands can have different effects. However, G4 motifs are present all over the human genome, meaning that the treatment with G4 ligands can potentially affect other cellular pathways and also the binding of NCL to key DNA or RNA G4s, as for example to the DNA G4s present in the oncogene c-Myc which can have an effect on cancerogenesis. Hence, it could be interesting to develop transcriptomic analysis in order to determine the effect of PhenDC3 on the transcription of cellular genes and also of EBV genes. In addition, it could be interesting to design a new G4 molecule able to specifically affect GAr mRNA G4-NCL interaction without affecting NCL binding to other targets.

In conclusion, in order to prevent GAr mRNA-NCL interaction, the better option seems to target the formation of the G4s present inside the GAr-encoding mRNA, either by the use of ASO or small G4 ligands. Of note, the use of ASO seems to be more specific for the different G4s motif and thus would avoid the recognition of other G4s present inside the human genome.

NCL implication in other viral infections

Several human viruses like HIV-1 or HPV are also able to specifically interact with NCL, this interaction being involved in viral entry and infection. This is the case in cervical cancer where NCL overexpression enhances the expression of the oncogenic proteins E6 and E7 from HPV 151 which are involved in tumor growth and malignancy, and inhibition of the tumor suppressor genes pRb and p53. In addition, NCL interacts and stabilizes the G4 motifs present in the LTR promotor of HIV-1 and inhibits the transcription of the integrated provirus 131 . Furthermore, cell surface NCL acts as a receptor of HIV-1 and favors cell attachment and entry 134 . Moreover, HIV-1 infected patients present a hyperlocalization of NCL at the surface of infected cells 134 .

These results suggest a common mechanism between human viruses which take advantage of a ubiquitous and essential cellular protein and the formation of a particular mRNA structure in order to favor viral entry, replication, transcription or translation of viral proteins. In the case of EBV, the GAr domain G4s would act as a recognition signal for NCL binding in order to ensure a minimal synthesis level of EBNA1 protein to assure EBV episome replication and maintenance, but low enough to avoid recognition of EBV-infected cells by specific T cells.

Is NCL interaction with GMPs mRNAs a common mechanism of oncogenic herpesviruses to avoid immune system elimination?

As described above, NCL was reported to be capable of interfering with viral infections. Due to its numerous functions in various cellular pathways, viruses have taken advantage of NCL binding in order to favor virus cell cycle and infection. In particular, the two human γ-herpesviruses EBV and To conclude, during this thesis we characterized a novel function for the cellular protein NCL on EBV EBNA1-based mechanism of immune evasion which involves the binding of NCL to the GAr domain G4 motifs. We showed for the first time that NCL, besides its already described functions as a 5´-and 3´-UTR RNA-binding protein altering mRNA translation and stability, is also able to alter the translation of some mRNAs by direct binding to their coding regions. Furthermore, we demonstrated that this interaction takes place in the nucleus, due to the normal location of NCL in the cells, and that small G4 ligands are able to disrupt NCL binding from EBNA1 mRNA which enhances EBNA1 expression and antigen presentation, making EBV-infected cells susceptible to T cell recognition and elimination.

As it was mentioned at the beginning of this discussion, at the moment I started my PhD two models were proposed to explain the mechanism involved in GAr-based translation inhibition: the GAr polypeptide and the GAr mRNA structure. Our results proved the influence of GAr mRNA G4s as a recognition signal for NCL binding, but we cannot excluded a possible additional influence of GAr polypeptide in the final mechanism.

In the course of this work, we identified the first cellular factor involved in the GAr-based inhibition of translation effect and a G4 ligand, PhenDC3, which is capable of disrupting the binding of NCL to EBNA1 mRNA, thereby leading to an increase in EBNA1 expression and antigen presentation. Taken together, these results indicate that NCL-GAr mRNA interaction constitutes a new therapeutic target to treat EBV-associated cancers.

Figure 1 . 22 Figure 2 - 24 Figure 3 - 27 Figure 4 - 35 Figure 5 - 36 Figure 6 - 39 Figure 7 - 43 Figure 8 - 45 Figure 9 - 47 Figure 10 - 52 Figure 11 - 56 Figure 12 - 58 Figure 13 - 60 Figure 14 -Figure 15 -Figure 16 -Figure 17 -

 122224327435536639743845947105211561258136014151617 Figure 1. Diagram showing an EBV virion and the localization of EBV latent genes on the viral DNA episome. ............................................................................................................................................. 22 Figure 2 -Scheme explaining the mechanism by which EBV is able to successfully infect and persist lifelong in host cells. (Figure extracted from 26 ) ................................................................................. 24 Figure 3-Epstein-Barr virus life cycle. Figure extracted from 42 . ........................................................ 27 Figure 4-EBNA1 protein organization. ............................................................................................... 35 Figure 5-The GMP of EBV, EBNA1, assures the replication and maintenance of the viral genome on every S phase of the cell cycle. Figure extracted from 2 . ..................................................................... 36 Figure 6-Defective ribosomal products (DRiPs) model. Figure extracted from 80 . ............................. 39 Figure 7-EBV´s EBNA1-based mechanism of MHC class I immune evasion. ...................................... 43 Figure 8-Proposed model for GAr-based inhibition of translation. Figure extracted from 5 . ............. 45 Figure 9-Schematic representation of G-quadruplex structures. Figure extracted from 102 . ............. 47 Figure 10-Human and yeast nucleolin protein organization. ............................................................ 52 Figure 11-Model representing the mechanism of action by which the G4 motifs present inside the GAr-encoding mRNA of EBNA1 allow immune evasion of the EBV-infected cells in latent phase. Figure extracted from 99 . ............................................................................................................................... 56 Figure 12-The yeast reporter system ADE2. ...................................................................................... 58 Figure 13-Yeast-based assay for EBNA1-based immune evasion. Figure adapted from 2, 3 . ............. 60 Figure 14-Nucleolin binding to GAr-carrying RNAs doesn't affect ribosomal assembly. ................. 147 Figure 15-The different cellular locations of NCL. Figure extracted from 1 . .................................... 149 Figure 16-Promoting G4 folding of the histone variant H2AFY. Figure extracted from 150 . ............. 151 Figure 17-Predicted G4-forming sequences of herpesvirus GMPs repetitive domain. .................... 154

