Claude Universite

Bernard -Lyon

M Le Professeur

Frédéric Fleury

Hamda Ben

Hadid M Le Professeur Didier

Revel M Le Professeur

Philippe Chevalier

M Fabrice

Vallée Mme

Dominique Marchand

Composantes Sante

G Rode

La Mme

C Professeure

Burillon

D Bourgeois

Vinciguerra

M X Perrot

A-M Professeure

Schott

Ph.D David Ourania Durantel
email: david.durantel@inserm.fr

Ph.D Andrisani
email: andrisao@purdue.edu

M.D Michael M C Lai

Adrien Foca

Ammen P Dhillon

Thomas Lahlali

Julie Lucifora

Anna Salvetti

Michel Rivoire

Amy C H Lee

Ahmed Diab

Fabien Zoulim

Ourania Andrisani

Antiviral activity of PLK1-targeting siRNA delivered by lipid nanoparticles in HBV-infected hepatocytes

Keywords: Hepatitis B virus, Polo-like-kinase 1, small-interfering RNA, lipid nanoparticle, antiviral, host-targeting agent, hepatocellular carcinoma Hepatitis B virus, Polo-like-kinase 1, small-interfering RNA, lipid nanoparticle, antiviral, hosttargeting agent, hepatocellular carcinoma

Identification of PLK1 as a proviral factor for the hepatitis B virus replication: A promising target for antiviral and anticancerous drugs development.

COMPOSANTES ET DEPARTEMENTS DE SCIENCES ET TECHNOLOGIE

In highly HBV endemic regions, 70-80% of hepatocellular carcinoma cases are attributable to this virus. Despite the existence of an HBV vaccine, the World Health Organization estimates 240 million individuals are chronically infected with HBV worldwide. Current antiviral treatments to control chronic HBV infections, and consequently reduce the incidence of liver cancer, are ineffective. New and effective therapies are needed not only for fighting the virus but also to prevent HCC emergence or progression.

The polo-like-kinase 1 (PLK1), which plays pivotal roles in mitosis and is over-expressed in many human cancers, represents a promising druggable target in oncology. Beside its role during cell division, PLK1 is also thought to be involved in gene expression regulation during interphase. It was shown that the X protein (HBx) could activate PLK1 in murine cell transformation models. Yet it remained to be determined whether PLK1 could also play a role for HBV replication in non-dividing hepatocytes. Our, and collaborators, recent studies have identified a positive link between PLK1

activation and HBV replication. The goal of this thesis project was to investigate the mechanism(s) by which PLK1 exerts a positive effect on HBV replication, with the future goal of exploring PLK1 as an antiviral target.

The interplay between PLK1 and HBV replication was firstly described using the HepAD38 cellular model of HBV replication. In this context, the HBV DNA is stably integrated into the host genome, under control of a Tet-off expression system. Transcription of HBV pregenomic RNA (pgRNA), the template of viral replication, is initiated by tetracycline removal. It has been shown that in HBV-replicating HepAD38 cells, increased PLK1 expression correlates with down-regulation of two proteins that are components of chromatin modifying complexes; SUZ12 protein of the PRC2 complex, and ZNF198 of the LSD1-CoREST-HDAC1 complex. PLK1 inhibition was described to inhibit HBV replication by reducing viral transcription. How PLK1 regulates HBV transcription remains unknown. On the other hand, in HBV replication models that resemble physiologic HBV infection, comprised of Primary Human Hepatocytes (PHH) and non-transformed/differentiated HepaRG cells (dHepaRG), where HBV replicates in non-transformed and non-dividing cells, thus enabling the study of the inter-phasic role of PLK1, irrespective of its well-established cell division implication, we have demonstrated that:

1) A pharmacological inhibition of PLK1 suppressed HBV replication by a different mechanism, likely targeting the packaging of pgRNA by the HBV core antigen (HBc).

2) Knocking-down PLK1 using siRNA delivered by lipid nanoparticles (LNP siPLK1) results in a strong drop of HBV DNAs, RNAs and HBe/HBsAg secretion without affecting the cell viability.

This thesis project brought the proof of concept that PLK1 could be a drug target in HBV infection. Furthermore, the use of LNP allowed us to improve the delivery of siPLK1 to hepatocytes.

Significantly, PLK1 inhibition is not toxic to quiescent cells in comparison to fast growing cancer cells, rendering PLK1 an attractive therapy target. High level of viremia in chronic HBV patients is a risk factor for progression to liver cancer. PLK1 specific inhibitors are already in clinical trials for other types of cancer (e.g., acute myeloid leukaemia) and could serve as bimodal therapy in HBV infected patients, by inhibiting virus replication as well as preventing emergence and spreading of neoplastic cells.

This project was part of a full-working group of experts and thus, has beneficiated of a strong support. The proximity of the oncology-specialized hospital, the Centre Léon Bérard provided us with fresh hepatic biopsy. Moreover, our project had access to liver-humanized mice, a closer model to physiological behaviors, to validate our approach.

Inhibition of cellular PLK1 is a novel antiviral approach, which in combination therapies with interferon (IFN-α) or nucleoside analogs offers great promise for curing chronic HBV infection and could even prevent emergence of cell transformation.

Résumé

Identification de PLK1 comme facteur proviral dans la réplication du virus de l'hépatite B: Une cible thérapeutique attrayante pour le développement de molécules antivirales et anticancéreuses.

Mots-clés: Hépatite B virus ; Polo-like-kinase 1 ; ARN interférents ; nanoparticules lipidiques ; antiviraux ; carcinome hépatocellulaire.

Travaux de thèse réalisés par Adrien Foca au sein du Centre de Recherche en Cancérologie de Lyon (CRCL), UMR INSERM 1052, CNRS 5286.

Dans les régions de fortes endémicités, 70-80% des carcinomes hépatocellulaires sont induits par le VHB. Bien qu'un vaccin prophylactique très efficace existe, il n'est d'aucune utilité pour les 250 millions de personnes chroniquement infectées. Les traitements actuels pour contrôler l'infection chronique par le VHB montrent des limites et le besoin de nouvelles thérapies se fait ressentir.

La Polo-like kinase-1 (PLK1), qui joue un rôle essentiel dans la mitose et est surexprimée dans de nombreux cancers, représente une cible prometteuse. Outre son rôle lors de la division cellulaire, PLK1 est impliquée dans la régulation de l'expression des gènes en interphase. Il a été montré que la protéine X du VHB (HBx) active PLK1 dans des modèles de cellules murines. Cependant, il restait à déterminer si PLK1 jouait un rôle au niveau de la réplication du VHB dans des hépatocytes quiescents. Des études récentes ont mis en évidence un lien positif entre l'activation de PLK1 et la réplication du VHB. Le but de ce projet de thèse a été d'étudier le(s) mécanisme(s) par le(s)quel(s)

PLK1 jouait un rôle positif sur la réplication virale, avec pour objectif futur d'explorer l'inhibition de PLK1 comme cible antivirale.

L'interaction entre PLK1 et la réplication du VHB a d'abord été décrite à l'aide du modèle HepAD38. Dans ce contexte, l'ADN viral est intégré dans le génome hôte, sous le contrôle d'un système d'expression Tet-off. La transcription de l'ARN prégénomique (pgRNA), à la base de la réplication virale, est initiée par la suppression de tétracycline. Dans ce contexte, l'augmentation de l'expression de PLK1 est corrélée avec la régulation négative de deux protéines; SUZ12 et ZNF198, faisant partie de complexes de remodelage de la chromatine. L'inhibition de PLK1 bloque la réplication du VHB, en agissant au niveau de la transcription virale. D'autre part, dans les modèles de réplication du VHB qui miment au mieux une infection, comprenant les hépatocytes primaires humains (PHH) et les cellules non transformées/différenciées HepaRG (dHepaRG), où le VHB se réplique dans des cellules quiescentes, nous avons mis en évidence que:

1) L'inhibition pharmacologique de PLK1 bloque la réplication virale, semblablement en perturbant l'encapsidation du pgRNA via une interaction avec la protéine core du VHB (HBc).

Thesis Adrien FOCA

2) Un knocking-down de PLK1 en utilisant des ARN interférents délivrés par nanoparticules lipidiques résulte en une forte baisse de la production de pgRNA et dans la sécrétion des antigènes HBeAg/HBsAg, sans impact sur la viabilité cellulaire.

Ce projet a donc permis la preuve de concept que PLK1 pouvait être une cible thérapeutique afin de controler la réplication du VHB. De plus, grâce à la technologie de délivrance par nanoparticules lipidiques d'ARN interférents, nous avons pu cibler spécifiquement les hépatocytes, augmentant de ce fait la spécificité et l'efficacité de nos traitements. Un travail sur la compréhension précise des méchanismes cellulaires impliqués permettra de mieux cerner cette interaction hôte/virus afin de poursuivre le développement de stratégies antivirales innovantes portant sur l'inhibition de PLK1.

De manière significative, l'inhibition de PLK1 est non toxique pour les cellules quiescentes par rapport à des cellules cancéreuses à fort taux réplicatif, ce qui fait de PLK1 une cible thérapeutique attrayante. Des inhibiteurs spécifiques sont déjà en essais cliniques pour certains cancers (e.g.,

Volasertib pour le traitement de la leucémie myéloïde aiguë) et pourraient servir de thérapie bimodale dans le cadre de patients infectés par le VHB, en inhibant la réplication virale, ainsi qu'en prévenant l'émergence de cellules néoplasiques.

L'inhibition de la PLK1 est une approche antivirale innovante, qui, en combinaison avec les thérapies actuelles de type IFN-α ou analogues nucléotidiques offre de grandes promesses pour endiguer l'infection chronique par le VHB mais également prévenir les événements carcinogéniques. The first descriptions of jaundice epidemics, jaundice being a common symptom of hepatitis B virus (HBV) infections, were reported in the time of ancient China and Babylonia. In Occident, Hippocrate documented an outbreak of jaundice that occurred in the Greek island of Thassos, in the fifth century B.C. Other episodes were observed in the Middle Ages, due to crowded and unsanitary conditions, mostly during times of war [START_REF] Martin | The discovery of viral hepatitis: a military perspective[END_REF]. The oldest evidenced case of hepatitis B was found in a 450-year-old mummy, but studies tends to indicates that HBV genotypes diversified long before the 16th century, between at least 3,000 years and possibly as long as 100,000 years ago (Kahila [START_REF] Bar-Gal | Tracing hepatitis B virus to the 16th century in a Korean mummy[END_REF][START_REF] Patterson Ross | The paradox of HBV evolution as revealed from a 16th century mummy[END_REF].

List of Figures and Tables

As early as 1940, medical researchers had determined that there were two different forms of virus-induced jaundice; one that is transmitted as an intestinal infection (i.e., feco-oral transmission), and the other spread mainly by blood transfusion/contact. They were respectively named Hepatitis A and B viruses.

The HBV discovery came by an indirect route. In 1963, Dr Baruch Blumberg, working at the NIH, was tracking inherited polymorphic serum proteins in the blood of patients worldwide.

Blumberg and colleagues used sera from multi-transfused individuals as sources of antibody to human serum proteins. The idea was that these sera could contain antibodies bound to proteins differing in sequence from those of the transfusion recipients. They screened over 20,000 sera specimens and identified a new antigen, named "Australia antigen" (AuAg) from an Australian Aborigine blood [START_REF] Blumberg | A "NEW" ANTIGEN IN LEUKEMIA SERA[END_REF]. This antigen was found to be very stable and demonstrated a relatively rare occurrence in the United States population compared to people originating from Asia and pacific islands. Moreover, sera from multi-transfused and leukemic patients exhibited high frequency of AuAg over anti-AuAg antibody. These led Blumberg and colleagues to hypothesize that AuAg predicted leukemia risk. However, in 1966, James Blair, a patient with Down syndrome, initially negative for AuAg was observed to seroconvert concomitantly with "chronic anicteric hepatitis" [START_REF] Blumberg | A serum antigen (Australia antigen) in Down's syndrome, leukemia, and hepatitis[END_REF]. In the mean time, a member of Blumberg's laboratory experienced an hepatitis due to contact with contaminated materials, again with the appearance of AuAg in the blood. These interesting observations allowed the possible association between a viral infection and AuAg, which is now named HBsAg. The ability to carry out retrospective studies with assays for AuAg confirmed a long-held suspicion that HBV was responsible for a chronic hepatitis leading to cirrhosis and liver cancer in many parts of the world [START_REF] Okochi | Observations on Australia antigen in Japanese[END_REF][START_REF] Prince | An antigen detected in the blood during the incubation period of serum hepatitis[END_REF]. Eventually, in 1970, Dane observed complete HBV particle as virus-like entities in serum of patients with AuAgrelated hepatitis [START_REF] Dane | Virus-like particles in serum of patients with Australia-antigen-associated hepatitis[END_REF](Figure 1). Since its discovery, research works on the virus have lead to a better understanding of its life cycle and natural history, as well as to the development of viro-suppressive treatments. Work is yet to be done to cure patients from HBV infections (Figure 2).

Classification and phylogeny of Hepadnaviridae

Viruses belonging to the Hepadnaviridae family, together with the Spumaviridae (foamy viruses), are the only known DNA viruses to replicate their DNA by reverse transcription of a viral pregenomic RNA template. HBV is considered as a prototype of the Hepadnaviridae family, which contains two genera, the orthohepadnaviruses that infect mammals, and avihepadnaviruses that infect birds (Chapter Version: ICTV Ninth Report; 2011 Taxonomy Release). This two genera assignment is based, in addition to the host specificity spectrum, on rather weak sequence homology between the two groups, despite the overall similar genome organisation. Duck hepatitis B virus (DHBV) and Heron hepatitis B virus (HHBV) are prototypes of the avihepadnavirus group. It is of importance to indicate here that DHBV served as an excellent model to study the molecular and cellular biology of Hepadnaviridae and develop nucleoside analogues, before the advents of transgenic/humanized animals allowing infection with human HBV. The orthohepadnavirus genus is mainly represented by four species, exhibiting a sequence divergence <35%, with HBV, WHV, GSHV, and WMHV as the prototypes (Figure 3). Another virus related to GSHV has been isolated in Arctic ground squirrel (ASHV) but has not been assigned yet due to lack of host range studies [START_REF] Testut | A new hepadnavirus endemic in arctic ground squirrels in Alaska[END_REF]. Naturally, this classification is not absolute and may evolve according to future findings as well as to progress in species-to-species infection characterization. As an example of phylogenetic classification complexity, the woolly monkey hepatitis B virus is closely related in DNA sequence to HBV, with less than <20% sequence variation, but do not infect human or chimpanzee, which is highly susceptible to human HBV genotypes.

To date, eight well-characterized HBV genotypes have been identified (A to H) showing pairwise differences between 8 to 17%. Two new genotypes (i.e., I and J) have been identified in Vietnam/Laos and in the Ryukyu Islands in Japan respectively, but are not yet approved by the ICTV (International committee on the taxonomy of viruses) [START_REF] Olinger | Possible New Hepatitis B Virus Genotype, Southeast Asia[END_REF][START_REF] Tatematsu | A Genetic Variant of Hepatitis B Virus Divergent from Known Human and Ape Genotypes Isolated from a Japanese Patient and Provisionally Assigned to New Genotype[END_REF]Yu et al., 2010)(Figure 4). Interestingly, all these genotypes tend to exhibit different geographical distributions (Figure 5) and distinct clinical manifestations. Hence, genotype C is known to favour rapid progression to HBV-related cirrhosis, while genotype G is lacking HBeAg production due to mutation in the PreCore region [START_REF] Stuyver | A new genotype of hepatitis B virus: complete genome and phylogenetic relatedness[END_REF]. Finally, genotypes A-D, F, H and I are classified further into sub-genotypes with between 4% and 8% intergroup nucleotide divergences. Among them, the HBV F1b was recently described in Alaska native population to exhibit several core mutations, which are strongly associated with HCC development [START_REF] Hayashi | A novel association between core mutations in hepatitis B virus genotype F1b and hepatocellular carcinoma in Alaskan Native People[END_REF]. This diversity leads to different natural history of infection and modes of transmission, thus delineating the need of a large arsenal of therapeutic options [START_REF] Liu | Therapeutic implications of hepatitis B virus genotypes[END_REF].