I 1 T

 1 IF2α: translation initiation factor 2 alpha IFN-γ: interferon gamma Ig: immunoglobulin IκB: inhibitor of NF-κB IM: infectious mononucleosis IRES: internal ribosomal entry site K KS: Kaposi sarcoma KSHV: Kaposi sarcoma-associated herpesvirus L LCL: lymphoblastoid cell line LMP: latent membrane protein LP: lymphocyte predominant LTR: long terminal repeat M MHC: major histocompatibility complex miRNA: microRNA MIIC: MHC class II loading compartment ML: multicentric Castleman´s disease mRNA: messenger RNA N NCL: nucleolin NHE: nuclease hypersensitive element NLPHL: nodular lymphocyte predominant HL NLS: nuclear localization signal NPC: nasopharyngeal carcinoma NSR1: nucleolin yeast homologue O ORC: origin recognition complex OriP: origin of plasmid replication OVA: ovalbumin P PEL: primary effusion lymphoma PRK: phosphoribulokinase R RBD: RNA-binding domain rDNA: ribosomal DNA RPA: replication protein A rRNA: ribosomal RNA S SHL8: SIINFKEL antigenic peptide derived from ovalbumin STAT1: signal transducer and activator of transcription TAP: transporter associated with antigen processing TCR: T-cell receptor TRAIL: TNF-related apoptosis inducing ligand TSS: transcription start site TPA: 12-O-tetradecanoylphorbol-13-acetate U UTR: untranslated region V VEGF: vascular endothelial growth factor VZV: varicella-zoster virus Z ZRE: ZEBRA response element

Figure 1 .

 1 Figure 1. Diagram showing an EBV virion and the localization of EBV latent genes on the viral DNA episome. (a) Electron microscopy of the Epstein-Barr virus (EBV) virion, (b) EBV viral episome, (c) representation of EBV latent proteins open reading frames. The origin of replication (oriP) is shown in orange, the ORFs encoding each of the latent proteins are represented by violet arrows and the direction represents the transcription sense. Scheme extracted from 22 .

Figure 2 -

 2 Figure 2 -Scheme explaining the mechanism by which EBV is able to successfully infect and persist lifelong in host cells. (Figure extracted from 26 ) EBV first makes contact and infects epithelial cells where it initiates a lytic program causing an amplification of the viral paticles. Then, the virus infects naive B cells present in lymphoid tissues. These activated B cells undergo the latency III program. In order to escape from this program the family of EBNA3 latent genes negatively regulates this program, allowing the infected B cells to migrate and start a germinal center (GC) reaction where it establishes the latency II program. This latency program allows the cell to exit from the GC center and thus to become a resting memory B cell in the periphery. This program is called latency 0 and is characterized by the switch off of all latent genes. When this cell enters in S phase it expresses the EBNA1only program (latency I) in order to maintain the viral episome.

Figure 3 -

 3 Figure 3-Epstein-Barr virus life cycle. Figure extracted from 42 .

  (BamHI A rightward transcripts) and BHRF1 (BamHI H rightward ORF1). EBV´s miRNAs are encoded from two different regions of EBV genome: BARTs from the BamHI A region rightward transcript and BHRF from the 5´and 3´-UTRs of BamHI H rightward ORF1. BART region encodes 40 different miRNAs whereas BHRF region encodes 4 other miRNAs.

Figure 4 -

 4 Figure 4-EBNA1 protein organization.

Figure 5 -

 5 Figure 5-The GMP of EBV, EBNA1, assures the replication and maintenance of the viral genome on every S phase of the cell cycle. Figure extracted from 2 .

Figure 6 -

 6 Figure 6-Defective ribosomal products (DRiPs) model. Figure extracted from 80 .

Figure 7 -

 7 Figure 7-EBV´s EBNA1-based mechanism of MHC class I immune evasion.

Figure 8 -

 8 Figure 8-Proposed model for GAr-based inhibition of translation. Figure extracted from 5 .

Figure 9 -

 9 Figure 9-Schematic representation of G-quadruplex structures. Figure extracted from 102 . A) G-quartet arrangement, B) G-quadruplex secondary structure and C) the intermolecular hydrogen bonding between RNA G-quartet forming residues.

  the team of L.H Hurley showed by footprinting experiments that the NHE-III element forms G4 structures which are involved in c-MYC transcription inhibition. The disruption of one nucleotide critically involved in G4 formation increased c-MYC transcription and inversely, TMPyP4 (a known G4 ligand) treatment in lymphoma cell lines showed antitumor activity due to G4 stabilization and thus transcription repression of c-MYC 106 .b) G4 in RNADuring the past few years several studies have shown the importance of G4 inside RNAs. Due to the fact that RNA does not contain a complementary strand, G4 formed inside RNA are usually more stable than their DNA counterparts 107 .

Figure 10 -

 10 Figure 10-Human and yeast nucleolin protein organization.In green is represented the acidic N-terminal domain, in violet all RNA-binding domain (RBD) and in orange the C-terminal RGG domain.

Finally

  , regarding EBV, G4 forming sequences have been identified in the EBNA1-encoding mRNA. P.Murat et al demonstrated that the mRNA sequence of EBNA1 encoding the central GAr domain may form several G4. In vitro studies have shown that these G4 present inside the GAr domain coding sequence of EBNA1 are able to inhibit translation, thereby leading to immune evasion during the latent phase[START_REF] Murat | G-quadruplexes regulate Epstein-Barr virus-encoded nuclear antigen 1 mRNA translation[END_REF] . P. Murat et al proposed that EBNA1 G4 may cause ribosome stalling and therefore inhibit translation. The treatment with pyridostatin (PDS), a G4 ligand known to stabilize G4, causes a decrease in EBNA1 inhibition of translation whereas the treatment with antisense oligonucleotides that destabilize G4 structure enhances EBNA1 translation and promotes T CD8 + recognition (Figure11).These experiments showed that targeting the GAr domain with G4 ligands may interfere with EBNA1 translation inhibition and would thus modify the rate by which EBNA1-derived antigenic peptides are presented by MHC class I.As of today, both GAr polypeptide-based and G4-based hypotheses are still topical and the cellular factors involved in GAr-based immune evasion are still unknown.

Figure 11 -

 11 Figure 11-Model representing the mechanism of action by which the G4 motifs present inside the GArencoding mRNA of EBNA1 allow immune evasion of the EBV-infected cells in latent phase. Figure extracted from 99 .

Figure 12 -

 12 Figure 12-The yeast reporter system ADE2.

Figure 13 -

 13 Figure 13-Yeast-based assay for EBNA1-based immune evasion. Figure adapted from 2, 3 .

ABSTRACT The oncogenic Epstein -

 Epstein Barr virus (EBV) evades the immune system but has an Achilles heel: its genome maintenance protein EBNA1. Indeed, EBNA1 is essential for viral genome maintenance but is highly antigenic. Hence, EBV evolved a system in which the glycine-alanine repeat (GAr) of EBNA1 self-limits the translation of its own mRNA at a minimal level to ensure its essential function thereby, at the same time, minimizing immune recognition. Here, we show that nucleolin (NCL) is required for this process via a direct interaction in the nucleus with G-quadruplexes formed in GAr-encoding mRNA sequence. Overexpression of NCL exacerbates GAr-based translation inhibition whereas its downregulation relieves the suppression of both translation and antigen presentation. Moreover, the G-quadruplex ligand PhenDC3 prevents NCL binding on EBNA1 mRNA and reverses GAr-mediated repression of translation and antigen presentation. Hence the NCL-EBNA1 mRNA interaction is a relevant therapeutic target to trigger an immune response against EBV-carrying cancers.

  naive CD8 + T cells (OT1 cells) recognizing specifically the OVA 257-264 SIINFEKL epitope on the murine k b MHC class I molecule. The OT1 cells were isolated from peripheral and mesenteric lymph-nodes of 12-week-old mice and stained with the CellTrace TM Violet fluorescent dye. Then, OT1 cells were mixed with H1299 cells expressing 235GAr-OVA and the k b molecule. As a control, H1299 cells expressing OVA and the k b molecule were used. As expected and due to the GAr inhibitory effect on both translation and antigen presentation, 235GAr-OVA-expressing H1299 cells (Fig. 3c, left panel) led to a much weaker activation of OT1 cells as compared to OVA-expressing H1299 cells (Fig. 3d, left panel), as determined by evaluating the number of dividing OT1 cells by FACS analysis. However, siRNA-mediated NCL knockdown in 235GAr-OVAexpressing H1299 cells significantly increased proliferation of OT1 cells (Fig. 3c, right panels)

  PCR were ADE2-forward: 5 -ATTGTGCAAATGCCTAGAGGTG-3 , ADE2-reverse: 5 -AATCATAA -GCGCCAAGCAGTC-3 ; Actin-forward: 5 -ATGGTNGGNATGGGNCARAAR-3 , Actin-reverse: 5 -CTCCATRTCRTCCCAGTTGGT-3 ; EBNA1-forward: 5'-GGCAGTGGACCTCAAAGAAGAG-3'; EBNA1reverse: 5'-CAATGCAACTTGGACGTTTTT-3'; OVA-forward: 5'-GAGGAGGCTTGGAACCTAT-3'; OVAreverse: 5'-CAGTTTGAGAATCCACGGAG-3'. All the experiments were performed in triplicates and were repeated at least three times.NCL siRNA downregulation1, 75 x 10 5 H1299 cells were transfected with 0.75 µg of EBNA1, EBNA1 GAr, 235GAr-OVA or OVA vectors using standard procedures and incubated at 37°C for 8 hours. Cells were then transfected either with 40 nM of control siRNA or FlexiTube GeneSolution for NCL (Qiagen). siRNA transfection were performed using HiPerFect transfection reagent (Qiagen) following the manufacturer's protocol. 40 hours after siRNA transfection, cells were collected for western blot or flow cytometry analyses.Flow cytometry analysis48 hours after the transfection, cells were collected using trypsin and washed twice with 1X PBS. Cells were suspended in 50 µL of 1X PBS and incubated with 0.4 µg of anti-Mouse OVA257-264 (SIINFEKL) peptide antibody bound to H-2Kb PE or Anti-Mouse MHC Class I (H-2Kb) antibody bound to PE (Ebioscience) for 30 min at room temperature. Cells were then washed with 1X PBS and analyzed by FACS on a CANTO II flow cytometer (BD Biosciences, USA).

Figure 1 :

 1 Figure 1: Identification and confirmation of the critical role of nucleolin in GAr-based translation inhibition in yeast. (a) Rationale of the yeast-based genetic screen. Contrary to yeast cells expressing ADE2 gene (left panel) that forms white colonies on rich medium (YPD), those expressing the 43GAr-ADE2 fusion (right panel) form pink colonies due to the ability of GAr to self-limit the translation of its own mRNA in yeast as in human cells. Based on this model, we looked for yeast genes whose overexpression from high copy number plasmids leads to a redder phenotype, suggesting an exacerbation of the GAr-based translation inhibition. (b) Effect of NSR1 overexpression on 43GAr-Ade2p protein level. The overexpression of NSR1 gene, which encodes the yeast nucleolin, led to a redder phenotype associated to a decrease in 43GAr-Ade2p level as evidenced by SDS-PAGE and western blot analysis (left panel). GAPDH was used as a loading control. The mean 43GAr-Ade2p/GAPDH ratio from 3 independent experiments is shown in the right panel. (c) Effect of NSR1 gene deletion on 43GAr-Ade2p and Ade2p protein level. SDS-PAGE and western blot analysis showing that deletion of NSR1 (nsr1 ) had no effect on Ade2p level, whereas it strongly increased the level of 43GAr-Ade2p. GAPDH was used as a loading control. The mean 43GAr-Ade2p/GAPDH or Ade2p/GAPDH ratios from 3 independent experiments are shown in the right panel. (d) Human nucleolin (NCL) is able to complement NSR1 deletion. SDS-PAGE and western blot analysis of the yeast nsr1 strain expressing 43GAr-Ade2p (left) or Ade2p (right) and overexpressing (right lanes), or not (left lanes), HA-tagged human nucleolin (HA-NCL).