Epidemiology 1.3.1. HBV: a worldwide healthcare issue

The World Health Organization (WHO) reports there are around 250 million people chronically infected by HBV (CHB) worldwide, associated with a high co-morbidity due to hepatic complications. Of these 250 million, 25% could eventually die of chronic liver disease or hepatocellular carcinoma if left untreated (WHO Global hepatitis report, 2017). HBV is unevenly distributed worldwide with two highly-endemic areas, i.e., Africa and South-East Asia (Figure 6). The highest HBsAg seroprevalence is found in South Soudan with 22% of individuals being chronic carriers, while lowest-prevalence areas are observed in Northern and Western Europe. Globally, the prevalence of HBV decreased over the two past decades and now is estimated to be 3.6% of the world population (MacLachlan and Cowie, 2015;Schweitzer et al., 2015).

In the Global Burden of Disease Study 2010 (GBD 2010), 786,000 deaths were attributable to HBV, of which 341,400 were estimated to be caused by liver cancer, 312,400 by cirrhosis and 132,200 by acute hepatitis B [START_REF] Lozano | Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study[END_REF].

Entry into the host and transmission pathways

HBV is found in many body fluids and viral load may reach >10 10 copies per mL in blood. Thus, the rate of direct transmission is very high compared to other viruses such as HCV or HIV. Basically, HBV can be transmitted following two schemas: Vertical transmission: defined by perinatal infection from infected mothers to the newborn or very young child. This route represents the greatest source of new infections worldwide but exhibits a clear dichotomy, depending on geographic areas. In Asia, the rate of perinatal transmission can reach 90% due to high rate of HBeAg positivity and high HBV viral load in mothers' sera. In Western Europe, North America and Africa, the risk of vertical transmission is approximately 10%, explained by lower HBeAg positivity in sera and better healthcare systems (from prevention to treatment accessibility). The high rate of chronicity in many parts of Africa is attributable to horizontal spread to young children from playmates and adults involved in their care. Hyper-immune globulin to HBsAg injection and vaccination at birth can reduce the risk of chronic infection by >90% [START_REF] Beasley | Prevention of perinatally transmitted hepatitis B virus infections with hepatitis B immune globulin and hepatitis B vaccine[END_REF][START_REF] Lo | Combined passive and active immunization for interruption of perinatal transmission of hepatitis B virus in Taiwan[END_REF][START_REF] Stevens | Prospects for control of hepatitis B virus infection: implications of childhood vaccination and long-term protection[END_REF]. Some data suggest that HBeAg may cross the placenta, although, no evidences that this causes immune tolerance and facilitates chronic infection has yet been obtained.

Horizontal transmission: it regroups blood transfusion, IV (intravenous) drug use, sexual transmission and nosocomial infections. Since the 1970s, HBV infection through blood transfusion has drastically decreased due to the abolition of paid donors and systematic screening of volunteer blood donors for serum HBsAg and anti-HBc immunoglobulins.

Ongoing implementation of specific HBV-qPCR screening should decrease the risk of undetected rare variants escaping the serologic assay for HBsAg and solve the problem of occult HBV infections, in which neither HBsAg nor anti-HBc are detected. Sexual transmission still represents 30-40% of HBV infection new cases in developed countries while the role of intravenous drug use seems to be decreasing with time, currently representing only 6% of reported new cases. Nosocomial transmission is estimated to be 30% from highly viremic patients, but appears to be very rare (WHO, guidelines for the prevention, care and treatment of persons with chronic hepatitis B infection, 2015). Some cases have been reported in dialysis centers or with HBV infection from infected surgeons [START_REF] Harpaz | Transmission of hepatitis B virus to multiple patients from a surgeon without evidence of inadequate infection control[END_REF]. Finally, percutaneous infection is still a major mode of HBV transmission, due to lifestyle in developing/developed countries (e.g., intravenous drug use, tattoos, acupuncture, and piercings) [START_REF] Goldstein | Incidence and risk factors for acute hepatitis B in the United States, 1982-1998: implications for vaccination programs[END_REF][START_REF] Alter | Epidemiology and prevention of hepatitis B[END_REF][START_REF] Yang | Transmission of Hepatitis B and C Virus Infection Through Body Piercing: A Systematic Review and Meta-Analysis[END_REF][START_REF] Rempel | Outbreak of acute hepatitis B virus infection associated with exposure to acupuncture[END_REF].

Unraveling the complexity of Hepatitis B virus 1.4.1. Viral structure and subviral particles

Infectious HBV virions (Dane particles) are spherical entities of ≈42nm in diameter (depending on genotype) with no evidence of outer projections after negative staining (Transmission Electron Microscopy) observations. However, in cryo-EM, projections are distinguishable outside the envelope and are evidences of spike-like features composed of L, M, and S proteins (respectively Large, Medium and Small forms of HBsAg) (Figure 7). This envelope, formed by HBs in a 1:1:4 ratio of L, M and S, and host-cell lipids acquired from budding into multivesicular bodies (MVBs), contains a nucleocapsid of 36nm. A hundred and twenty core protein (HBc) dimers, organized in a triangulation T=4, compose this icosahedral capsid [START_REF] Marion | Hepadna viruses: hepatitis B and related viruses[END_REF][START_REF] Heermann | Large surface proteins of hepatitis B virus containing the pre-s sequence[END_REF]. A small fraction of capsids (<5%) consists of only 90 dimers with a triangulation number T=3 but may represent dead-end products caused by an aberrant assembly process [START_REF] Newman | Stability and Morphology Comparisons of Self-Assembled Virus-Like Particles from Wild-Type and Mutant Human Hepatitis B Virus Capsid Proteins[END_REF].

Capsids contain a single copy of the partially double-stranded relaxed-circular DNA (rcDNA) genome of ≈3.2kb, which is covalently linked to the viral polymerase (P) at the 5ʹ end of the complete strand (negative strand). The viral titre of Dane particles can vary significantly among patients and can be as high as 10 10 /mL of serum.

As mentioned, infected hepatocytes also produce/secrete subviral non-infectious particles, composed only by HBsAg. These latter are either spherical (16-25nm of diameter) or filamentous (20nm of diameter and variable in length) and represent the most abundant particle released in the blood from infected hepatocytes (up to 10 14 /mL for spheres). Filamentous structures contain mainly L and M forms of HBsAg while M and S proteins, at a ratio of about 1:2, compose spheres. They may have various role in the HBV life cycle, including neutralization of anti-HBV antibodies or immune escape by ligand/receptor antagonization processes, thus facilitating virus spread and maintenance [START_REF] Chai | Properties of subviral particles of hepatitis B virus[END_REF].

Eventually, in patient sera, various particles and intermediates of complete virions can be found. There are either secreted by infected hepatocytes or released upon apoptosis, but their roles are not totally deciphered [START_REF] Bardens | Alix regulates egress of hepatitis B virus naked capsid particles in an ESCRT-independent manner[END_REF]Hu and Liu, 2017;[START_REF] Caballero | Hepatitis B virus: The challenge of an ancient virus with multiple faces and a remarkable replication strategy[END_REF](Figure 8).

HBV genome structure and organization within Dane particles

In virions, the viral genome, as rcDNA, is about 3.2kb in length. The 5' of the negative strand is covalently linked to the viral polymerase while the 5' of the positive strand has a covalently attached 19nt oligoribonucleotide primer, which served for the minus-strand DNA synthesis. The 3' end of the positive strand terminates at a variable position, thus creating a single stranded gap that may account for up to 60% of the viral genome [START_REF] Summers | Genome of hepatitis B virus: restriction enzyme cleavage and structure of DNA extracted from Dane particles[END_REF][START_REF] Lien | Evidence that a capped oligoribonucleotide is the primer for duck hepatitis B virus plus-strand DNA synthesis[END_REF][START_REF] Seeger | Hepatitis B virus biology[END_REF]. Minus strand exhibits a small 8 to 9nt-long terminal redundancy, which is required for the formation of rcDNA upon plus-strand DNA synthesis [START_REF] Seeger | Biochemical and genetic evidence for the hepatitis B virus replication strategy[END_REF])(Figure 9). A ≈250nt overlapping between the two strands ensures circularization of rcDNA. In this overlapping, we found several regulatory elements, such as the two direct repeat regions (DR1 and DR2), which are mandatory for viral replication [START_REF] Seeger | Biochemical and genetic evidence for the hepatitis B virus replication strategy[END_REF][START_REF] Wang | Function of direct repeat sequences in DNA replication of hepatitis B virus[END_REF].

A significant fraction of Dane particles (≈10%) contains double-stranded linear DNA (dslDNA) instead of rcDNA. These virions can enter cells but frequently lose important sequences from their ends during initiation of infection, thus playing a minor role in HBV replication [START_REF] Caballero | Hepatitis B virus: The challenge of an ancient virus with multiple faces and a remarkable replication strategy[END_REF]. This incoming dslDNA is likely involved in the early integration of HBV DNA into host genome [START_REF] Yang | Integration of hepadnavirus DNA in infected liver: evidence for a linear precursor[END_REF].

The HBV genome is small and condensed, thus exhibiting a certain complexity towards its organization. The genome contains four overlapping open reading frames (ORFs), four promoters, two enhancer elements, and a single polyadenylation signal to regulate transcription of viral RNAs (Figure 9). All viral transcripts are encoded by the minus strand, are capped and polyadenylated.

Transcription regulatory regions are present within ORFs and are active following the transport of the genome into the cell nucleus [START_REF] Seeger | Biochemical and genetic evidence for the hepatitis B virus replication strategy[END_REF]. HBV cccDNA is formed into the nucleus of infected hepatocytes from rcDNA. This latter is converted into cccDNA by host machinery following a multiple-steps process, which is not fully characterized yet, but likely involves tyrosyl-DNA-phosphodiesterase, endonuclease, DNA polymerase, polynucleotide kinase, phosphatase and DNA ligase activities [START_REF] Qi | DNA Polymerase κ Is a Key Cellular Factor for the Formation of Covalently Closed Circular DNA of Hepatitis B Virus[END_REF][START_REF] Kitamura | Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus[END_REF]. Under physiologic conditions, cccDNA is associated with histones and other proteins to form a mini-chromosome and serves as template for viral RNAs transcription [START_REF] Newbold | The covalently closed duplex form of the hepadnavirus genome exists in situ as a heterogeneous population of viral minichromosomes[END_REF].

Although the exact location of the cccDNA in the nucleus is still elusive, studies suggest that it localizes preferentially near to active chromatin enriched in H3K4me3, H3K4me2, as well as H3K27ac marks (ENCODE Project Consortium, 2012). In a paper published recently in Nature Communications by Moreau, Cournac and colleagues, it was reported that HBV cccDNA contacts preferentially CpG islands enriched in Cfp1, which is a factor required for its transcription [START_REF] Moreau | Tridimensional infiltration of DNA viruses into the host genome shows preferential contact with active chromatin[END_REF].

Theoretically, the cccDNA is persistently sustained into quiescent cell without affecting viability as the cellular Pol II considers it as a normal and functional chromosome. Furthermore, the cccDNA pool can be maintained through a rcDNA recycling loop from neosynthesized capsids [START_REF] Ko | Hepatitis B virus genome recycling and de novo secondary infection events maintain stable cccDNA levels[END_REF].

However, the cccDNA can be eliminated, during apoptosis of hepatocytes and/or successive mitosis, which leads to cccDNA dilution and loss as it has no centromere. Up to now, no therapeutic is available to directly target the cccDNA in an efficient manner, highlighting the need of fundamental research efforts towards understanding its biology.

The cellular Pol II carries out the transcription of the cccDNA, leading to the main production of 2 genomic and 3 sub-genomic viral RNAs. The major transcripts that are detected in HBV-infected livers are 3.5kb, 3.5kb, 2.4kb and 2.1kb in length and termed pregenomic (pg), pre-C/C, pre-S and S mRNAs respectively. A minor transcript, the 0.7kb X mRNA, has occasionally been detected in infected tissues and evidenced more consistently in cells transfected with subgenomic HBV DNA [START_REF] Moolla | Regulatory elements of hepatitis B virus transcription[END_REF][START_REF] Shiromoto | IL-1β/ATF3-mediated induction of Ski2 expression enhances hepatitis B virus x mRNA degradation[END_REF](Table I). New tools, such as deep RNA sequencing or 5' RACE-PCR should help deciphering further theses viral RNAs in vivo.

Table I: List of HBV viral mRNAs transcripted from cccDNA.

Table II: List of the eight HBV proteins.

HBV viral proteins

The HBV genome harbours four ORFs, encoding 8 viral proteins. Seven are described as principal and the eighth, the hepatitis B spliced protein (HSBP), which is produced by alternative splicing of pgRNA was solely recently described and found in liver biopsies [START_REF] Soussan | In vivo expression of a new hepatitis B virus protein encoded by a spliced RNA[END_REF].

1.4.4.1. Large, Medium and Small envelope proteins; How to build HBV envelope the right way

The three viral envelope proteins L, M and S are synthetized from two mRNAs, both originating from the S ORF (Table I andII). The 2.4kb mRNA encodes L-HBsAg while the 2.1kb encodes M-or S-HBsAg, depending on the AUG used. The initiation codon for S-HBsAg is preferentially recognized by the ribosomal machinery, explaining the large excess of S-compared to M-HBsAg (4:1 ratio) [START_REF] Sheu | Preferential ribosomal scanning is involved in the differential synthesis of the hepatitis B viral surface antigens from subgenomic transcripts[END_REF]. The three forms of HBsAg share the same STOP codon, thus differing only in their N-ter domains. S-HBsAg is a 226aa polypeptide; M has a 55aa additional domain called PreS2 and L possesses S, PreS2 and a 108-119aa PreS1 domain in N-ter (Figure 10).

These three proteins are synthetized in the ER and matured in the Golgi apparatus as their fate is to be eventually secreted. Regarding principal post-translational marks, L-, M-and S-HBsAg are either N or O-glycosylated. Furthermore, the L form is myristoylated on its second aa, which favours a topology of L-HBsAg that makes the preS1 attachment site less accessible to neutralizing antibodies [START_REF] Bremer | N-terminal myristoylation-dependent masking of neutralizing epitopes in the preS1 attachment site of hepatitis B virus[END_REF]. The three forms of HBsAg harbour four transmembrane domains organized as described in Figure 10B.

The S domain is cysteine-rich, resulting in disulfure bridges formation, mandatory for homoheterodimerization and subsequent envelope assembly. The 124-147 region is known as the "a" determinant, which is the main target of neutralizing antibodies [START_REF] Wounderlich | Characterization of early hepatitis B virus surface protein oligomers[END_REF]. The M form of HBsAg seems to be unnecessary for neither formation nor infectivity of HBV particles [START_REF] Bruss | The role of envelope proteins in hepatitis B virus assembly[END_REF]. The L protein is found under two topologies at the ER membrane:

i-preS: the first transmembrane domain is not translocated the right way and stays cytosolic, thus allowing interaction with newly assembled capsids.

-e-preS: the first transmembrane domain is translocated through the ER, facilitating the downstream interaction with the NCTP HBV receptor [START_REF] Prange | Host factors involved in hepatitis B virus maturation, assembly, and egress[END_REF].

Beside the involvement of L, M and S proteins in envelope formation, they are also found in subviral particles. These latter are known as bait and hold a key role in immunity control. Spheres are mostly composed by M and S forms whereas filaments harbour also the L protein [START_REF] Gilbert | Hepatitis B small surface antigen particles are octahedral[END_REF][START_REF] Chai | Properties of subviral particles of hepatitis B virus[END_REF].

"A review I have written and published in Antiviral Research details the diverse functions of the core protein in the viral life cycle. Please confer to Appendix"

The capsid protein or HBc, is a 183aa long polypeptide of 21kDa, which has been classically viewed as a structural protein that self-assembles to form the viral nucleocapsid. However, accumulated evidences support that HBc functions extend well beyond this structural role. HBc seems to be involved in nearly every stage of the HBV life cycle, including subcellular trafficking and release of HBV genome, RNA metabolism and trafficking, capsid assembly and transport, and reverse transcription (Nassal, 1992;Rabe et al., 2003;Perlman et al., 2005;Basagoudanavar et al., 2007).

Moreover, recent findings implicate an active role for HBc in epigenetic regulation of the viral and host genomes. Collectively, this multiplicity of functions elevates HBc as an excellent target for mechanism-based antiviral therapeutics (Durantel and Zoulim, 2016).