Figure 2 :

 2 Figure 2: Overexpression of NCL exacerbates GAr effect on protein expression whereas its downregulation reduces its inhibitory effect on translation. (a) SDS-PAGE and western blot analysis of the level of endogenous EBNA1 in three EBV-infected B cell lines overexpressing (right lanes), or not (left lanes), HA-tagged nucleolin (HA-NCL). Actin was used as a loading control. EBNA1/actin ratios are indicated below the gels. (b) SDS-PAGE and western blot analysis of the level of EBNA1 or EBNA1 GAr in response to NCL knockdown. H1299 cells were transfected with EBNA1 or EBNA1 GAr expressing vectors and with control siRNA or siRNA targeting NCL, as indicated. GAPDH was used as a loading control. EBNA1/GAPDH or EBNA1 GAr/GAPDH ratios are indicated below the gels. The efficiency of NCL downregulation was estimated by determining the level of remaining NCL in cells treated by siRNA targeting NCL versus cells treated by control siRNA. (c) Same experiment as in (b) except that H1299 cells were transfected with chicken ovalbumin (OVA) or 235-GAr-OVA. (d) Autoradiographs showing relative mRNA translation efficiencies of 235GAr-OVA versus OVA in response to NCL knockdown. H1299 cells transfected with 235GAr-OVA (upper panel) or OVA (lower panel) constructs and with control siRNA or siRNA targeting NCL, as indicated, were pulse-labelled with [ 35 S] methionine, and lysates were subjected to immunoprecipitation with antibodies raised against OVA (left lanes) or IgG as a control (right lanes), and subjected to SDS-PAGE and autoradiography. Quantification of 235GAr-OVA or OVA signals are indicated.

Figure 3 :

 3 Figure 3: NCL downregulation activates GAr-limited antigen presentation and recognition by specific T lymphocytes. (a) Effect of NCL knockdown on antigen presentation. H1299 were transfected with 235GAr-OVA and murine MHC class I K b plasmids and with control siRNA or siRNA targeting NCL, as indicated, and the levels of Kb/OVA-derived antigenic peptide complexes determined using

Figure 4 :

 4 Figure 4: NCL directly interacts with G4 formed in the GAr-encoding mRNA and NCL-EBNA1 mRNA interaction takes place in the nucleus. (a) Schematic representation of G4 structure. A guanine tetrad (G-tetrad) formed by the planar arrangement of four guanines held together by Hoogsteen hydrogen bonds (dashed lines) and stabilized by a cation (usually potassium) stacks with other G-tetrads to form G4.(b) RNA pull-down using G4 forming RNA oligonucleotides covalently linked to biotin and streptavidin-coupled sepharose beads. Lysate from H1299 cells was applied to the following matrices: streptavidin-coupled beads either alone (Empty), or together with GQ (containing the most probable G4 of GAr mRNA), GM (same sequence except that G critical for G4 formation were replaced by adenines or uridines) or ARPC2 (containing a G4 present in ARPC2 mRNA and that has been shown to bind NCL) RNA oligonucleotides. The sequence of these oligonucleotides is given in the Methods section. The proteins still bound to the beads after an 800 mM KCl wash were eluted and analyzed by SDS-PAGE and western blot.

Figure 5 :

 5 Figure 5: PhenDC3 prevents GAr inhibition of protein expression and NCL binding to GAr's Gquadruplexes. (a) SDS-PAGE and western blot analysis of the level of 235GAr-OVA or OVA in response to pyridostatin (PDS) treatment. H1299 cells transfected with 235GAr-OVA (left) or OVA (right) plasmids were treated with 5 M PDS (right lanes) or, as a control, with DMSO (left lanes). GAPDH was used as a loading control and 235GAr-OVA/GAPDH and OVA/GAPDH ratios are indicated below the gels. The chemical structure of PDS is depicted on the right (b) SDS-PAGE and western blot analysis of the level of 235GAr-OVA or OVA in response to PhenDC3 treatment. Same experiments as in (a) except that cells were treated with 5 M PhenDC3 which chemical structure is depicted on the right. (c) The effect of 5 M PhenDC3 treatment on 235GAr-OVA or OVA mRNA level was determined by qRT-PCR. (d) PhenDC3 competes for the binding of NCL on GAr G-quadruplexes. Same experiment than in Figure 4 (c) in the presence of 5 M PhenDC3 (second and fourth lanes) or DMSO as a control (first and third lanes).

Figure 6 :

 6 Figure 6: PhenDC3 activates GAr-limited antigen presentation. (a) PhenDC3 increases T cell proliferation. Same experiment as in Figure 3 (c) & (d) except that 235GAr-OVA (upper panels) and OVA (lower panels) expressing H1299 cells were treated with 5 M PhenDC3 or, as control, with DMSO as indicated. Quantification of proliferating T lymphocytes following PhenDC3 treatment as compared to DMSO-treated cells is shown in the graph on the right. (b) SDS-PAGE and western blot analysis of cells used in (a).

RNA staining 1 x 10 4

 4 H1299 cells were plated on 12 mm-diameter coverslips in 24-well plates and transfected with the indicated constructs or empty pCDNA3. 24 h post-transfection, cells were fixed with 4% paraformaldehyde for 20 min, permeabilized with PBS 1X 0.4% -Triton X-100 and 0.05% CHAPS for 10 minutes at room temperature and saturated with PBS 1X -3% BSA for 30 minutes. Samples were incubated overnight at 4 ºC with rabbit polyclonal anti-NCL (ab22758, Abcam) antibody or mouse 4 monoclonal anti-HA antibody conjugated to Alexa Fluor® 488 (6E2, Cell Signaling). Goat anti-rabbit Ig antibody conjugated to Alexa Fluor® 647 (Sigma) was used as secondary antibody when necessary.RNA-FISH assays were performed by using globin and OVA probes obtained from Biosearch Technologies according to manufacturer s protocol. For protein immune-detection coupled to RNA-FISH, samples were incubated with chicken polyclonal anti-HIV1 Rev antibody (ab36623, Abcam) after the FISH probes hybridization step and immunocomplexes were detected under standard conditions using goat anti-chicken IgY conjugated to Alexa Fluor® 568 (Abcam). Samples were examined in an LSM 800 confocal laser microscope (Carl Zeiss MicroImaging GmbH, Jena,Germany).Proximity Ligation Assay (PLA) for RNA/protein interactions H1299 cells were grown, fixed and permeabilized as described above. Samples were hybridized ) conjugated at digoxigenin in the end.Afterwards, samples were saturated with the blocking solution (PBS 1X -3% BSA -0.1% saponine) and incubated for 2 hours with mouse monoclonal anti-digoxigenin (DI-22, Sigma) and rabbit polyclonal anti-NCL (ab22758, Abcam) primary antibodies diluted in blocking solution. The proximity ligation assay (PLA) was carried out using the Duolink PLA in situ kit (Sigma) according the manufacturer s protocol. For co-staining of HIV-Rev protein in PLA samples, chicken polyclonal anti-HIV1 Rev antibody (ab36623, Abcam) and goat anti-chicken IgY antibody conjugated to Alexa Fluor® 568 (Abcam) were added to primary antibodies and Duolink secondary antibodies mixes, respectively. For co-staining of NCLΔNLS, samples were incubated for minutes with mouse monoclonal anti-HA antibody conjugated to Alexa Fluor® 488 (6E2, Cell Signaling) after the PLA amplification step.

  ). Because GAr-based translation inhibition affects the production of antigenic peptides and thus the recognition of infected cells by specific T CD8 + cells, we next measured the effect of GAr RNA localization on T CD8 + cells proliferation. For this purpose, we determined the proliferation rate of naïve T CD8 + cells (OT1 cells) recognizing specifically the OVA257-264 SIINFEKL epitope complexed to MHC class I molecule. The OT1 cells were first isolated from peripheral and mesenteric lymph-nodes of 12-weeks-old mice and stained with the CellTrace TM Violet fluorescent dye. OT1 cells were then mixed with HEK-293 T Kb expressing nuclear or cytoplasmic GAr-globin and GAr-OVA RNAs. As controls, cells expressing globin and OVA RNAs were used. As expected, antigenic peptides derived from GAr nuclear RNAs (GGC and GOC) are poorly detected by T cells (3,87% ± 1,96; p=0,0055** for GGC and 19,80% ± 3,80; p=0,0002*** for GOC) whereas cytoplasmic GAr RNAs (GGG and GOG)-derived antigenic peptides lead to a strong activation of T CD8 + cells (70,17% ± 17,11; p=0,9204 ns for GGG and 83,97% ± 4,73; 7 p=0,1496 ns for GOG) (Figure 3B-C and E-F), similarly as what is observed for GAr free globin controls

Figure 1 -

 1 Figure 1 -The nuclear or cytoplasmic localization of RNA is driven by the presence of the GAr domain.

9 (

 9 D) FISH staining of ovalbumin (OVA) RNA. Fusion of GAr to the OVA cDNA shifts the localization of the RNA from predominantly cytoplasmic (OC) to largely nuclear (GOC). In contrast, GAr has no effect on the localization of the spliced RNA (OG and GOG). Red and blue staining represent RNA and DAPI, respectively. Scale bar = 10 µm. (E) Cells transfected with the indicated constructs were fractionated for analysis of RNA content of the nucleus and cytoplasm by RT-qPCR. The ratio of nuclear to cytoplasmic RNA was calculated showing a relative nuclear accumulation of intronless GAr-OVA RNA (GOC). Histogram bars represent the mean of three independent experiments. *p<0.05, **p<0.01, ns: not significant.

Figure 2 -

 2 Figure 2 -Only nuclear GAr-carrying RNAs are able to interact with nucleolin.

10

 10

Figure 3 -

 3 Figure 3 -GAr suppresses protein synthesis and MHC class I-restricted antigen presentation only from GAr-carrying RNAs present in the nucleus.

Figure 4

 4 Figure4 Changing GAr RNA location from nucleus to cytoplasm prevents its interaction with nucleolin and disrupts the capacity of GAr to suppress RNA translation and antigen presentation.

Figure 5 - 12 (Figures 3 , 4 and 7 Globin

 51237 Figure 5-Interaction of nucleolin with GAr-carrying RNAs is sufficient to suppress RNA translation and antigen presentation.

Figure 1 .

 1 Figure1. Overview of the EBV life cycle. Following a primary infection of naive B-cell by EBV, a latent infection settles down in most cases (A). In this situation the virus has to replicate and maintain its genome (B) thanks to its genome maintenance protein (GMP) EBNA1 which is highly antigenic. EBNA1 is thus the Achilles heel of EBV and the virus evolved a mechanism to tightly control its level, thereby allowing the immune evasion of EBV-infected cells and, at the same time, the replication and maintenance of the viral genome (C). In a few cases, the virus is reactivated (D) which leads to the activation of the lytic cycle by ZEBRA1 and thus to the lysis of the infected B cells, thereby releasing many viral particles that can infect some more naive B-cells. Because of its oncogenic activity, EBV can also, in a few cases, promote tumors (E), in particular some lymphomas like the Burkitt's and Hodgkin's lymphomas. The budding yeast Saccharomyces cerevisiae has been successfully used to study all these aspects of EBV biology.

Figure 2 .

 2 Figure 2. Principles of the yeast-based assays for identification of ARS and mechanisms allowing the maintenance of EBV episome. (A)The yeast assay that allowed the identification of ARS in EBV episome. The EBV episome was first digested and the resulting fragments were cloned into a plasmid that contained the yeast URA3 gene which allows an ura3yeast strain to grow on a minimal medium deprived of uracil (Step 1). This plasmid lacks of a eukaryotic origin of replication and is thereby rapidly lost by dividing yeast cells making them unable to grow on minimal medium not supplemented with uracil. The resulting library of vectors was then used to transform a recipient ura3yeast strain and the transformed cells were spread on a minimal medium not supplemented with uracil (Step 2). The colonies which appeared on this selective medium should, in principle, have been transformed by a vector that contains a fragment of EBV episome that acts like a eukaryotic origin of replication. The plasmids from these colonies were extracted and the activity of the EBV ARS was confirmed by the retransformation of the same yeast strain, thereby allowing the ARS characterization (Step 3). (B) Yeast model for the maintenance of EBV episome. The rationale is similar to the one of the assay presented in (A), except that the designed plasmid contains the TRP1 selection marker as well as its own origin of replication and yeast centromeric sequences (Yeast CEN, left) or the EBV FR sequence (right) which is known to be important for the maintenance of EBV episome, presumably by anchoring it to the host cell chromosomes via EBV-encoded EBNA1 and the human host cell-encoded hEBP2 proteins (Step 1). These plasmids were introduced in a recipient trp1 -yeast strain that expresses, or not, EBNA1 and/or hEBP2 (Step 2). Whereas the yeast vector that contains a yeast CEN sequence stably propagates irrespectively of the presence or absence of EBNA1 and/or hEBP2, the yeast vector that contains the EBV FR sequence is stably maintained only in yeast cells that expressed both EBNA1 and hEBP2 (Step 3). Hence, the crucial role of these two proteins in the EBV genome maintenance was recapitulated in yeast.