HBeAg: the HBV precore protein

HBeAg is a non-structural protein, which is related to the HBV core protein and circulates in HBV infected patients' bloodstream. HBeAg is encoded by the 3.5kb Pre-C/C mRNA, giving rise to a 25kDa precursor, similar to HBc but exhibiting an additional 29aa at its N-ter [START_REF] Messageot | Proteolytic processing of the hepatitis B virus e antigen precursor. Cleavage at two furin consensus sequences[END_REF]. The initiation AUG codon involved in HBeAg translation is located 87nt prior to the AUG used for HBc synthesis. This 25kDa precursor is addressed to the ER, where a furin cleaves the peptide signal in N-ter, leading to a 22kDa intermediate. This latter is then cleaved in the C-ter arginine rich domain and HBe secreted as a 17kDa protein that assembles in dimers [START_REF] Ito | Characterization of genotype-specific carboxylterminal cleavage sites of hepatitis B virus e antigen precursor and identification of furin as the candidate enzyme[END_REF].

HBeAg is not directly involved into HBV replication as HBe defective HBV genotype G is infectious and replicates in vitro alone [START_REF] Chudy | Hepatitis B virus genotype G monoinfection and its transmission by blood components[END_REF] and in human in the presence of genotype A [START_REF] Stuyver | A new genotype of hepatitis B virus: complete genome and phylogenetic relatedness[END_REF]. However, HBeAg is found among all Hepadnaviridae, highlighting its importance. Indeed, as HBeAg mimics HBc, it serves as a tolerogenic bait, extinguishing anti-HBV immune response through a saturation of lymphocytes T MHC-I receptors. HBeAg may cross the placenta, leading to tolerogenicity in child while HBeAg negative status in the mother can lead to fulminant hepatitis for the child [START_REF] Liang | A hepatitis B virus mutant associated with an epidemic of fulminant hepatitis[END_REF][START_REF] Chen | Pediatric fulminant hepatic failure in endemic areas of hepatitis B infection: 15 years after universal hepatitis B vaccination[END_REF].

Clinically, the level of circulating HBeAg witness the HBV replication rate and the seroconversion of HBeAg to anti-HBeAg evokes the end of active replication.

The viral polymerase (Pol)

The viral polymerase is the largest HBV protein, with a length of 845aa for 90kDa, and is the sole enzyme coded by HBV. Currently, in clinic, direct acting antivirals are focused on targeting this protein (e.g., nucleo-side/tide analogues NUCs). The Pol contains four distinct domains and exhibits several enzymatic activities:

-Terminal protein domain: aa 1-180. Mandatory for the covalent attachment to the Ɛ packaging signal of pgRNA. The Y63 residue of the TP domain and the minus strand 5' end of the pgRNA are engaged in a phosphodiester bond. This domain has a primase activity, allowing the reverse-transcription initiation through the synthesis of a 3-4nt primer [START_REF] Zoulim | Reverse transcription in hepatitis B viruses is primed by a tyrosine residue of the polymerase[END_REF][START_REF] Lanford | Transcomplementation of nucleotide priming and reverse transcription between independently expressed TP and RT domains of the hepatitis B virus reverse transcriptase[END_REF] -Spacer domain: aa 181-349. Functions not well elucidated yet. Could be of importance for flexibility of HBs proteins by interacting with the PreS domain and thus responding to immune pressure.

-Pol/RT domain: aa 350-693. The domain polymerase/reverse-transcriptase holds two functions. In the one hand, the RNA-dependent polymerase which ensure the pgRNA reverse-transcription into the minus strand DNA and in the other hand, the DNA-dependent polymerase require for the subsequent plus strand DNA synthesis. This domain is divided in several subdomains, where the so-called "C" contains the YMDD catalytic site, which is involved in resistance to NAs (nucleoside analogues, i.e., LAM, TDF, TAF) [START_REF] Liaw | Management of YMDD mutations during lamivudine therapy in patients with chronic hepatitis B[END_REF][START_REF] Cai | Compensatory variances of drug-induced hepatitis B virus YMDD mutations[END_REF][START_REF] Calica Utku | Treatment of chronic hepatitis B patients with tyrosinemethionine-aspartate-aspartate mutations[END_REF].

-RNaseH domain: aa 694-845. Is responsible for pgRNA degradation after completion of the minus strand DNA synthesis [START_REF] Wei | Expression, purification, and characterization of an active RNase H domain of the hepatitis B viral polymerase[END_REF]. HBx is a non-structural protein of 17kDa, involved in establishment and maintenance of infection. Indeed, a HBV mutant lacking HBx exhibits a lower amount of transcripts synthetized from the cccDNA and is unable to maintain the infection in a stable fashion [START_REF] Lucifora | Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection[END_REF]. HBx is an enigmatic protein found in high copy numbers in the cytoplasm of infected cell (up to 80,000 copies) but can also be found in the nucleus, especially in close vicinity to the cccDNA. HBx indirectly increases the cccDNA transcription by recruiting histones acetylases, leading in fine to better HBV replication. Recently, a study has shown that X protein is involved in the degradation of the SMC5/6 complex through a DDB1/E3 ubiquitin ligase-dependent mechanism. This way, the cccDNA is no more "recognized" by the SMC5/6 complex and can be transcriptionally active [START_REF] Decorsière | Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor[END_REF].

Eventually, HBx seems to be involved in the gene expression modulation of several host cell genes, involved especially in cell-cycle, apoptosis and DNA repair pathways (Figure 12). Thus, HBx seems to be a good candidate as pro-oncogenic protein even if the limits of its perimeter of action are still blurry [START_REF] Andrisani | The transcriptional function of the hepatitis B virus X protein and its role in hepatocarcinogenesis (Review)[END_REF][START_REF] Ma | Epigenetics in hepatocellular carcinoma: an update and future therapy perspectives[END_REF]. Please refer to section "HBV putative onco-proteins: the trans-effect" for details. As human transcripts, HBV RNAs are subjected to splicing events, which increase the genetic diversity. Among all the HBV spliced RNAs, the variant SP1 of the pgRNA is the best characterized.

The SP1 RNA, of 2.2kb, results from 1.3kb intron removal from the pgRNA and accounts for up to 30% of total pgRNAs amount. SP1 is translated into a 93aa long protein of 12.4kDa, the hepatitis B splicing-regulated protein (HBSP) [START_REF] Soussan | In vivo expression of a new hepatitis B virus protein encoded by a spliced RNA[END_REF]. HBSP and the viral polymerase share the same AUG codon and thus exhibiting a 46aa homology at their N-ter. The following sequence of HBSP is encoded by the original nucleotidic arrangement found in the spliced variant SP1 (Figure 13).

As SP1 shares homology with pgRNA, it can be encapsidated the same way and reverse transcripted, yet leading to defective HBV particles formation. These latters are found to be enhanced prior to development of HCC [START_REF] Bayliss | Hepatitis B virus splicing is enhanced prior to development of hepatocellular carcinoma[END_REF]. HBSP seems to be involved in reducing HBV-induced liver inflammation, lowering the efficacy of immune responses and in fine, promoting HBV infection and maintenance [START_REF] Soussan | The expression of hepatitis B spliced protein (HBSP) encoded by a spliced hepatitis B virus RNA is associated with viral replication and liver fibrosis[END_REF][START_REF] Pol | Alternative splicing-regulated protein of hepatitis B virus hacks the TNF-α-stimulated signaling pathways and limits the extent of liver inflammation[END_REF]. However, the roles of spliced HBV RNAs and HBSP need still to be further defined [START_REF] Chen | Hepatitis B virus spliced variants are associated with an impaired response to interferon therapy[END_REF].

"Involvement of HBc in HBV replication cycle is not extensively detailed hereafter as it is the focus of a review I have written in Antiviral Research. Please confer to the Appendix section."

Hepatitis B virus mainly infects liver cells and replicates exclusively in quiescent hepatocytes.

The entry is mediated through a low affinity interaction between HBV virion and heparan sulfate proteoglycans present at the hepatocytes cell surface [START_REF] Schulze | Hepatitis B virus infection initiates with a large surface protein-dependent binding to heparan sulfate proteoglycans[END_REF]. The following step is characterized by a higher affinity interaction involving the PreS1 domain of the HBV large envelop protein and its receptor, the sodium taurocholate cotransporting polypeptide (NTCP) on the basolateral side of hepatocytes (Yan et al., 2012). The downstream internalisation of virions is poorly understood although glypican 5 has been recently identified as co-entry factor (Verrier et al., 2016a).

After viral entry, nucleocapsids are transported to the nuclear pore, where the viral rcDNA is released and then converted into cccDNA, which is responsible for viral persistence. This latter serves as template for viral mRNAs transcription and proteins are synthetized in the cytosol. HBV can also be integrated into host genome, preferentially from dslDNA-containing particles, which facilitate homologous recombination [START_REF] Tu | HBV DNA Integration: Molecular Mechanisms and Clinical Implications[END_REF]. Some genes, as hTERT have been highlighted as nonrandom integration site of HBV genome, which are of importance in viral oncogenic events occurrence (confer section "2.3.4.1.1. HBV dslDNA integration: the cis-effect") [START_REF] Horikawa | Transcriptional regulation of the telomerase hTERT gene as a target for cellular and viral oncogenic mechanisms[END_REF]. Once exported to the cytosol, the pgRNA is both used for the translation of the core (HBc) and viral polymerase (Pol) and as the template for reverse transcription within nucleocapsid.

The polymerase would preferentially bind to the pgRNA used for its synthesis, on the packaging signal epsilon (Ɛ) at the 5ʹ end of the mRNA, while the translation is still ongoing. This process warrants a good stoichiometry between the genetic template and HBV pol before encapsidation of this ribonucleoprotein complex. The covalent attachment of the polymerase to the Ɛ domain of the pgRNA leads to HBc dimers recruitment and capsid formation (Figure 14). Cellular chaperones, such as HSP90, and the arginine-rich CTD domain of HBc are required for correct interaction with the pol-Ɛ complex (Nassal, 1992;[START_REF] Hu | Requirement of heat shock protein 90 for human hepatitis B virus reverse transcriptase function[END_REF]. Capsid formation and subsequent reverse transcription of the pgRNA into rcDNA are two intimately linked processes, dependent on a dynamic phosphorylation of HBc CTD. Several kinases as, SRPK1, CDK2 and PLK1 are known to phosphorylate HBc CTD in a kinetic-dependent manner, thus regulating subcellular localization of Core protein and reversetranscription of pgRNA in the capsid (Daub et al., 2002;Ludgate et al., 2012;Diab et al., 2017). For more details, see the dedicated review I have written focusing on the diverse functions of HBc in the viral life cycle (confer Appendix review (Diab et al., 2018)).

The first step in pgRNA reverse transcription is the minus-strand DNA synthesis, beginning from the bulge of the Ɛ loop. The polymerase remains covalently linked to the 5ʹ end of minus-strand DNA upon all subsequent steps of DNA synthesis, virus assembly and virions release. The RNA template is digested by the RNaseH activity present near the C-terminus of the polymerase, except of the 5' region, which serves as priming for the positive-strand synthesis [START_REF] Summers | Replication of the genome of a hepatitis B--like virus by reverse transcription of an RNA intermediate[END_REF].

Due to the location of DR1 within the terminal redundancy on the pregenome, the minus-strand DNA exhibits a short terminal redundancy region, which is crucial for rcDNA circularization (Figure 9). The plus-strand DNA synthesis is incomplete and reaches approximately 60% the genome in length, probably due to steric factors imposed by the nucleocapsid and the polymerase itself or a lack of incoming dNTP within the capsid [START_REF] Summers | Replication of the genome of a hepatitis B--like virus by reverse transcription of an RNA intermediate[END_REF][START_REF] Clark | Unveiling the roles of HBV polymerase for new antiviral strategies[END_REF]. The fate of freshly formed rcDNA containing capsids is dual. On the one hand, they can enter back to the nucleus, thereby amplifying the cccDNA pool, in what is called the "recycling loop". On the other hand, rcDNA containing capsids bud from intracellular membrane containing envelope proteins to form virions that are then secreted.

The budding process of mature rcDNA capsids throught the interaction with envelope proteins (L, M and S) is still a poorly understood mechanism. The N-terminal domain of L-HBsAg seems to play an important role in this interaction as M and S proteins alone cannot support translocation of capsids across membranes [START_REF] Halverscheid | Transgenic mice replicating hepatitis B virus but lacking expression of the major HBsAg[END_REF]. Reports provided evidence for a mechanism in which rcDNA containing capsids interact with envelope proteins and bud at MVB membranes [START_REF] Watanabe | Involvement of host cellular multivesicular body functions in hepatitis B virus budding[END_REF]. Indeed, the envelopment and secretion of Dane particles appear to be dependent on vacuolar protein sorting proteins AIP1 and VPS4B. The L-HBsAg binds to α-taxilin mediating thus a connection with tsg101 component of the ESCRT complex. Surprisingly, secretion of subviral particles occurs through the ER-Golgi compartment and do not depend on MVBs [START_REF] Patient | Hepatitis B virus subviral envelope particle morphogenesis and intracellular trafficking[END_REF]. However, as filaments contain a significant amount of L-HBs, some data suggest a possible release of these latters by MVBs [START_REF] Jiang | Subviral Hepatitis B Virus Filaments, like Infectious Viral Particles, Are Released via Multivesicular Bodies[END_REF].

A schematic HBV replication cycle highlighting the principal steps is available in

In vivo and in vitro models for HBV replication study

In this part, we will discuss in a non-exhaustive way, the different models used for the study of HBV. I decided to focus on historically important breakthroughs as well as on in vivo and in vitro systems that have been used during my PhD.

Natural infection models

As explained in the part "1.2. Classification and phylogeny of Hepadnaviridae", solely differentiated hepatocytes of few species are susceptible to a HBV infection followed by a complete replication cycle. Among them, higher primates, and in particular the chimpanzee has been employed historically to study virus transmission, host response and vaccination. Studies showed that these apes were protected from HBV infection by the vaccination comprised of HBsAg purified from virus carriers as well as from recombinant HBsAg produced in yeast. More recently, chimpanzees were used to show that vaccination was not protecting against some NA-escape HBV variants. Particularly, they helped confirming observations made in vaccinated infants who experienced breakthrough infections due to LAM resistance, which induce mutation in the overlapping S gene [START_REF] Kamili | Efficacy of hepatitis B vaccine against antiviral drug-resistant hepatitis B virus mutants in the chimpanzee model[END_REF]. Finally, in this model, studies have demonstrated the key roles of CD4 and CD8 T cells in the clearance of HBV infections and further documented lack of induction (or rapid suppression) of innate immune responses by the primary infection [START_REF] Thimme | CD8(+) T cells mediate viral clearance and disease pathogenesis during acute hepatitis B virus infection[END_REF]Wieland et al., 2004aWieland et al., , 2004b)). However, improvement in ethical domain as well as the need of highly specialized personal and expensive infrastructures led to decay in the use of those animals [START_REF] Dandri | Animal models of HBV infection[END_REF].

Other surrogate models, based on host-adapted viruses have been of importance for the study of HBV. Among them, Woodchuck infected by WHB was the first model suggesting causality between HBV infection and progression to HCC. Indeed, 100% of neonatally infected woodchucks developed liver cancer within 2 to 4 years. Strikingly, 20% of those that resolved the infection after neonatal inoculation were subjects to HCC, highlighting that HBV leaves a suitable landscape for cancer development, even after the resolution of the infection [START_REF] Korba | Hepatocellular carcinoma in woodchuck hepatitis virus-infected woodchucks: presence of viral DNA in tumor tissue from chronic carriers and animals serologically recovered from acute infections[END_REF].

From transgenic mice to humanized infectable mice

As mice are useful models in biology (inexpensive, easy-handling, rapid growth, tools at disposition …), several transgenic mice have been developed to study the HBV liver-specific pathogenesis, either by expression of the entire virus or by individual gene products [START_REF] Kim | HBx gene of hepatitis B virus induces liver cancer in transgenic mice[END_REF]. However, this unusual route of partial infection led to the introduction of bias and resulted in rapid resolution of HBV infection [START_REF] Iannacone | Mouse Models of Hepatitis B Virus Pathogenesis[END_REF]. About 25 years ago, Brinster and colleagues created transgenic mice that express the serine protease urokinase-type plasminogen activator (uPA) in hepatocytes. This allowed the repopulation of the murine hepatic liver-shape with human hepatocytes, rendering mice susceptible to HBV infection. Based also on repopulation of mice livers by human hepatocytes, the FRG model was developed. These mice are immune-deficient Fah knockout lacking the genes for Rag-2 and the common gamma chain of the IL receptor. These animals breed normally while on nitisinone (NTBC).