Figure 3 .

 3 Figure 3. Flowchart of the role of EBNA1/EBP2 in EBV episome maintenance. Resting memory B-cells (left) are EBNA1 positive but no active transcription nor translation of EBNA1 (which is highly antigenic) can be detected, thereby EBV infected cells evade the immune system. At the same time most of the host cell-encoded hEBP2 protein is sequestered into the nucleolus. Dividing/mitotic memory B-cells (right) express the minimal level of EBNA1 which allows to tether the EBV episome to the host cell chromosomes. This binding also requires the host cell hEBP2 which is released from the nucleolus following its disassembly as cells enter into mitosis, thus allowing the EBNA1-hEBP2 complex to form and link the EBV episome to the host cell chromosomes. EBV has evolved a mechanism to express EBNA1 at a functional but minimal level to allow the maintenance of its episome and, at the same time, to evade the immune system.

Figure 4 .

 4 Figure 4. Principle of the yeast-based assay for EBNA1-based immune evasion of EBV. (A)The GAr domain of EBNA1 which is responsible for its self-limiting expression (thereby allowing the immune evasion of EBV) was fused to the yeast ADE2 reporter gene which encodes Ade2p, a very stable protein, like the EBNA1 protein. Full expression of Ade2p in yeast leads to the formation of white colonies, whereas, due to the inhibition of its translation in cis by the GAr domain, the GAr-ADE2 construct leads to the formation of pink colonies. This effect, which is GAr length-dependent as in human cells, demonstrates that the ability of the GAr domain to inhibit the translation of its own mRNA is also operant in yeast. This assay based on the pink phenotype of the GAr-Ade2p expressing yeast cells was used to perform drug screening aiming at isolating compounds able to interfere with the GAr effect on translation (A). Drugs that suppress the inhibitory effect of GAr on translation lead to the formation of whiter colonies that are easily monitored on Petri plates. The effect of these drugs on the GAr-dependent inhibition of the protein expression can then be evaluated both in yeast (B) and in human cells (C). Finally, their effect on the GAr-mediated restriction of the antigen presentation can also be evaluated using a T-cell assay (D). At each step it is important to check if the effects observed are GAr-dependent.

Marc Blondel ,

 Blondel María José Lista and Cécile Voisset work in the YMCD Inserm UMR1078 team at the University School of Medicine in Brest, France. Their main focus of interest is to develop yeast (Saccharomyces cerevisiae)-based chemobiological models for various human diseases that include prion-based diseases, mental retardation observed in the Down syndrome or cancer linked to infection by the Epstein-Barr virus. Once established, these models are used to isolate modifiers that can either be genes to decipher cellular mechanisms involved in the disease of interest, or drugs that can be developed toward a use in the clinic. Hence, yeast is a starting point which is used both as a tool and a model in these chemobiological approaches. Frédéric Bihel works in the department of medicinal chemistry (CNRS, University UMR7200) at the Faculty of Pharmacy in Strasbourg. His main focus of interest is to develop new chemical entities based on heterocyclic scaffolds targeting various human diseases that include neurodegenerative disorders or cancers. He also develops pharmacological tools (biotin, fluorescent probes, etc) designed to get an insight in the biological mechanisms involved in these pathologies. The work of the team of Robin Fåhraeus and Chrysoula Daskalogianni in Inserm UMR1162 at Hôpital Saint Louis in Paris has been focused on different aspects of basic cancer research and more recently on the physiological aspects of mRNA translation control in the p53 and in the MHC class I restricted pathways. They are interested to see how signaling pathways use mRNAs within the p53 pathway to help differentiate p53 tumor suppressor activity. They are also interested to know the physiological importance during cancer and viral infection of an alternative source of peptides for the MHC class I pathway that is produced during the processing and maturation of the nascent mRNA. Discussion Oncogenic viruses have evolved several strategies which allow them to escape from host immune system recognition and elimination. They have developed various mechanisms that target all the steps of the MHC class I and class II pathways. They can affect MHC class I synthesis and degradation, peptide-loading, peptide transport into the ER, mRNA translation, proteasome degradation, etc. As presented in the introduction, EBV expresses different proteins during the latent phase and the lytic cycle in order to avoid MHC class I and class II responses. During latency, the EBNA1 protein is able

Figure 14 -

 14 Figure 14-Nucleolin binding to GAr-carrying RNAs doesn't affect ribosomal assembly.

  Furthermore, in glioblastoma a glycosylated form of cell surface NCL is associated with the grade of cancer malignancy, indicating that different post-translational modifications of NCL can be involved in different aspects of the oncogenic process 148 .

Figure 15 -

 15 Figure 15-The different cellular locations of NCL. Figure extracted from 1 .

Figure 16 -

 16 Figure 16-Promoting G4 folding of the histone variant H2AFY. Figure extracted from 150 . Graphical representation of the ASO-based strategy used to promote the folding of the G4 motif present inside the 5´-UTR of the histone variant H2AFY. Under normal conditions the structure cannot be formed because the G-tracts are already part of another nucleotide structure.

  of PDS treatment in cell-based systems (Figure5A, chapter 1 of the results section). This can be due to the fact that P. Murat's group only performed these experiments in vitro which conditions are very different in comparison to cell-based assays. Next we tested several other compounds among which PhenDC3. Surprisingly, PhenDC3 treatment enhances 235GAr-OVA and endogenous EBNA1 expression contrary to what was expected (Figure5B, D-E, chapter 1 of the results section). We

Figure 17 -

 17 Figure 17-Predicted G4-forming sequences of herpesvirus GMPs repetitive domain. EBNA1 GAr domain (A), LANA1 QED domain (B), Papiine herpesvirus EBNA1ba GAr-like domain (C) and Macacine herpesvirus 4EBNA1rh (D) GAr-like GAr domain predicted G4s. By using the software QGRS mapper (http://bioinformatics.ramapo.edu/QGRS/index.php) we identified the predicted G4-forming sequences in the 711 bp nucleotidic sequence of the GAr mRNA (according to the B95.8 strain), the 1377 bp nucleotic sequence of the QED mRNA, the 147 bp corresponding to EBNA1ba and the 141 bp domain corresponding to EBNA1rh. All possible G4 motifs were identified.