These FRG mice can be readily repopulated with human hepatocytes after pre-treatment with a vector expressing urokinase 1-3 days prior to transplantation (Azuma et al., 2007). The selective pressure to regenerate the liver is controllable in the FRG model but is lacking in the uPA/SCID animals. Indeed, as the mice breed normally under NTBC, no liver damages are observable, thus their handling is easier compared to uPA models. Utilization of these systems has been limited because of the technical challenge of acquiring and transplanting human hepatocytes, which is done by injection of hepatocytes into the spleen. However, improvement in techniques and handling led now to >95% of primary human hepatocytes (PHH) replenishment with low mortality rate and incoming double humanized-mice model (liver and immune system) is a trend on which research efforts are made [START_REF] Dusséaux | Viral Load Affects the Immune Response to HBV in Mice With Humanized Immune System and Liver[END_REF][START_REF] Kremsdorf | Modeling hepatitis virus infections and treatment strategies in humanized mice[END_REF].

In vitro models for the study of HBV

Historically, most of the studies on the HBV life cycle were performed in hepatoblastoma cell lines where HBV DNA was transfected, due to a lack of susceptibility towards HBV infection. In 2002, Gripon and colleagues developed and characterized a human hepatoma cell line infectable by HBV, called HepaRG (Gripon et al., 2002). Up to recently, this latter, with PHH cultures, were the only two in vitro models allowing studies of the HBV complete life cycle, from virions entry to release. However, the identification, in 2012, of the sodium-taurocholate cotransporting polypeptide (NTCP) as the main HBV receptor led to engineered cell line development, allowing proper infection by HBV (i.e., HepG2-NTCP, Huh7-NTCP) (Yan et al., 2012;Verrier et al., 2016b).

Chapter 2: From HBV-infected liver to hepatocellular carcinoma

Clinical manifestations of chronic HBV infections and pathogenesis

Natural history of the disease

Following acute infection, approximately 95% of adults and 5% to 10% of newborns recover, while the remainder evolve towards chronic viral infection. Not all patients with chronic HBV infection, which is characterized by the persistence of HBsAg for more than 6 months, have chronic hepatitis (CHB), which is evidenced by elevated ALT/ALAT, inflamation and fibrosis progression. The natural history of chronic HBV infection has been schematically divided into five phases, taking into account the presence of HBeAg, HBV DNA levels, alanine aminotransferase (ALT) values and eventually the grade of liver inflammation (Figure 16). Since 2017, guidelines from EASL recommend a new nomenclature based on the description of infection vs. hepatitis. However, the global HBV community is not united as one and the ancient nomenclature is presented here (EASL 2017 Clinical Practice Guidelines):

-Phase 1 "Immune tolerant": characterized by the presence of HBeAg in the serum, high viral load, no to low liver necroinflammation and normal ALT (<40 IU/L). The rate of spontaneous HBeAg loss is very low in this phase. These patients are highly contagious due to the high levels of HBV DNA. The high level of HBV DNA integration events and clonal hepatocyte expansion suggest that hepatocarcinogenesis could be already underway in this phase.

-Phase 2 "Immune (re)active": presence of serum HBeAg, high levels of HBV DNA and elevated ALT. The liver is moderately inflamed and subject to a rapid fibrosis progression. The outcome of this phase is variable. Most patients achieved HBeAg seroconversion and HBV DNA suppression and enter the HBeAg negative infection phase. Those patients who fail to control the infection progress to the HBeAg-negative CHB phase for many years.

-Phase 3 "HBeAg-negative chronic HBV infection": previously known as "inactive carrier" phase, is characterized by serum anti-HBe antibodies, low viral replication, HBV DNA <2,000 IU/mL and normal ALT. HBsAg loss and/or seroconversion may occur spontaneously in 1-3% of cases per year, typically in patients with HBsAg <1,000 IU/mL. Nevertheless, viral infection is not resolved, as cccDNA remains active in hepatocytes.

-Phase 4 "HBeAg-negative chronic hepatitis B": characterized by detectable anti-HBe in the serum and fluctuating moderate to high levels of serum HBV DNA and ALT levels. The liver shows fibrosis and necroinflammation. Most patients harbour HBV variants with precore and/or the basal core promoter (BCP) mutants, abolishing HBeAg production.

-Phase 5 "HBsAg-negative": also known as "occult infection". Serum negative HBsAg with or without anti-HBsAg and presence of anti-HBcAg. Normal ALT levels and undetectable HBV DNA. However, the cccDNA is frequently detected in the liver but could be silenced somehow [START_REF] Levrero | Control of cccDNA function in hepatitis B virus infection[END_REF]. If cirrhosis has developed before HBsAg loss, patients remain at risk of HCC, therefore the surveillance should continue (Figure 17).

Prevention and Control

Vaccines and other prevention strategies

The first generation of vaccines containing S alone were produced by processing of HBsAg purified from plasma of HBV carriers. Nowadays, vaccines are obtained from the yeast Saccharomyces Cerevisiae expressing S from recombinant DNA. In Europe a vaccine, containing both the S and M envelope proteins expressed in a Chinese hamster ovary cell line, is also available. This latter seems to result in lowering the frequency of nonresponders to the classical vaccine [START_REF] Holzer | Overexpression of hepatitis B virus surface antigens including the preS1 region in a serumfree Chinese hamster ovary cell line[END_REF]. These vaccines are administered by intramuscular injection in the deltoid region and are highly protective with an efficacy > 95% for children or young adults. About 5% of those vaccinated fails to respond with development of antibodies to the vaccine [START_REF] Hollinger | Hepatitis A and B vaccination and public health[END_REF][START_REF] Zanetti | The global impact of vaccination against hepatitis B: a historical overview[END_REF]. The protection last for more than 10 years and although anti-HBs antibodies titer may decline to undetectable levels, the immunity persists, indicating the existence of an immunologic memory [START_REF] Osiowy | From infancy and beyond… ensuring a lifetime of hepatitis B virus (HBV) vaccineinduced immunity[END_REF]. An universal vaccination program has been recommended by the WHO in order to eradicate HBV by 2030. As example, the implementation of such a program in Taiwan resulted in a loss of HBsAg prevalence: from 9.8% in 1988 to 1.3% in 1994 in children [START_REF] Chen | Seroepidemiology of hepatitis B virus infection in children: Ten years of mass vaccination in Taiwan[END_REF][START_REF] Ni | Hepatitis B vaccination in children: the Taiwan experience[END_REF]. In Italy, the same mass vaccination program was implemented with similar results [START_REF] Tosti | Acute Hepatitis B After the Implementation of Universal Vaccination in Italy: Results From 22 Years of Surveillance (1993-2014)[END_REF]. In France, since January 1 st of 2018, the vaccination is mandatory again for <18 months child, due to resurgence of HBV, imputable to population movements and migration (LOI n°2017-1836 Art.L.3111-2.-I.) [START_REF] Coppola | Hepatitis B virus, hepatitis C virus and human immunodeficiency virus infection in undocumented migrants and refugees in southern Italy, January 2012 to[END_REF].

Furthermore, vaccination campaigns have not only decreased the incidence of HBV infection and the prevalence of chronic carriers, but also the incidence of HCC, thereby making HBV vaccine the first to have proven efficacy in cancer prevention [START_REF] Stroffolini | The impact of the hepatitis B mass immunisation campaign on the incidence and risk factors of acute hepatitis B in Italy[END_REF].

Eventually, other prevention strategies, such as screening the blood products, proper sterilization of medical instruments, antenatal screening of pregnant women and avoidance of risky behaviours may preclude HBV transmission. General education should be advocated in addition to vaccination as in highly endemic regions, the knowledge awareness among the healthy population is not satisfying yet [START_REF] Yasobant | Knowledge of hepatitis B among healthy population: A community-based survey from two districts of Gujarat, India[END_REF].

Antiviral therapies

The main goal of therapy is to improve survival and quality of life by preventing disease progression and subsequent HCC development. The suppression of HBV DNA to undetectable levels and normalisation of ALT levels as well as HBeAg seroconversion are considered as clinical endpoints of therapy. The loss of HBsAg is perceived as the optimal treatment endpoint, termed "functional cure", but is only rarely achieved with the current antiviral arsenal and should be an overall objective for HBV eradication.

Currently, there are two main therapeutical options for CHB patients: treatment with a NA or with IFN-α. This latter has been used since the 1970s [START_REF] Greenberg | Effect of human leukocyte interferon on hepatitis B virus infection in patients with chronic active hepatitis[END_REF]. Although IFN-α has a direct antiviral effect, suppressing HBV replication, its major effect is considered immunologic.

Nowadays, IFN-α is used under a pegylated form, thus increasing its stability [START_REF] Craxi | Pegylated interferons for chronic hepatitis B[END_REF]. However, this antiviral strategy tends to lose fields toward more specific and well-tolerated NAs. In Europe, six NAs have been approved for the treatment of HBV infection, classified into:

-Low barrier against HBV resistance: Lamivudine (LAM), Adefovir dipivoxil (ADV) and Telbivudine (TBV).

-High barrier to HBV resistance: Entecavir (ETV), Tenofovir disoproxil fumarate (TDF) and Tenofovir alafenamide fumarate (TAF).

The main advantage of treatment with a NA exhibiting a high barrier to resistance is its predictable long-term antiviral efficacy as well as its favourable safety profile [START_REF] Lok | Antiviral therapy for chronic hepatitis B viral infection in adults: A systematic review and meta-analysis[END_REF][START_REF] Terrault | and American Association for the Study of Liver Diseases[END_REF]. Currently, management of HBV infection with antiviral therapy is recommended only for patients with active liver disease and not for patients that do not have elevated ALTs and/or evidence of inflammatory liver disease. This has been mediated by the fact that NAs at disposition, until recently, exhibited a low barrier resistance (i.e., LAM). The higher barrier to entecavir and tenofovir resistance may lead to a change in treatment guidelines (Table III).

However, a long life treatment is mandatory to prevent HBV reactivation upon treatment arrest, as NAs are ineffective on supressing the cccDNA. CccDNA loss under NAs therapies is believed to reflect the death of infected hepatocytes rather than intracellular instability, at least in the context of chronic infections. NAs, which act by inhibiting HBV DNA synthesis, thus indirectly lower the amount of cccDNA, as some infected hepatocytes die and others proliferate to maintain the overall liver cell population [START_REF] Summers | Hepadnavirus envelope proteins regulate covalently closed circular DNA amplification[END_REF][START_REF] Fourel | Evidence that hepatocyte turnover is required for rapid clearance of duck hepatitis B virus during antiviral therapy of chronically infected ducks[END_REF][START_REF] Zhu | Kinetics of hepadnavirus loss from the liver during inhibition of viral DNA synthesis[END_REF].

TDF 0 0 0 0 0 TAF 0 0 - - -

Future insights in anti-HBV drug development

The currently approved therapies exhibit an overall satisfactory antiviral effect by decreasing the HBV DNA to undetectable levels, good HBeAg seroconversion and limited side-effects. The issue concerning resistance to NAs (up to 80% of patients treated for 5 years with lamiduvine) is partially solved with the availability of innovative, high barrier resistance molecules such as ETV, TDF and TAF.

Nowadays, the life-long treatment need and low HBsAg seroconversion are the two remaining problems and need to be addressed by the scientific/medical HBV community.

In this respect, the step forward in anti-HBV drug development is focused on targeting the cccDNA, by preventing its formation or promoting its degradation/silencing. Another trendy way to moving forward antiviral strategy is to focus on the HBc protein, which is involved in several HBV life cycle steps, as detailed in the review I have written (confer Appendix review (Diab et al., 2018)).

Regarding pre-clinical efforts, you can refer to the study I get involved in, that deciphers the anti-HBV activities of two CpAMs being trialed by the Janssen pharmaceutical group (confer Appendix paper Lahlali et al., 2018)).

An innovative approach resides in the use of siRNA, targeting either HBV directly or proviral host factors. In this regard, the compound ARB-1467 from Arbtutus Biopharma, comprises three siRNA triggers that target all four HBV transcripts and has been shown in preclinical studies to reduce all viral antigen levels as well as HBV DNA. Furthermore, these siRNA utilizes Arbutus' proprietary lipid nanoparticle (LNP) platform to improve proper delivery. The ARB-1467 is currently under phase 2a clinical trial in subjects with CHB infection receiving nucleos(t)ide analogue therapy (NCT02631096). In my second study, we took advantage of the LNP technology to deliver siRNA targeting PLK1, a host kinase considered as a HBV proviral factor (confer Second study: Antiviral activity of PLK1-targeting siRNA delivered by lipid nanoparticles in HBV-infected hepatocytes).

Finally, other anti-HBV strategies such as restoring immunity (i.e., TLR-7/8 agonists, reverse T-cell exhaustion), targeting proviral host factors (i.e., HTA), genomic approaches (siRNA or CRISPR/Cas9) and RNA destabilizers are under early-drug development and could reinforce the anti-HBV arsenal, allowing a more personalized medicine [START_REF] Gane | Future anti-HBV strategies[END_REF][START_REF] Gilani | The implication of CRISPR/Cas9 genome editing technology in combating human oncoviruses[END_REF]. The management of HBV infection should come from either a breakthrough innovation or more probably from combining already approved NAs with new molecules.

According to the WHO and following model previsions, the incidence of chronic infections should reduce by 90% and mortality by 65% (compared to 2015), which would eliminate hepatitis B as public health threat by 2030.

However, as mentioned earlier, available treatments are limited and the cccDNA elimination seems far to be reachable. Although potent inhibition of viral replication could be completed, the risk of developing HCC is still a major threat. Moreover, we still do not know how to manage HBV integration events, which account also for HCC development.

Thesis Adrien FOCA 54 2.3. HBV-induced HCC: a threat to global health

Generalities

HCC management is a global challenge for the WHO, with 800,000 deaths/year worldwide (Figure 18). Among them, >340,000 are related to HBV etiology with an increasing incidence in developing countries. HCC is currently the main concern for CHB patients with up to a 100 fold increased risk of developing liver cancer compared to general population. Of the 257 million people chronically infected, <5% are estimated to be efficiently treated [START_REF] Rapti | Risk for hepatocellular carcinoma in the course of chronic hepatitis B virus infection and the protective effect of therapy with nucleos(t)ide analogues[END_REF]. The first line of defense towards HBV-induced HCC is thereby improvements in prevention/diagnosis and proper access to antiviral therapies; especially in HBV highly-endemic regions.

However, patients who have been treated with appropriate NAs have still a risk of developing HCC of 5 fold the normal (El-Serag, 2012). This latter point highlights the need to put efforts in fundamental research in order to better understand HBV biology (i.e., role of cccDNA and integarated DNA, host/virus interactions, long-lasting immunity modulation…). Thus, the ultimate goals in the treatment of chronic HBV infection are to suppress viral replication, eliminate the cccDNA and in fine prevent the life-limiting outcomes of liver failure and liver cancer.

Epidemiology

Soon after HBV discovery, the scientific community aimed for a potential link between HBV and HCC. In a cohort of 17 chronically infected patients, Sherlock and colleagues found five HCC positives, which lead the HBV community to focus further on a possible link between the virus and liver carcinogenesis. HCC develops rarely during the first 4 decades of life, except in HBV hyper endemic regions, bringing a new clue in the HBV-HCC correlation (El-Serag, 2012). Definitive support in favor to the hypothesis that HBV plays a role in HCC development came from epidemiologic studies. Particularly, a prospective study on >22,000 Taiwanese men has revealed that HBsAg positive individuals have a relative lifetime risk of developing HCC multiplied by 20 compared to uninfected [START_REF] Beasley | Hepatocellular carcinoma and hepatitis B virus. A prospective study of 22 707 men in Taiwan[END_REF]. Finally, the strongest evidence was found in WHV-infected woodchucks, where, as explained before, 100% of individuals develop HCC within 3 to 4 years postinfection [START_REF] Korba | Hepatocellular carcinoma in woodchuck hepatitis virus-infected woodchucks: presence of viral DNA in tumor tissue from chronic carriers and animals serologically recovered from acute infections[END_REF].