A 6 .

 6 Concluding remarks

Table 1 -

 1 EBV-associated cancers. Table extracted from 4 . .................................................................

Table 2 -

 2 EBV latency programs. The different viral products expressed during each latency phase and the diseases associated to each programs.Table extracted from 42 . .................................................

Table 3 -

 3 Homologies between γ-herpesviruses GMPs. Table extracted from 93 . ................................

Table 4 -

 4 G-quadruplex structures found in pathogens. Table adapted from 129 . ...............................

	HCMV: human cytomegalovirus
	HHV: human herpesvirus
	HIV: human immunodeficiency virus
	HL: Hodgkin lymphoma
	HLA: human leukocyte antigen
	HLC: classic hodgkin lymphoma
	hnRNP: heterogeneous nuclear ribonucleoprotein
	HPV: human papilloma virus
	HRS: Hodgkin-Reed Sternberg
	HSV: herpes simplex virus

Table 1 -

 1 EBV-associated cancers.

  Table extracted from 4 .

Table 2 -

 2 EBV latency programs. The different viral products expressed during each latency phase and the diseases associated to each programs.

  Table extracted from 42 .

Table 3 -

 3 Homologies between γ-herpesviruses GMPs. Table extracted from 93 .

Table 4 -

 4 G-quadruplex structures found in pathogens. Table adapted from 129 .

		Pathogen	G4 localization	G4 function	Virulence phenotype
	Viruses	HPV	LTR, L2, E1, E4 (Tlučková et al 2013 130 )	DNA transcription regulation	VL
		EBV	EBNA1 (Murat et al 2014 99 )	mRNA translation, antigenic presentation	VL
		HIV-1	LTR (Perrone et al 2013 131 )	DNA/RNA transcription	VL/AD
	Prokaryotic N. meningitidis	pilE (Wörmann et al 2014 132 )	DNA breakpoints	Av
	pathogens N. gonorrhoeae	pilE (Kuryavyi et al 2012 127 )	DNA breakpoints	Av
	Eukaryotic	P. falciparum	Var (Smargiasso et al 2009 128 )	DNA transcription regulation	Av / AD
	pathogens

  cells are derived from metastatic lymph node from lung carcinoma. Raji cells are type III

latency Burkitt's lymphoma. HCT116 cells are derived from colorectal carcinoma. B95.8 cells are derived from cotton-top Tamarin Monkey peripheral blood lymphocyte. Mutu-1 cells are derived from an EBV-positive Burkitt's lymphoma biopsy specimen from a Kenyan patient. NPC-6661 cell line was established from a xenografted NPC in the early 90's 35 and was kindly provided by Prof. Kwok-Wai Lo from the Chinese University of Hong Kong. H1299, Raji, B95.8 and Mutu-1 cells were cultured in RPMI-1640 media supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine. HCT116 cells were cultured in McCoy's 5A Glutamax media supplemented with 10% FBS, and NPC-6661 cells in RPMI-1640 media supplemented with 25 mM HEPES (Gibco) and 2 mM L-glutamine and 10% FBS. All cells were cultured at 37°C with 5% CO 2 . Transient transfections were performed using Genejuice reagent (Merck Bioscience)

  1,* , Cécile Voisset 1,* , Marie-Astrid Contesse 1 , Gaëlle Friocourt 1 , Chrysoula Daskalogianni 2 , Frédéric Bihel 3 , Robin Fåhraeus 2 and Marc Blondel 1 1 Institut National de la Santé et de la Recherche Médicale UMR1078; Université de Bretagne Occidentale, Faculté de Médecine et des Sciences de la Santé; Etablissement Français du Sang (EFS) Bretagne; CHRU Brest, Hôpital Morvan, Laboratoire de Génétique Moléculaire, Brest, France 2 Institut National de la Santé et de la Recherche Médicale UMR1162, Université Paris Diderot, Sorbonne Paris Cité, Institut Universitaire d'Hématologie, Paris, France 3 Laboratoire d'Innovation Thérapeutique, UMR7200, CNRS, Université de Strasbourg, Faculté de Pharmacie, 74, Illkirch, France

Acknowledgments

ACKNOWLEDGEMENTS

We thank all group members for valuable discussion and G. Friocourt, L. Meijer and B. Abiven for critical proofreading of the manuscript. We are indebted to F. Lacroute for the kind gift of the pFL44L-based yeast genomic DNA library and to F. Mahuteau-Betzer for helpful discussions and for having seeded our collaborative network. The work on EBV in the teams of M.B. and R.F. was supported by the following grant agencies: "La Ligue contre le cancer", "La Ligue contre le cancer CSIRGO", CREATE from the Région Bretagne, "Fondation ARC", "Institut National du cancer (INCa)" and IBSAM. M.J.L. is a recipient of a joint PhD grant from "La Ligue contre le cancer Bretagne" and Région Bretagne and of a 6 month-extension from "Fondation ARC".

We thank Béatrice Abiven for her critical reading of the manuscript. The work on EBV in the teams of RF and MB was supported by the following grant agencies: La Ligue contre le Cancer, La Ligue contre le cancer CSIRGO, CREATE from the Région Bretagne and Institut National du Cancer (INCa). A "Programme labellisé 2012" from ARC supported the collaborative work of RF, MB and FB groups.

The authors declare no financial or commercial conflict of interest.

COMPETING FINANCIAL INTERESTS

The authors declare no competing financial interests.

ADDITIONAL INFORMATION

Supplementary information is available in the online version of the paper. Reprints and permissions information is available online. Correspondence and requests for materials should be addressed to M.B.

ONLINE METHODS

SUPPLEMENTARY FIGURE LEGENDS

Chapter 2:

Unraveling NCL involvement in the GAr-based translation inhibition: nuclear trapping, translation inhibition or both?

• Graphical abstract:

• Manuscript presentation:

In the first part of this thesis we identified NCL as the first cellular factor able to interact directly with the GAr-encoding mRNA and affecting its translation inhibition effect. Furthermore, we determined that this interaction takes place in the nucleus and that a well-known G4 ligand (PhenDC3) is able to partially inhibit the binding of NCL to the G-quadruplex (G4) sequences of GAr mRNA and thereby prevent the GAr-based inhibition of translation and antigen presentation. We next wondered how this mechanism occurs. Is NCL trapping EBNA1 mRNA in the nucleus, thus explaining why the mRNA is poorly translated? Or does NCL itself inhibit translation through its binding to G4?