Most of HBV-infected patients who develop HCC have cirrhosis (70-90% of HBV-induced HCC)

secondary to the necroinflammation. The seroprevalence of HBsAg among patients with HCC varies globally with 3% in Sweden, 55% in Greece and can reach up to 70% in South Korea. In accordance to several studies, the HCC incidence rate is 0.02 per 100 person-years in inactive carriers, 0.3 in subjects with chronic HBV without cirrhosis, and 2.2 in subjects with cirrhosis (El-Serag, 2012).

Several factors have been reported to increase the HCC risk among HBV-infected patients such as demographic ones, viral (HBV genotypes, replication level and co-infection) and environmental (alcohol, tobacco and exposure to aflatoxin). It is well known that men have an overall increased risk for HCC development (sex ratio maleto-female up to 5), which is partially explained by the higher exposure to alcohol and viral hepatitis (Liu et al., 2017)(Figure 19). However, the risk remains increased when these two confounders are adjusted and men have an increased risk for cirrhosis and subsequent HCC, especially in HBVinfected group.

Moreover, the viral load is usually higher in men. A study focusing on >18,000 Taiwanese and Shanghainese HBV-carriers has shown that high serum level of testosterone is associated with HCC, suggesting an involvement of androgens in both viral replication intensity and carcinogenesis [START_REF] Yuan | A cohort study of serum testosterone and hepatocellular carcinoma in Shanghai, China[END_REF]. In transgenic mice model, studies have shown that HBV can increase the transcription of androgen receptors through the HBx protein and conversely, androgens bind directly the viral genome, leading to higher HBV transcription [START_REF] Chiu | Hepatitis B virus X protein enhances androgen receptor-responsive gene expression depending on androgen level[END_REF][START_REF] Yeh | Gender disparity of hepatocellular carcinoma: the roles of sex hormones[END_REF].

Finally, some papers reported that oestrogen can be favourable and protects against HBV infection progression. On the one hand, estradiol has been described as a potent antioxidant, which suppresses hepatic fibrosis and decreases induction of redox sensitive transcription factors, hepatocyte apoptosis and hepatic stem cells activation by inhibiting reactive oxygen species production in primary cultures [START_REF] Shimizu | Female hepatology: Favorable role of estrogen in chronic liver disease with hepatitis B virus infection[END_REF]. On the other hand, the frequency of NK cells in the liver of chronically HBV-infected females was higher than in males and correlated with plasma level of estradiol [START_REF] Macek Jilkova | Sex Differences in Spontaneous Degranulation Activity of Intrahepatic Natural Killer Cells during Chronic Hepatitis B: Association with Estradiol Levels[END_REF]. These two latter points could partially explain the ability of females to better deal with HBV disease.

The impact of HBV genotypes on HCC development

As largely developed in the section "1.2. Classification and phylogeny of Hepadnaviridae", due to genetic variability, the human HBV is further classified in eight (A to H) distinct, wellestablished genotypes. These latter seem to affect both natural course of HBV infection and clinical outcomes.

Genotypes B and C are more common in Asian population and studies have shown that HBV genotype C was strongly associated with progression of chronic infection to severe liver diseases, such as cirrhosis and HCC. In a meta-analysis investigating the correlation between HBV genotypes and HCC, HCC detection was 12% for genotype B and 25% for genotype C, with an odd ratio of 2.05 [START_REF] Wong | Meta-analysis: The association of hepatitis B virus genotypes and hepatocellular carcinoma[END_REF].

However, a study from Taiwan demonstrated that genotype B led more rapidly to HCC development, with most of patients under 50 years old and no evidence of cirrhosis [START_REF] Kao | Basal core promoter mutations of hepatitis B virus increase the risk of hepatocellular carcinoma in hepatitis B carriers[END_REF]. Another Taiwanese study, focusing on children HBV carriers highlighted that genotype B was involved in 74% of HBV-related HCC [START_REF] Ni | Clinical relevance of hepatitis B virus genotype in children with chronic infection and hepatocellular carcinoma[END_REF].

These different clinical outcomes depending on HBV genotypes are mostly due to sub types variations or well-identified mutations in the HBV genome. Focusing on the BCP, T1762 and A1764 are known to increase HCC incidence while mutations found in the precore region such as G1896A are associated with a decreased risk of HCC [START_REF] Liu | Role of hepatitis B viral load and basal core promoter mutation in hepatocellular carcinoma in hepatitis B carriers[END_REF][START_REF] Yang | Associations between hepatitis B virus genotype and mutants and the risk of hepatocellular carcinoma[END_REF]. A study from China, conducted on HBeAg-positive patients infected with the C1 sub-genotype, reported that, in addition to T1762/A1764 BCP mutations, V1753 and A1768 mutations were closely correlated with HCC.

Moreover, the increased risk of HCC mediated by these BCP mutations could be partially based on modifications of HBx biological functions [START_REF] Li | Accumulation of the mutations in basal core promoter of hepatitis B virus subgenotype C1 increase the risk of hepatocellular carcinoma in Southern China[END_REF].

Furthermore, antiviral drug-selected mutants such as the hepatitis B virus rtA181T/sW172* which has a dominant negative secretion defect of HBsAg and alters the profile of viral rebound, may lead to missed diagnosis if viral load is used as the only criterion for drug failure. Thus, implementing HBV polymerase chain reaction sequencing is mandatory to detect these drug-selected mutant, in order to prevent undetected HBV infection and subsequent risk for HCC progression [START_REF] Warner | The antiviral drug selected hepatitis B virus rtA181T/sW172* mutant has a dominant negative secretion defect and alters the typical profile of viral rebound[END_REF].

Finally, a recent study published in Hepatology identified a specific mutation in the core region of HBV genotype F1b within an Alaskan native population. The A2051C mutant led to upregulation of proliferation and/or carcinogenesis related-genes such as MYC, GAB2, BDKRB2, FST and MAP3K8 in human hepatocytes infected with genotype F1b. Moreover, this mutation was associated with better viral replication and an enhanced stability of HBc dimerization. This specific HBV genotype is then associated with quicker progression to fibrosis and HCC than other genotypes [START_REF] Hayashi | A novel association between core mutations in hepatitis B virus genotype F1b and hepatocellular carcinoma in Alaskan Native People[END_REF](Figure 20).

Genetic factors

Several genetic factors have been associated with an increased risk of HBV-related HCC.

Amon them, the p53 mutant R249S is the most widely described and believed to directly cooperate with HBx for HCC development, in a pathway that bypasses cirrhosis [START_REF] Ortiz-Cuaran | Association between HBX status, aflatoxin-induced R249S TP53 mutation and risk of hepatocellular carcinoma in a case-control study from Thailand[END_REF]. This p53 specific mutant is found to be induced mainly by aflatoxin exposure and its involvement in liver carcinogenesis is discussed in a further extent in the following section "2.3.3.4. Aflatoxin B1 and HBV: the two-headed monster".

Regarding TNF-, IL-23R, CD3G, MiR-106b-25 cluster, non-classical HLA, IL-18, and CTLA-4, specific SNP alleles have been described to increase the HBV-related HCC risk albeit the mechanisms have still to be determined [START_REF] Xu | Hepatitis B virus-induced hepatocellular carcinoma[END_REF].

Recently, new tools such as genome-wide association studies (GWAS) helped to identify five independent loci that are significantly associated with HBV-related HCC: rs17401966 at KIF1B, rs9272105 at HLA-DQA1/DRB1, rs455804 at GRIK1, rs7574865 at STAT4, and rs9275319 at HLA-DQ [START_REF] Zhang | Genome-wide association study identifies 1p36.22 as a new susceptibility locus for hepatocellular carcinoma in chronic hepatitis B virus carriers[END_REF]Li et al., 2012;[START_REF] Jiang | Genetic variants in STAT4 and HLA-DQ genes confer risk of hepatitis B virus-related hepatocellular carcinoma[END_REF]. However, these studies were Asia-centric and descriptive thus, the biological process underlying is yet to be deciphered. Moreover, the role of these loci in carcinogenesis may be controversial, as the rs17401966 at KIF1B was not found to be associated with HBV-related HCC development in Thaiwanese patients [START_REF] Sopipong | The KIF1B (rs17401966) single nucleotide polymorphism is not associated with the development of HBV-related hepatocellular carcinoma in Thai patients[END_REF].

Interestingly, familial predisposition as a risk factor for HBV-related HCC has been suggested.

In a study published in 2000 by Yu and colleagues, the HCC risk associated with having parents and/or siblings with HCC was evaluated in 4808 male HBV-positive patients. The main conclusion was that first-degree relatives of patients with HBV-related HCC appear to be at increased risk for liver cancer development [START_REF] Yu | Familial risk of hepatocellular carcinoma among chronic hepatitis B carriers and their relatives[END_REF]. Shared environment, habits but also genetic background are regrouped under this "familial predisposition" term. Regarding genetics, a GWAS has identified GLUL and SLC13A2/FoxN1 haplotypes for familial HBV-related HCC. To date, this is the sole genome-wide association study to identify genetic factors for familial HBV-related HCC, which differ from sporadic HCC [START_REF] Lin | Genomewide association analysis identifies a GLUL haplotype for familial hepatitis B virus-related hepatocellular carcinoma[END_REF].

Aflatoxin B1 and HBV: the two-headed monster

Aflatoxin B1 (AFB1) is a Mycotoxin produced by A. flavus and A. parasiticus fungi and is mostly found in contaminated food, due to warm and damp storage conditions of corn and peanuts. AFB1 is a powerful hepatocarcinogen, leading the International Agency for Research on Cancer to classify it as a class-1 carcinogen (Overall evaluations of carcinogenicity IARC). Once ingested, AFB1 is metabolized within the hepatocytes in AFB1-exo-8,9-epoxide, which can bind to and damage DNA, leading especially in a mutation at the serine codon 249 of the tumour suppressor p53. This specific event is detected in ≈50% of HCC from aflatoxin-endemic areas patients, most of whom had HBV infections [START_REF] Bressac | Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa[END_REF][START_REF] Turner | The role of aflatoxins and hepatitis viruses in the etiopathogenesis of hepatocellular carcinoma: A basis for primary prevention in Guinea-Conakry, West Africa[END_REF]. Areas where aflatoxin is a recurrent environmental problem correlate with highly endemic HBV regions (Figure 21).

There is evidence from both epidemiological studies and animal models that the two factors can act synergistically to increase the HCC risk, but the underlying mechanisms of interaction are yet undefined. As example, studies in China have shown that AFB1 increased the risk of HCC by 4 fold and HBV alone by 7 fold. However, when the two factors are presents, the carriers had as much as a 60fold increase in risk of HCC [START_REF] Qian | A follow-up study of urinary markers of aflatoxin exposure and liver cancer risk in Shanghai, People's Republic of China[END_REF]. Thereby, the prevention of HBV-induced HCC by establishment of vaccination programs in highly endemic areas would reduce the effects of AFB1 on liver carcinogenesis risk (El-Serag, 2012).

Regarding the mechanism involved, albeit being not well deciphered yet, it was assumed that most of the pathogenesis was imputable to the R249S p53 mutant. Indeed, R249S has lost ability to bind p53 response elements and to transactivate downstream genes, in fine increasing proliferation and clonogenic properties in HCC. The mutant p53 R249S was found to form a complex with HBx in PLC/PRF/5 cells, but the interplay between these two factors is still unprecised [START_REF] Gouas | Effects of the TP53 p.R249S mutant on proliferation and clonogenic properties in human hepatocellular carcinoma cell lines: interaction with hepatitis B virus X protein[END_REF].

In 2010, Jiang and colleagues has demonstrated that HBx variants may cooperate with p53 R249S mutant, inducing aneuploidy, anchorage-independent growth and proliferation in a telomeraseimmortalized normal human hepatocyte-derived cell line (Jiang et al., 2010).

Thesis Adrien FOCA 60

However, the increased risk of HCC found in HBV-carriers and patients exposed to AFB1 concomitantly, is not only driven by the p53 axis. Recently, the autophagy-related protein p62 has been pointed out as a possible prognosis factor for HCC in AFB1-HBV population. Indeed, p62 expression was found to be correlated with high NRF2 and Nqo1, promoting tumour recurrence.

Finally, this pathway downregulates AKR7A3, which is mandatory for liver detoxification of aflatoxin B1 [START_REF] Xiang | Expression of P62 in hepatocellular carcinoma involving hepatitis B virus infection and aflatoxin B1 exposure[END_REF].

From a fundamentalist point of view, meta-analysis of correlation between AFB1 and HBV genotypes in HCC risk would be usefull to further decipher the underlying mechanism involved. Aristolochic acids are known to be carcinogenic, mutagenic and nephrotoxic phytochemicals since the early 2010s [START_REF] Shaw | Toxicological risks of Chinese herbs[END_REF]. They are commonly found in birthwort plants which are used in an extensive manner in Chinese traditional medicine. Recently, it was suspected that aristolochic acid-induced mutations may be associated with HBV to fasten the development of HCC. A retrospective, population-based (>800,000 individuals) study conducted in Taiwan by Chen and colleagues highlighted a significant dose-response relationship between the consumption of aristolochic acid and HCC in patients with HBV infection [START_REF] Chen | Herbal medicine containing aristolochic acid and the risk of hepatocellular carcinoma in patients with hepatitis B virus infection[END_REF]. Although the use of herbal medicine containing aristolochic acid has been largely banned worldwide, the community highlights the need of a close follow-up of HBV positive patients who have taken those herbs.

Mechanisms of HBV-induced HCC

The pathogenesis of HBV-induced HCC is a multifactorial process with still elusive mechanisms. However, the field has been driven by two hypotheses; on one hand, the virus is thought to act directly on carcinogenesis while on the other hand, HCC formation might be a consequence of chronic necroinflammation due to the immune response, unveiling an indirect role of HBV. Actually, both contribute to HCC development.

HBV direct-associated hepatocarcinogenesis

HBV dslDNA integration: the cis-effect

A significant fraction of virions (≈10%) contains dslDNA as incoming genomic material, instead of rcDNA. These entities can both give rise to cccDNA from being converted by cellular

enzymes, yet, dslDNA can also integrate into the host cell genome at double-strand DNA breaks by non-homologous end joining (NHEJ) [START_REF] Bill | Genomic DNA double-strand breaks are targets for hepadnaviral DNA integration[END_REF][START_REF] Tu | Hepatitis B virus DNA integration occurs early in the viral life cycle in an in vitro infection model via NTCP-dependent uptake of enveloped virus particles[END_REF](Figure 22). Although the reimport of dslDNA-containing nucleocapsids occurs under HBV replication, it appears to play minor role in HBV integration events. Interestingly, complete HBV genome integration virtually never occurs, thus, in most of the cases, these inserts cannot serve as template for viral replication.

Up to 85% of HCC from HBV etiology contains HBV integrated DNA and through clinical progression of chronic hepatitis B, dslDNA virions fraction increases and can reach up to 35%, suggesting a correlation between these two factors [START_REF] Bréchot | Molecular bases for the development of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC)[END_REF]. Integration events are observed in all known hepadnaviruses, including woodchuck, ducks, as well as in chimpanzees and humans [START_REF] Yang | Integration of hepadnavirus DNA in infected liver: evidence for a linear precursor[END_REF][START_REF] Summers | Residual integrated viral DNA after hepadnavirus clearance by nucleoside analog therapy[END_REF][START_REF] Mason | Detection of clonally expanded hepatocytes in chimpanzees with chronic hepatitis B virus infection[END_REF][START_REF] Mason | HBV DNA Integration and Clonal Hepatocyte Expansion in Chronic Hepatitis B Patients Considered Immune Tolerant[END_REF]. Since hepatocytes are the only liver cells clearly susceptible to HBV infection/replication, it was hypothesised that HCC arise from dedifferentiation of hepatocytes. The first evidence for a direct role of HBV in the liver carcinogenesis was fuelled by the observation of clonal HCC tumours with respect to the viral integration site [START_REF] Esumi | Clonal origin of human hepatoma determined by integration of hepatitis B virus DNA[END_REF].

Nowadays, integration of HBV DNA, also named the cis-effect, is one of the reported drivers of HCC development by inducing pro-carcinogenic pathways. Among them, chromosomal instability, insertional mutagenesis in key cancer-associated genes, transcription of downstream cancerassociated cellular genes, and formation of a persistent source of viral protein expression were reported [START_REF] Budzinska | Cellular Genomic Sites of Hepatitis B Virus DNA Integration[END_REF]. However, the contribution of each of these mechanisms is still unclear, as no sequence consensus integration sites have been found. HBV seems to be able to integrate widely into the host genome, but occurrence in chromosomes 13, X and Y appeared to be less frequent [START_REF] Murakami | Large scaled analysis of hepatitis B virus (HBV) DNA integration in HBV related hepatocellular carcinomas[END_REF]Jiang et al., 2012a). Finally, the number of HBV integrations is associated with lower patient survival as it maximizes the risk of insertion within critical genes.

Nevertheless, several hot point sites of HBV DNA integration into the host genome have been reported. Indeed, large-scale analysis using the Alu-PCR approach revealed that integration events occur frequently in actively transcripted regions. Recurrent HBV insertions nearby the putative cancer-related TERT, the epigenetic regulator MLL4 and the cell-cycle CCNE1 genes have been highlighted [START_REF] Paterlini-Bréchot | Hepatitis B virus-related insertional mutagenesis occurs frequently in human liver cancers and recurrently targets human telomerase gene[END_REF][START_REF] Murakami | Large scaled analysis of hepatitis B virus (HBV) DNA integration in HBV related hepatocellular carcinomas[END_REF][START_REF] Sung | Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma[END_REF](Figure 23).

Regarding the rate of integration in different stages of infection, a recent study has shown that HBV insertion in human genome is an event frequently found in mild phases of chronic hepatitis.

Using Alu-PCR followed by a downstream Sanger sequencing of the amplicons, they found integration events in 39/74 biopsies (52%), which is far below the 85-90% expected. However, the use of a single primer spawning a human-HBV region could have missed several insertional events [START_REF] Larsson | Integration of hepatitis B virus DNA in chronically infected patients assessed by Alu-PCR[END_REF].

Moreover, HBV integration has been associated with important genomic alterations, such as chromosomal translocations, inverted duplication events and large deletions [START_REF] Yaginuma | Hepatitis B virus integration in hepatocellular carcinoma DNA: duplication of cellular flanking sequences at the integration site[END_REF][START_REF] Hino | Hepatitis B virus integration site in hepatocellular carcinoma at chromosome 17;18 translocation[END_REF][START_REF] Tokino | Chromosomal translocation and inverted duplication associated with integrated hepatitis B virus in hepatocellular carcinomas[END_REF]. Some of these latter could explain the relatively rare occurrence of HCC on non-cirrhotic liver.

At last, HBV integration can act in trans on carcinogenesis. Although integrated viral sequences are defective for replication, it can result on HBx and/or HBs mutants' production, which is described to disrupt and redirect cellular functions, leading to HCC development. Indeed, several studies have shown than >40% of the integration breakpoints on the HBV genome occurred within a 1,800bp range, where the regulatory DR1 region is located and near the 3' end of HBx gene, leading to virus-human fusion protein and/or mutated HBx production (Jiang et al., 2012b;[START_REF] Sung | Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma[END_REF](Figure 24). In this regard, a study published in 2015 by Quetier and colleagues reported that Cterminal-truncated HBx proteins (Ct-HBx) production that is due to HBV integration is a potent cell transforming factor. In mice treated with DEN in order to initiate the carcinogenesis, Ct-HBx expression was correlated with higher incidence and number of liver lesions and associated with 63 inflammation. Thus, they speculated that truncated HBx may therefore facilitate HCC development in chronically infected patients [START_REF] Quetier | C-terminal-truncated hepatitis B virus X protein enhances the development of diethylnitrosamineinduced hepatocellular carcinogenesis[END_REF]. This second HBV direct-associated hepatocarcinogenesis, called the trans-effect, highlights the involvement of viral gene products as potential onco-proteins. Although HCC development occurs decades after chronic infection, indicating that HBV cannot transform hepatocytes in a single hit event, the hypothesis gained momentum with the discovery that transgenic mice expressing the entire coding sequence of HBx developed HCC [START_REF] Kim | HBx gene of hepatitis B virus induces liver cancer in transgenic mice[END_REF]. From this study, it appears that HCC development under HBx overexpression was mostly due to the selection of the mouse strain, CD1, known to develop spontaneous hepatomas at an increased rate. Thus, HBx was described to promote but not necessarily initiate carcinogenesis. Supporting this hypothesis, in another background, HBx transgenic mice treated with diethylnitrosamine (highly mutagenic/oncogenic compound) were more susceptible to HCC than nontransgenic individuals were [START_REF] Slagle | Increased sensitivity to the hepatocarcinogen diethylnitrosamine in transgenic mice carrying the hepatitis B virus X gene[END_REF].

Furthermore, in 2003, a study has shown a collaborative influence of HBx and c-Myc that result in HCC development after a prolonged latent period [START_REF] Lakhtakia | Hepatocellular carcinoma in a hepatitis B "x" transgenic mouse model: A sequential pathological evaluation[END_REF]. Finally, several cell-culture based studies have demonstrated effects of HBx on proliferation and cell viability, strengthening observations made in vivo [START_REF] Gearhart | The hepatitis B virus HBx protein modulates cell-cycle regulatory proteins in cultured primary human hepatocytes[END_REF]Zhang et al., 2015;[START_REF] Fan | Hepatitis B virus X protein induces EpCAM expression via active DNA demethylation directed by RelA in complex with EZH2 and TET2[END_REF]Wang et al., 2017b).

However, conflicting results emerged from these studies, describing opposite effects and suggesting that HBx could be cytostatic, or even induces apoptosis [START_REF] Schuster | Induction of apoptosis by the transactivating domains of the hepatitis B virus X gene leads to suppression of oncogenic transformation of primary rat embryo fibroblasts[END_REF]. The main caveat regarding these studies is the use of irrelevant systems, where HBx is highly expressed while in chronically infected liver, its level is barely detectable [START_REF] Su | Expression of hepatitis B virus X protein in HBV-infected human livers and hepatocellular carcinomas[END_REF][START_REF] Neuveut | Mechanisms of HBV-related hepatocarcinogenesis[END_REF]. Thus, the expression of HBx protein was described to potentiate the induction of liver disease in a long run manner, thereby classifying HBx as a weak oncogene.

Focusing on the underlying mechanism of HBx-induced HCC, albeit being not precisely welldefined, it is believed to involve multifunctional aspects of HBx, acting in a timely fashion in order to support HCC initiation and/or development. HBx is able to directly sequester p53, leading to p53mediated apoptosis inhibition, which is thought to be one of the main mechanisms responsible for HBx-induced hepatocarcinogenesis (Chan et al., 2016). Another major axis is the CREB/ATF pathway activation, which is involved in liver metabolism, cell proliferation and hepatocarcinogenesis [START_REF] Abramovitch | A pivotal role of cyclic AMP-responsive element binding protein in tumor progression[END_REF]. In HBx-transfected cells, a study from Cougot and colleagues has shown that HBx interacts with CBP/p300, playing a decisive role in the downstream CREB-dependent transcription. In this model, HBx seems to increase CREB/ATF DNA-binding affinity as well as to enhance the recruitment of CBP/p300 to CREB/ATF bound to cellular DNA. Furthermore, a microarray-based screen for HBx target genes allowed the authors to unveiled activation of two CREB-responsive genes; IL-8 and PCNA in primary human hepatocytes and confirmed their results in HepaD38, where the level of HBx is significantly increased after tetracycline removal [START_REF] Cougot | The hepatitis B virus X protein functionally interacts with CREB-binding protein/p300 in the regulation of CREB-mediated transcription[END_REF]. As CBP/p300 recruitment represent the limiting event for activating CREB target genes, HBx could overwhelm this issue and therefore predispose cells to transformation.

HBx was also found to interact with DNA repair protein such as DDB1, however studies tend to conclude that HBx regulates the function of DDB1 in both positive and negative manners, thus playing different roles in viral replication and carcinogenesis. In 2016, Decorsière and colleagues uncover a novel role for the Smc5/6 complex, which can act as a restriction factor blocking specifically extrachromosomal DNA transcription. HBx, by hijacking the DDB1-containing E3 ubiquitin ligase complex to target Smc5/6, promotes the cccDNA transcription, and therefore contributes to HCC development [START_REF] Decorsière | Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor[END_REF].

The group of Dr Andrisani has been largely involved in the understanding of HBx-induced transformation. Notably, they have shown that HBx and PLK1 where inherently linked and playing crucial functions in oncogenic transformation, mostly by echancing the degradation of repressing factors involved in the inhibition of cellular proliferation. This axis is extensively developed in the section "3.3.9. PLK1 as a keystone in virus-induced oncogenesis? The example of HBV-related HCC".

At last, HBx has been shown to inactivate BubR1, an effector of several kinases, including PLK1, involved in centrosome dynamics and mitotic spindle formation. Several studies have linked HBx expression to DNA re-replication, leading to partial polyploidy, which is highly associated with carcinogenesis [START_REF] Andrisani | The transcriptional function of the hepatitis B virus X protein and its role in hepatocarcinogenesis (Review)[END_REF][START_REF] Rakotomalala | Hepatitis B virus X protein increases the Cdt1-to-geminin ratio inducing DNA re-replication and polyploidy[END_REF]Studach et al., 2009a).

In summary, despite a wealth of information about biological activities of HBx, it is still hard to claim that HBx is involved as a strong oncogene in HCC development.

Interestingly, HBs has also been suggested as a potential oncogene, yet, to a lesser extent than HBx. Notably, overexpression of M truncated protein in transgenic mice induced the activation of the c-Raf/Erk2 pathway, leading in fine to an increased hepatocyte proliferation. After 1-2 years, these mice developed small liver tumours [START_REF] Hildt | The PreS2 activator MHBs(t) of hepatitis B virus activates c-raf-1/Erk2 signaling in transgenic mice[END_REF]. However, these observations are still on debate regarding their relevance and transposition to human carcinogenesis.

In addition, the intact L protein also has been shown to transactivate viral and host genes.

Recently, a study published in The Journal of Pathology by Li et al., has shown that L-HBsAg is involved in HCC development through induction of cytokinesis failure, leading to aneuploidy in hepatocytes. This phenotype appears to be mediated via DNA damage and G2/M checkpoint override. Interestingly the authors have shown that this mechanism was supported by a PLK1 upregulation and that inhibiting PLK1 resulted in G2/M checkpoint restauration and in a decrease of tumour formation incidence, tumour volume and tumour weight (Li et al., 2018). This study strengthens evidences that HBs could act as an oncogene but also identifies a potential target to interfere with the L-HBsAg-induced HCC, which may be full of interest for patient failing to seroconvert after antiviral therapy.

HBV indirect-associated hepatocarcinogenesis

HCC development in patients is explained mostly by regeneration of hepatocytes, a compensatory effect to HBV-induced death. Hepatocytes are a self-renewing population with no impact on carcinogenesis as it occurs on a sporadic basis to maintain the liver mass. However, under persistent injuries, such as those observed in chronic hepatitis B, the high regeneration rate leads to fibrosis and cirrhosis, a favourable ground for HCC development. Of note, all factors inducing chronic liver disease are associated with an increased risk of HCC, and not necessarily due to HBV etiology only.

Regarding HBV, several studies have suggested that high levels of L-HBsAg are toxic for hepatocytes and transgenic mice developed HCC under this condition [START_REF] Chisari | Molecular pathogenesis of hepatocellular carcinoma in hepatitis B virus transgenic mice[END_REF]. Due to high apoptosis rate, macrophages recruitment is increased and leads to free radicals generation, which are known to further expand hepatocyte DNA damages. In support to this hypothesis of HBVinduced carcinogenesis, Hagen and colleagues have detected deoxyguanosine adducts on DNA isolated from the livers of these mice [START_REF] Hagen | Extensive oxidative DNA damage in hepatocytes of transgenic mice with chronic active hepatitis destined to develop hepatocellular carcinoma[END_REF]. In summary, L-HBsAg overexpression leads to high death and compensatory proliferation but also to hepatocyte DNA damage attributable to free radicals generated by macrophages.

The detail of HBV-induced liver carcinogenesis is still to be deciphered, as it remains elusive.

The combination of viral gene expression, modification of cell physiology and changes in the microenvironment of the hepatocytes results in increased cell proliferation, which is one of the main hallmarks of cancer. However, the time course of occurrence of these events is still fuzzy. In one hand, viral mechanisms that contribute to hepatocyte transformation include modulation in gene expression caused by HBx and/or HBV insertions, HBsAg (mainly truncated M and full size L), along with inhibition of DNA repair and apoptosis pathways. On the other hand, cellular mechanisms include hepatocyte killing by CTLs, induction/propagation of DNA damage via free radicals generated by macrophages and expression of cytokines providing a proliferative stimulus to hepatocytes.

Prevention and treatments of HBV-related HCC

As already discussed, the prevention of HBV-related HCC depends mainly on the management of HBV infection. Considerable progress has been made in the last decades, especially thanks to large-scale programs of vaccination against hepatitis B virus. One of the most striking examples is found in Taiwan, where a universal infant vaccination program was introduced in the 80s. The decrease in the rate of HBsAg-positivity among children was associated with a significant childhood HCC reduction from 0.52/100,000 for those born between 1974 and 1984 to 0.13 for those born between 1984 and 1986 [START_REF] Ni | Hepatitis B vaccination in children: the Taiwan experience[END_REF]). However, childhood chronic hepatitis B infection and subsequent HCC have not been eliminated by the universal vaccination program, thus efficient treatments for hepatocellular carcinoma are still needed.

Staging and prognostic assessment

As for all oncologic pathology, the HCC staging is crucial for the choice of the adapted therapeutic protocol to follow and should help define outcome prediction. Since most of patients have associated liver disease, the prognostic assessment should not only focus on the tumour stage but also on the level of liver necroinflammation and the degree of liver function impairment. In this respect, the Barcelona Clinic Liver Classification (BCLC) system established in 1999 is the most commonly used staging system for hepatocellular carcinoma. Since its publication, it has been extensively validated and updated according to the results of investigations that have incorporated [START_REF] Yau | Development of Hong Kong Liver Cancer staging system with treatment stratification for patients with hepatocellular carcinoma[END_REF]. Basically, all these classification methods are constructed on the same basis albeit Asian systems tend to be oriented with personalized systemic therapies instead of chirurgical approaches. Recently, the community tried to improve staging system with implementation of tumour biomarkers, such as the level of -fetoprotein in serum, microvascular invasion and tumour differentiation status [START_REF] Llovet | Plasma biomarkers as predictors of outcome in patients with advanced hepatocellular carcinoma[END_REF][START_REF] Forner | Hepatocellular carcinoma[END_REF].

These latter might have prognostic power but to date have no value in modifying treatment decisions.

Treatments

As HCC is considered chemorefractory and we still have no efficient enough molecules, the aim of treatment is to increase survival while maintaining the highest quality of life. This reason highlights the difficulties to evaluate the strength of any treatment approach as they are compared to untreated patients and effectiveness is always a reflec of a survival gain of few months. The potentially useful treatment number is large and goes from liver transplantation to selective tyrosine kinase inhibitors. The next chapters' goal is to give an overview of existing options for HCC management and to point out issues regarding HCC cure.