Our first question was to determine if the GAr translation inhibition is still effective when GAr-mRNA is mislocalized in the cytoplasm (intron-containing constructs or HIV-1 REV-based cytoplasmic transport). We demonstrated that these modifications of the localization of GAr mRNA from the nucleus to the cytoplasm abolishes its interaction with NCL as well as the GAr-based translation inhibition. We next showed that changing NCL localization from the nucleoplasm to the cytoplasm (by mutation of its nuclear localization signal) allows the interaction between the cytoplasmic GAr mRNA and the cytoplasmic NCL, and that this interaction reestablishes GAr-dependent inhibition of translation. These results indicate that NCL has a direct effect on the regulation of the GAr-based translation inhibition whatever the localization of its interaction with GAr mRNA. However, the fact that the nuclear localization of GAr mRNA correlates with the nuclear localization of NCL still suggests that nuclear trapping may also play a role. Hence, we have not formally determined yet if the GAr inhibition of translation involves both direct inhibition of translation and nuclear trapping, or if the nuclear trapping of GAr mRNA by NCL is solely responsible for GAr-based inhibition of translation. These data were generated in collaboration with R. Prado-Martins in the lab of Dr. Robin Fåhraeus.

The manuscript presented below represents my interpretation of the results. These results will be part of a publication that is currently in preparation, from which I will be the second author.

2

MATERIALS and METHODS

Plasmid constructions

Supplementary Table 1 corresponds to a list of plasmids, primers and restriction enzymes used in this study. We construct a set of reporters described in Figure 1. Briefly, full-length globin gene and OVA exons 5-7 fragment were amplified by PCR from the Glob-exon-SL8 construct 144 and chicken genomic DNA, respectively. The amplicons were cloned into pcDNA3 to create the control constructs (GG, OG).

The OVA ORF of the pcDNA3-GAr-OVA construct [START_REF] Tellam | Targeting of EBNA1 for rapid intracellular degradation overrides the inhibitory effects of the Gly-Ala repeat domain and restores CD8+ T cell recognition[END_REF] was replaced by the globin gene or OVA exons 5-7 fragment to generate plasmids in with the GAr domain is located upstream and in the same frame than the inserts (GGG, GOG). Afterwards, RNA from H1299 cells transfected with these gene constructs were used to generate cDNAs. The resultant cDNAs were used in a second round of cloning using the same primers and approach employed to produce the gene constructs to generate the cDNA constructs (GC, GGC, OC, and GOC). The GAr globin-RRE gene and GAr globin-RRE cDNA constructs were cloned according to the same method, using the plasmid Glob-exon-SL8-RRE 144 as template. The HA-NCL NLS plasmid was created by site-directed mutagenesis employing the pcDNA3-HA-NCL construct (Lista et al submitted) as a template. The NLS sequence KKRA was replaced by AARA using the primers described in the Supplementary Table 1. The Rev expression vector was a gift from Ali Saïb, Saint-Louis Hospital Paris All constructs were amplified in DH E. coli and sequenced by Sanger method.

Cell culture, transfection and drug treatments

The human cell line H1299 and the human HEK-293 T Kb stable cell line (kindly provided by L.

Eisenlohr, Thomas Jefferson University, Philadelphia, PA) were cultured in RPMI-1640 and DMEM, respectively, supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. Transient transfections were performed using Genejuice reagent Merck Bioscience according to manufacturer s protocol All cells were cultured at C with 5% CO2. For cell treatment with isoginkgetin (Sigma) and PhenDC3 (kindly provided by MP. Teulade-Fichou from Inserm U1196, Curie Institute, Orsay, France), cells were incubated with 30 µM and 5 µM of drug for 24h and 48h after transfection, respectively. Drug stock solutions were prepared in DMSO (Euromedex). All experiments, except for the antigen presentation assays, were carried out using H1299 cells.

Supplementary Table 1 -Plasmids, primers and restriction enzymes used in this study.

Supplementary Table 2 -Primers used for RT-PCR and RT-qPCR

Primer

Sequence (5'-3')

The Epstein-Barr Virus (EBV) is the first oncogenic virus described in humans. It is a ubiquitous virus which infects over 90% of the human population.

EBV causes a latent infection of B lymphocytes and remains asymptomatic in most of infected individuals. However, in some conditions like immunosuppression, EBV can cause uncontrolled cell proliferation responsible for some types of cancers like Burkitt lymphoma, Hodgkin lymphoma and nasopharyngeal carcinoma.

During latency, EBNA1 is the only protein expressed in all EBV-infected cells as it is essential for EBV genome replication and maintenance. EBNA1 is highly antigenic and T cells raised against EBNA1 exist; nevertheless EBV-infected cells are not eliminated by the host immune system. This is due to the central GAr (Gly-Ala repeat) domain of EBNA1 which is able to inhibit the translation of its own mRNA in cis, thereby preventing the recognition of EBV-infected cells by the immune system of the host. The mechanisms involved in this GAr-based translation inhibition were unknown when I started my PhD.

During this thesis, we identified the first cellular factor, Nucleolin (NCL), able to affect GAr-based translation inhibition. We showed that NCL overexpression enhances EBNA1 translation inhibition and inversely, that its downregulation decreases EBNA1 translation inhibition effect. Furthermore, we demonstrated that the interaction between NCL and GAr RNA occurs throught NCL binding to GAr-encoding mRNA via a particular secondary structure called G-quadruplex (G4). We showed that disrupting NCL-GAr RNA interaction with small G4 ligands enhances EBNA1 translation and thus antigen presentation, which constitutes a new therapeutic avenue to treat EBV-associated cancers.

Finally, we went deeper into the mechanism involved in this interaction by showing that NCL has a direct effect on the GAr-based translation inhibition and that the nuclear location of NCL-GAr RNA interaction is forced by the nuclear location of NCL in the cells.

Taken together, these results have shed light upon the mechanism involved in the GAr domain translation inhibition and immune evasion, and have revealed a possible therapeutic target to eradicate EBV-associated cancers.