Liver resection

For patients without cirrhosis, liver resection is the treatment of choice for hepatocellular carcinoma. Indeed, major liver resections can be done without main issues regarding necroinflammation and regeneration in this case. Usually, this option is of interest for early tumour and is associated with good survival. Although the tumour size is not an excluding criteria, the risk of vascular invasion and dissemination increases with diameter [START_REF] Fuks | Benefit of initial resection of hepatocellular carcinoma followed by transplantation in case of recurrence: an intention-to-treat analysis[END_REF]. In Europe and USA, level of bilirubin and portal pressure are the variables used to select appropriate candidates (European Association For The Study Of The Liver and European Organisation For Research And Treatment Of Cancer, 2012), while the japanese use the indocyanine green retention rate [START_REF] Kokudo | Evidence-based Clinical Practice Guidelines for Hepatocellular Carcinoma: The Japan Society of Hepatology 2013 update (3rd JSH-HCC Guidelines)[END_REF]. Patients without clinically significant portal hypertension (CSPH) and normal bilirubin reach 70% survival at 5 years, whereas survival decrease to <50% when both factors are present [START_REF] Ishizawa | Neither multiple tumors nor portal hypertension are surgical contraindications for hepatocellular carcinoma[END_REF]. The main issues with liver resection are the invasiveness of the procedure and the increased risk of recurrence due to dissemination. Indeed, at 5 years, de novo tumours complicate 70% of the cases. A less invasive alternative option to liver resection is laparoscopic surgery, which exhibits similar long-term survival but is not usefull regarding the recurrence issue [START_REF] Han | Laparoscopic versus open liver resection for hepatocellular carcinoma: Case-matched study with propensity score matching[END_REF](Figure 26). However, as HBV-related HCC occurs mostly on cirrhotic liver (>70%), the liver resection approach is uncommonly used and effectively manages a very small fraction of HBV-related HCC. Of course, liver transplantation is the best option but is limited by the availability of organs and only patients those exhibits high recurrence risk factors such as microvascular invasion or satellite nodules after liver resection are indicated for ab initio liver transplantation [START_REF] Ferrer-Fàbrega | Prospective validation of ab initio liver transplantation in hepatocellular carcinoma upon detection of risk factors for recurrence after resection[END_REF].

.2. Liver transplantation

The liver transplantation is the best therapeutic option available as it can theoretically cure both the tumour and underlying cirrhosis. The best post-liver transplantation survival, of 70% at 5 years with recurrence <15%, is found when the initial tumour is unresectable, <5cm or three nodules <3cm each, defined by the Milan criteria [START_REF] Mazzaferro | Liver transplantation for the treatment of small hepatocellular carcinomas in patients with cirrhosis[END_REF]. The main limitation regarding liver transplantation is donor shortage. Even with dedicating all transplants to HBV-related HCC patients, it will be sufficient for barely 25% of them. This limitation imposes waiting list, a time under which tumours might progress and impede transplantation. Thus, it is crucial to refine liver transplantation indications according to donor availability in each allocation area, the proportion of enlisted patients without hepatocellular carcinoma versus those with hepatocellular carcinoma as well as the dynamics of the waiting list [START_REF] Mazzaferro | Squaring the circle of selection and allocation in liver transplantation for HCC: An adaptive approach[END_REF][START_REF] Sapisochin | Liver transplantation for hepatocellular carcinoma: outcomes and novel surgical approaches[END_REF].

Percutaneous tumour ablation

For early-stage HCC, image-guided tumour ablation is a widely accepted and used treatment option. Ablation induces necrosis either by temperature shock (radiofrequency, microwave or cryoablation) or by chemical stress (mostly percutaneous ethanol injection) [START_REF] Forner | Ablation for hepatocellular carcinoma: Is there need to have a winning technique?[END_REF].

Among them, radiofrequency ablation is preferred and recommended as first-line technique as it allows better disease control and outcomes compared to others [START_REF] Germani | Clinical outcomes of radiofrequency ablation, percutaneous alcohol and acetic acid injection for hepatocelullar carcinoma: a meta-analysis[END_REF](Figure 27).

Regarding small tumours, especially for patients with Child-Pugh stage A liver disease, ablation exhibits non-inferior survival compared to surgical resection, challenging this latter, in terms of costefficiency and invasiveness [START_REF] Hasegawa | Comparison of resection and ablation for hepatocellular carcinoma: a cohort study based on a Japanese nationwide survey[END_REF]. However, focusing on HBV-related HCC, such therapies could result in HBV reactivation in 15-30% even in HBsAg negative patients, highlighting the need of expert-oriented choice for local ablation [START_REF] Yoshida | Safety and efficacy of lamivudine after radiofrequency ablation in patients with hepatitis B virus-related hepatocellular carcinoma[END_REF][START_REF] Jang | Hepatitis B virus reactivation in patients with hepatocellular carcinoma undergoing anti-cancer therapy[END_REF].

Transcatheter tumour therapy

Image-guided transcatheter tumour therapies take advantage of the highly-developed vascularization system of the tumour to deliver drugs, embolic particles, or radioactive devices [START_REF] Forner | Treatment of intermediate-stage hepatocellular carcinoma[END_REF]. Among them, solely transarterial chemoembolization has shown survival benefit [START_REF] Llovet | Systematic review of randomized trials for unresectable hepatocellular carcinoma: Chemoembolization improves survival[END_REF]. Polyvinyl alcohol spheres enabled the slow release of chemotherapy, increasing the survival time up to 40 months while reducing adverse events [START_REF] Burrel | Survival of patients with hepatocellular carcinoma treated by transarterial chemoembolisation (TACE) using Drug Eluting Beads. Implications for clinical practice and trial design[END_REF]. This kind of approach is particularly recommended for patients with compensated liver, unresecable large tumour [START_REF] Malagari | Chemoembolization with doxorubicin-eluting beads for unresectable hepatocellular carcinoma: five-year survival analysis[END_REF]. Finally, transarterial chemoembolization might be full of interest for patients on the waiting list for liver transplantation as it exhibits a nice delay in tumour progression.

Systemic therapies

Until 2008, no efficient systemic therapy for hepatocellular management was available. This is in part explained by the fact that somatic mutations found in HCC remain undruggable (TERT, CTNNB1, TP53 and ARID1A).

The understanding of molecular mechanisms driving liver carcinogenesis has led to the development of targeted therapies. A large number of molecules have been tested or are currently under development [START_REF] Nault | The end of almost 10 years of negative RCTs in advanced hepatocellular carcinoma[END_REF], but to date, only Sorafenib and

Regorafenib have been shown to improve survival versus placebo [START_REF] Llovet | Sorafenib in advanced hepatocellular carcinoma[END_REF][START_REF] Bruix | Regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment (RESORCE): a randomised, double-blind, placebo-controlled, phase 3 trial[END_REF]. Both are orally available multikinase inhibitors targeting RAF, KIT, RET, VEGFR1 and PDGFR, exhibiting antiproliferative and antiangiogenic effects, albeit underlying mechanisms are not well known yet. Sorafenib was the first evaluated one, in two positive randomised placebo-controlled trials: one multicentre trial done predominantly in Europe and the USA, and another trial done in the Asia-Pacific area [START_REF] Llovet | Sorafenib in advanced hepatocellular carcinoma[END_REF][START_REF] Cheng | Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial[END_REF]. Several promising agents with suggestive findings from early-stage studies failed to demonstrate survival benefit over placebo as first or second-line treatment for HCC (Table IV). Regorafenib was the only drug that demonstrated survival benefit as a second-line treatment and was evaluated in the RESORCE phase 3 trial for patients with hepatocellular carcinoma but were tolerant to Sorafenib [START_REF] Bruix | Regorafenib for patients with hepatocellular carcinoma who progressed on sorafenib treatment (RESORCE): a randomised, double-blind, placebo-controlled, phase 3 trial[END_REF].

Recently, Lenvatinib was described to be non-inferior to Sorafenib in first-line treatment but it targets the same population, thus limiting its interest [START_REF] Spallanzani | Lenvatinib as a therapy for unresectable hepatocellular carcinoma[END_REF].

Finally, promising results have been announced for Cabozantinib as a second-line treatment.

This is another tyrosine kinases inhibitor acting on VEGF receptors 1, 2, and3

Future of HBV-related HCC treatments

Although Sorafenib and Regorafenib are available for HCC treatment, outcomes remain poor as they allow a survival gain of few months only. Moreover, the management of HCC is mostly the same regarding etiology, thus it would be full of interest to identify key mechanisms of HBV-related carcinogenesis in order to develop efficient and well-tolerated therapies.

Chekmate for hepatocellular carcinoma may come from immune therapies. Past years, a better comprehension of the mechanisms underlying the ability of tumor to escape immune surveillance has led to impressive results with immunotherapy in several cancers, and this strategy is currently being developed for HCC [START_REF] Da Motta Girardi | Hepatocellular Carcinoma: Review of Targeted and Immune Therapies[END_REF]. Nivolumab, an anti-PD1 monoclonal antibody has received an accelerated approval from the FDA for the treatment of HCC.

The approval is based on a phase 1/2 clinical trial in which patients showed a better overall rate response, over Sorafenib. Moreover, Nivolumab showed no lower response in patients with chronic viral hepatitis [START_REF] El-Khoueiry | Nivolumab in patients with advanced hepatocellular carcinoma (CheckMate 040): an open-label, non-comparative, phase 1/2 dose escalation and expansion trial[END_REF][START_REF] Ray | Liver cancer: Nivolumab: checkmate for hepatocellular carcinoma?[END_REF].

The second promising axis reside in the development of chimeric antigen receptorengineered T-cell (CAR-T) and T-cell receptor (TCR-T) immune cell therapies. LioCyx, the first engineered TCR-T-cell therapy for treatment of liver cancer has received the approval for a phase 1/2 clinical trial. These strategies are at the dawn but results are encouraging and should offer a new approach for the treatment of HBV-related liver cancers [START_REF] Koh | Targeted Therapy of Hepatitis B Virus-Related Hepatocellular Carcinoma: Present and Future[END_REF])(Figure 28).

Finally, investigating multi-kinases inhibitors acting as Sorafenib with a spectrum extended to other molecular pathways is still a trend. The polo-like kinase 1, a cell-cycle regulating kinase which is also involved in DNA damage responses appears to be a first-choice target for HBV-related HCC. On the one hand, PLK1 was extensively studied and reported as a key driver of liver carcinogenesis and associated with poor prognosis [START_REF] He | Overexpression of polo-like kinase1 predicts a poor prognosis in hepatocellular carcinoma patients[END_REF]Mok et al., 2012). Inhibition of PLK1 was described to control and limit the progression of established HCC by inhibiting cell proliferation and promoting apoptosis [START_REF] Deng | Intravenous liposomal delivery of the short hairpin RNAs against Plk1 controls the growth of established human hepatocellular carcinoma[END_REF]Wang et al., 2017a). Recently PLK1 siRNA encapsulated in proprietary lipo nanoparticles (LNPs) were clinically trialed in patients with advanced HCC (NCT02191878); collected data showed an overall partial response according to Choi's criteria in 22% of subjects and tumour density reduction of up to 59%. On the other hand, our studies have highlighted PLK1 as a potent proviral factor for HBV and a specific inhibition of this kinase results in antiviral activity (confer First and Second studies). Thus, the use of specific PLK1 inhibitors or multi-kinases inhibitor such as rigosertib could be of merit for HBV-related HCC treatment [START_REF] Dietrich | Combined effects of PLK1 and RAS in hepatocellular carcinoma reveal rigosertib as promising novel therapeutic "dual-hit" option[END_REF]. Chapter 3: Polo-like kinase 1, a new target for anti-HBV and anti-HCC therapies?

Polo-like kinases: an extremely well-conserved family from Yeast to Human

In 1988, Sunkel and Glover reported in the Journal of cell science that Drosophila neuroblast cells with mutant alleles of the locus polo showed abnormal mitotic and meiotic divisions.

Chromosomes were randomly oriented, with a circular arrangement, leading to the production of polyploid cells. Moreover, a specific mutant named polo² was found to be lethal, highlithing the essential roles of the polo kinase in larvae cell division and development [START_REF] Sunkel | polo, a mitotic mutant of Drosophila displaying abnormal spindle poles[END_REF].

Latter, homologous genes were found in Saccharomyces cerevisiae and Schizosaccharomyces pombe, encoding CDC5 and Plo1 proteins respectively [START_REF] Kitada | A multicopy suppressor gene of the Saccharomyces cerevisiae G1 cell-cycle mutant gene dbf4 encodes a protein kinase and is identified as CDC5[END_REF][START_REF] Ohkura | The conserved Schizosaccharomyces pombe kinase plo1, required to form a bipolar spindle, the actin ring, and septum, can drive septum formation in G1 and G2 cells[END_REF]. Subsequent research showed that PLKs were present in all branches of the Eukarya, and that humans had five paralogues; PLK1-5 (de Cárcer et al., 2011)(Figure 29). All these related proteins share a specific motif called Polo-box (PB), which can be organized to dictate PLKs activity, localization and functions.

Although the original mutants were characterized by the presence of abnormal spindles formation, later studies indicated that PLKs are involved at multiple stages during mitosis [START_REF] Archambault | Polo-like kinases: conservation and divergence in their functions and regulation[END_REF].

Structural organization and features of human PLKs

PLKs members derived from the ancestral PLK1 and shared similar architecture. The N-ter contains a Ser/Thr catalytic kinase domain while in the C-ter region the Polo box domain (PBD) is located, this latter being composed by two or more PBs. PLK1-3 share a strong homology while PLK4 is divergent in the primary sequence and PLK5 is considered as a pseudo kinase as it lacks of residues for substrate recognition in the PBD (Carvalho-Santos et al., 2010)(Figure 30).

Focus on the human PLK1

General structure of PLK1

PLK1 is a 603 aa, 66kDa Ser/Thr kinase. It holds a kinase domain in C-ter, aa 53-305, followed by a linker where is located a D-box domain, of importance for APC/c mediated degradation of PLK1. This interdomain linker (IDL) is involved in the inactivation of PLK1 as it maintains inaccessible the activation loop located in C-ter due to steric hindrance. The IDL is followed by a PBD containing two distinct polo boxes, which serve as phospho-binding motifs (Figure 31). The region between the two PBs recognizes peptides containing phosphorylated Ser and Thr residues with the consensus motif Ser-[pSer/pThr]-[Pro/X] where X stand for any residue. As for the others PLKs members, the PBD of PLK1 located at the C-ter regulates the catalytic activity of PLK1, its localization and substrate binding [START_REF] Cheng | The crystal structure of the human polo-like kinase-1 polo box domain and its phospho-peptide complex[END_REF]Elia et al., 2003).

PLK1 activation: a multi-step and dynamic process

The activation of PLK1 is mediated through two phosphorylations within the C-ter kinase domain; the first is located at the Thr210 in the T-loop (also known as the activation loop) and the second at the Ser137 located near the end of the hinge. Thus, antibodies recognizing either pPLK1T210 or pPLK1S137 are useful to evidence PLK1 activation level.

Starting from the inactive conformation of PLK1, the kinase domain is inhibited by three different ways:

-The interdomain linker sequesters the T-loop where is located the Thr210 of the kinase domain -The PBD reduces the flexibility of the hinge where is located the Ser137 -Binding of the PBD to Map205 protein, thereby stabilizing the autoinhibited state These inhibitions can be release partially and/or totally, allowing a very fine control of PLK1 spatiotemporal activity [START_REF] Zitouni | Polo-like kinases: structural variations lead to multiple functions[END_REF]. The phosphorylation of the Ser137 is known to disrupt the intremolecular interaction between the PBD and the kinase domain. The interaction with the protein Bora is the most described regarding the freeing of the T-loop, allowing activating kinases such as Aurora A to phosphorylate the Thr210 of PLK1 [START_REF] Xu | Structural basis for the inhibition of Polo-like kinase 1[END_REF]. Furthermore, phosphopeptides interacting whit the hinge between the two PBs is another mechanism showed to disrupt the inhibitory interaction between the PBD and the kinase domain. Finally, phosphorylation of Map205 by CDK1 results in destabilization of the inhibited stage of PLK1 (Figure 32). However, the regulation of PLK1 activation model is based on studies conducted mainly in Drosophila and although being partially confirmed in human cells, structural studies of full-length PLK1 are needed to further clarify its regulation and mechanism of action.

Spatiotemporal regulation of PLK1 activity

Among PLKs members, PLK1 has the most dynamic localization, mostly due to its involvement in cell-cycle progression, which requires a timely-fashion regulation of its activity. In quiescent cells, PLK1 was not extensively studied albeit this kinase is found to be mainly cytosolic during the interphase with localization near the microtubules and centrosomes. During mitosis, PLK1 is present at kinetochores, central spindle, centrosomes, microtubules and midbody, depending the stage of cellular division [START_REF] Petronczki | Polo on the Rise-from Mitotic Entry to Cytokinesis with Plk1[END_REF]. This spatial organization of PLK1 is though to be dependent on phosphopeptides, harbouring the consensus motif Ser-[pSer/pThr]-[Pro/X], binding to the PBD. Moreover, PLK1 can also bind to nonphosphorylated peptides and phosphopeptides previously phosphorylated by PLK1 itself, which is known as self-priming. All these mechanisms highlight the complexity of PLK1 regulation and indicate the presence of a local feedback loop. As example, its localization to central spindle is mediated through PLK1-phosphorylated PRC1 while the localization to the kinetochores is dependent on CDK1phosphorylated INCENP [START_REF] Goto | Complex formation of Plk1 and INCENP required for metaphase-anaphase transition[END_REF][START_REF] Hu | Plk1 negatively regulates PRC1 to prevent premature midzone formation before cytokinesis[END_REF]. As explained before, the protein Map205 binds PLK1 in a phosphor-independent manner and allows relocalization of inactive PLK1 to microtubules, suggesting kinase-independent activity of PLK1.

Finally, other post-translational modifications seem to play a role in PLK1 localization. Among them, the non-proteolytic ubiquitylation of PLK1 through the CUL3/KLHL22 complex is likely to promote the release of PLK1 from kinetochores. Indeed, in the absence of KLHL22, PLK1 accumulates to kinetochores, leading to the activation of the spindle assembly checkpoint. The CUL3/KLHL22 complex, which ubiquitylates Lys492, located within the PBD, results in PLK1 dissociation from kinetochores, which is mandatory for mitosis [START_REF] Beck | Ubiquitylation-dependent localization of PLK1 in mitosis[END_REF](Figure 33).

Regulation of PLK1 level

PLK1 being mainly involved in the regulation of key steps of the cell-cycle, its expression is tightly regulated a both mRNA and protein levels. However, the post-transcriptional modifications of PLK1 seem to be crucial as they allow a fast and transient activation/inhibition of PLK1 kinase activity.

Regarding PLK1 protein level, it is maintained low in interphase and peaks during mitosis [START_REF] Winkles | Differential regulation of polo-like kinase 1, 2, 3, and 4 gene expression in mammalian cells and tissues[END_REF]. In G1, the PLK1 gene is under the control of transcriptional repressors such as the CDE-CHR (cell-cycle-dependent element -cell-cycle gene homology region) sequences within the PLK1 promoter, which are involved in PLK1 repression in coordination with p53/p21. During the G2 phase, the DREAM complex increases PLK1 transcription through the action of FoxM1 protein [START_REF] Reichert | Lin9, a subunit of the mammalian DREAM complex, is essential for embryonic development, for survival of adult mice, and for tumor suppression[END_REF].

PLK1 is involved in nearly every steps of the cell-cycle "For an overview of PLK1 involvement in the cell-cycle, please refer to Figure 34."

G2/M transition and the mitotic entry

PLK1 is an early trigger for G2/M transition. In the absence of PLK1 kinase functions, several proteins fail to be recruited in early mitosis, as shown with polo mutant in D.melanogaster [START_REF] Sunkel | polo, a mitotic mutant of Drosophila displaying abnormal spindle poles[END_REF]. The G2/M transition is allowed by the removal of inhibitory phosphates on CDK1, resulting in the activation of the M phase-promoting factor (MPF; also known as CDK1-CyclinB) complex. This state is regulated by the activity of two kinases, MYT1 (Myelin transcription factor 1) and WEE1 and one phosphatase, CDC25 (Cell-division cycle protein 25). Both WEE1 and CDC25 are phosphorylated on several sites, which could be attributed to CDK1, highlighting positive and negative-feedback loops. Data indicates that PLK1 can phosphorylate and thereby regulate both, CDC25 and MYT1.

MPF activity results in cyclin B translocation to the nucleus, which is followed by further activation of MPF and nuclear envelope breakdown. However, PLK1 is not mandatory for CDK1 activation, which seems to occur with no, or low level of PLK1 [START_REF] Toyoshima-Morimoto | Plk1 promotes nuclear translocation of human Cdc25C during prophase[END_REF].

Furthermore, inhibition of PLK1 in several organisms generally causes aberrations and/or delays during mitosis rather than a G2 arrest, indicating a regulatory action of PLK1 during this step [START_REF] Van Vugt | Getting in and out of mitosis with Polo-like kinase-1[END_REF]. On the other hand, PLK1 acts as a trigger kinase and controls CDC25 after a release from G2 arrest. This indicates that PLK1 has a key role for the entry in mitosis in this case. In summary, PLK1 activates MPF by activating CDC25 and by inhibiting both MYT1 and WEE1 [START_REF] Kumagai | Purification and molecular cloning of Plx1, a Cdc25-regulatory kinase from Xenopus egg extracts[END_REF].

PLK1 and the centrosome machinery

The centrosome is the main microtubule-organizing centre (MTOC) and is found to be very conserved in term of structures in Eukarya (Figure 29). It is composed of two centrioles, which are surrounded by the pericentriolar material. The centrosomes follow a cycle in parallele of the cellcycle, with duplication occurring in G1/S while assembly occurs during the S phase. They undergo maturation in late G2, and then separate during mitosis. In human, PLK1 was shown to be involved in the centrosomal localization of γ-tubulin during centrosome maturation [START_REF] Teixidó-Travesa | The where, when and how of microtubule nucleation -one ring to rule them all[END_REF]. The aim of this process is to increase the microtubule nucleation activity, which is required for spindle formation. A recently identified substrate of PLK1, Nlp (Ninein-like protein), interacts with γtubulin ring complexes [START_REF] Casenghi | Polo-like kinase 1 regulates Nlp, a centrosome protein involved in microtubule nucleation[END_REF]. At the onset of mitosis, the Nlp phosphorylation by PLK1 results in its dissociation from centrosome, allowing the recruitment of other proteins. This leads to increase the γ-tubulin level, required for mitosis.

The mitotic entry is a step being regulated by the association of PLK1 with the centrosome.

As discussed earlier, the complex CDK1-CyclinB, is a key regulator of M phase in eukaryotes and evidence showed that the activation of the MPF initially occurs at the centrosome [START_REF] Jackman | Active cyclin B1-Cdk1 first appears on centrosomes in prophase[END_REF]. In accordance with the cell-cycle clock, PLK1 coordinates centrosome cycle so that the right number of functional structures is assembled at the right time.

PLK1 is involved in kinetochore microtubules attachment and chromosomes segregation

During mitosis, PLK1 localizes to both spindles and kinetochores [START_REF] Van Vugt | Getting in and out of mitosis with Polo-like kinase-1[END_REF]. These centromere associations support two important related processes. First, they are the anchorage site for spindles and secondly, centromeres play a crucial role for the spindle checkpoint, including a control ensuring that all chromosomes have undergone bipolar attachment, before sister chromatids separation [START_REF] Van Vugt | Getting in and out of mitosis with Polo-like kinase-1[END_REF]. Aurora B is a kinase with well-established functions in destabilizing attachments and its substrates are highly phosphorylated in the lowtension state [START_REF] Liu | Polo-like kinase-1 regulates kinetochoremicrotubule dynamics and spindle checkpoint silencing[END_REF]. There is still a debate on how the attachment initially occurs, however, evidence has shown that PLK1 expression level is high during prometaphase to balance the destabilizing activity of Aurora B and that PLK1 is required for the initial stable attachment of kinetochore microtubules. Indeed, in metaphase, PLK1 phosphorylates BubR1, which stabilizes microtubule-kinetochore attachment. The non-proteolytic ubiquitylation of PLK1 PBD by CUL3-KLHL22 at Lys492 is a crucial step in promoting the release of PLK1 from kinetochores, which is mandatory for the separation of sister chromatids [START_REF] Beck | Ubiquitylation-dependent localization of PLK1 in mitosis[END_REF].

PLK1 and sister-chromatids separation

The separation of sister-chromatids occurs through the APC/c-dependent ubiquitylation and is highly dependent on correct chromosome alignment and on attachment of spindle microtubules to the kinetochores. The ubiquitylation of Securin leads to its degradation and releases the Separase, a protease that cleaves Cohesins, responsible of the sister-chromatids cohesion. Moreover, the APC/c degrades CyclinB, resulting in the inactivation of M-CDK (mitotic cyclin-dependent kinase) complexes and promoting exit from mitosis [START_REF] Eckerdt | Polo-like kinase 1: target and regulator of anaphase-promoting complex/cyclosome-dependent proteolysis[END_REF]. In this context, PLK1 is able to phosphorylate and activate components of the APC/c, thus, regulating sister-chromatids separation.

PLK1, centriole disengagement and cytokenesis; the end of a journey

Following the cell-cycle kinetic, the next step regards the centriole disengagement. It was thought that PLK1 alone was essential and necessary for this process [START_REF] Van Vugt | Getting in and out of mitosis with Polo-like kinase-1[END_REF].

However, the centriole disengagement is delayed without the activity of APC/c-Cdh1 and PLK1 was shown to be not mandatory. These two ways to complete the process allow the cell to control the kinetic of centriole disengagement. This ensures that centrioles will not prematurely separate in late G2 or in early mitosis, thereby risking multipolar spindle assembly, but rather disengage in a timely fashion. PLK1 and CDK1 activities on the APC/c allow a smooth regulation of this process [START_REF] Zitouni | Polo-like kinases: structural variations lead to multiple functions[END_REF].

Cytokinesis is known as the final step of the mitosis, resulting on the formation of two daughter cells. It involves rearrangement of the cytoskeleton to form a midzone that keeps sister chromatids apart, leading in fine to abscission of daughter cells. In eukaryotes, this process requires ingression of the actomyosin-based contractile ring between segregated sister genomes. The activity and relocalization to spindle the spindle midzone of PLK1 during late anaphase allows recruitment of RhoGEF Ect2 to the central spindle, which promotes the activation of the RhoA GTPase, inducing assembly and ingression of the contractile ring [START_REF] Ebrahimi | Polo kinase interacts with RacGAP50C and is required to localize the cytokinesis initiation complex[END_REF]. This overview of PLK1 roles in cell-cycle highlights its importance and enables a better understanding on its potential involvement in carcinogenesis processes. Another key role of PLK1 is its involvement in DNA repair mechanisms, which is also a process relevant for carcinogenesis. This point is developed in the part below.

PLK1 in DNA damage response

Cells are continually threatened with stresses conditions, either endogenous such as ER stress, replicative stresses, or exogenous factors; mainly UV exposure and genotoxic reagents. When DNA is damaged, a checkpoint mechanism is activated, allowing cells enough time to repair before going further into the cell-cycle. If damages are too severe, an apoptotic pathway takes place. At the onset of mitotic entry, a cooperation between Aurora B and Bora leads to the activation of PLK1 [START_REF] Van Vugt | Getting in and out of mitosis with Polo-like kinase-1[END_REF]. When double-strand breaks (DSB) occur, especially in G2 phase, the ATM (Ataxia telangiectasia mutated) protein is recruited by the MRN (Mre11/Rad50/Nbs1) complex (Figure 35). ATM directly phosphorylates Bora, leading to its degradation by the E3 ubiquitin ligase SCF-β-TRCP (Skp, Cullin, F-box containing complex). In this case, a G2 arrest occurs due to the nonactivation of PLK1. However, the mechanism described above could not explain the complete inhibition of PLK1. Until lately, it was unclear how PLK1 was totally shut down, allowing the G2 DNA damage checkpoint establishment. A recent paper uncovered the role of B55α in PLK1 dephosphorylation by mediating PP2A/PLK1 (Protein phosphatase 2A) association [START_REF] Wang | Regulation of polo-like kinase 1 by DNA damage and PP2A/B55α[END_REF]. Moreover, it has been reported that PLK1 directly phosphorylates the Rad51 recombinase and facilitates the homologous recombination (HR) [START_REF] Yata | Plk1 and CK2 act in concert to regulate Rad51 during DNA double strand break repair[END_REF].

PLK1 also plays a role when DNA damages occur during mitosis. This is a very short period comparing to other stages of cell-cycle but critical in term of genomic distribution. DNA damages at this step often result in mitotic arrest. In mitosis, PLK1 is activated and the cell has to shut down its activity in order to set up a mitotic arrest. The down-regulation of PLK1 occurs through the intervention of PP2A. PP2A activity on PLK1 dephosphorylation is dependent on ATM-Chk1 (ATM-Checkpoint kinase 1) pathway in mitotic DNA damage response [START_REF] Cholewa | The role of polo-like kinase 1 in carcinogenesis: cause or consequence?[END_REF].

Finally, evidences showed relationship between PLK1 and p53, responding together to fix DNA damages. The p53 protein is known as a key tumor suppressor, a genome guardian, by inducing cycle arrest when cells are damaged due to stress conditions. DNA damages leads to p53 activation through its phosphorylation by ATM/ATR and Chk1/Chk2. The p53 protein has opposite functions to PLK1 regarding its involvement in the cell-cycle. After DNA damages, the aim is to stop cell-cycle progression, mainly by upregulating p53 and downregulating PLK1. The accumulation of phospho-p53 allows the activation of downstream effectors, such as p21, which inhibits CDKs and induces DNA damage regulating proteins. PLK1 is inhibited by an ATM/ATR-dependent fashion, as seen before and p53 can transcriptionally regulate PLK1 by binding with E2F1 at PLK1 promoter. This complex p53-E2F1 suppresses the expression of PLK1 [START_REF] Zhou | p53 Suppresses E2F1-dependent PLK1 expression upon DNA damage by forming p53-E2F1-DNA complex[END_REF]. Moreover, p53 can act indirectly to repress PLK1. Indeed, p21/waf1, during DNA damage response, targets a CDE/CHR (Cellcycle-dependent element/Cell-cycle genes homology region) within PLK1 promoter, resulting in its downregulation. At last, p53 acts as a downregulator on FoxM1, which stimulates PLK1 expression [START_REF] Pandit | p53 negatively regulates expression of FoxM1[END_REF].

Here different mechanisms, occurring in DNA damage responses, have been presented. This allowed defining and understanding the pivotal role of PLK1. Cell has to act at genomic and proteomic levels to shut down drastically the activity of PLK1. Two main points are concerned by this inhibition. On one hand, this allows the cell-cycle arrest. On the other hand, avoiding the recovery of cell-cycle arrest during repairing damaged DNA is mandatory. In the part below, the involvement of PLK1 in carcinogenesis is discussed, in the light of actual knowledges and overviews done before.

Involvement of PLK1 in oncogenesis

PLK1 is considered as a proto-oncogene, overexpressed in up to 80% of malignancies, and has been associated with poor prognosis (Table V). As seen before, the expression of PLK1 is directly linked with the progression of the cell-cycle and its function goes even beyond this. In physiological conditions, PLK1 starts accumulating during S-phase, shows a peak at G2-M transition, stabilizes through mitosis and decreases upon mitotic exit. Of course, this pattern of expression is radically altered in cancer cells. As example, PLK1 can localize to nucleus before G2-M, and can even be detectable in the G1-S phase. These observations suggest that PLK1 must have cancer-cells specific functions. This is supported by the fact that PLK1 is involved in the G1-S transition and DNA replication in cancer cells, by phosphorylating Orc2, a major component of the pre-replicative complex. On the other hand, the overexpression of PLK1 leads cells to go through the cell-cycle, representing a high mitotic potential. Both, overexpression/deregulation of PLK1 activity and PLK1 cancer-cells specific functions are involved in oncogenesis phenomena.

Cancer Type

Fold Change PLK1 has been found interacting with a wide range of tumour suppressors. Among them, proteins involved in DNA damage repair pathways and progression through the cell-cycle such as BRCA1/2 [START_REF] Lee | Phosphorylation of BRCA2 by the Polo-like kinase Plk1 is regulated by DNA damage and mitotic progression[END_REF][START_REF] Chabalier-Taste | Polo-like kinase 1 mediates BRCA1 phosphorylation and recruitment at DNA double-strand breaks[END_REF], ATM [START_REF] Lee | Mitotic DNA damage response: Polo-like kinase-1 is dephosphorylated through ATM-Chk1 pathway[END_REF], ATR [START_REF] Deming | ATR enforces the topoisomerase II-dependent G2 checkpoint through inhibition of Plk1 kinase[END_REF], CYLD [START_REF] Stegmeier | The tumor suppressor CYLD regulates entry into mitosis[END_REF], BubR1 [START_REF] Izumi | BubR1 localizes to centrosomes and suppresses centrosome amplification via regulating Plk1 activity in interphase cells[END_REF]. The imbalance between these interactions, resulting in deregulation of important pathways may contribute to cancer development.

In the two next subparts, the focus is set on two majors' tumour suppressors that have been shown

to be downregulated by PLK1; p53 and PTEN.

PLK1, a major foe for the "guardian of the cell"

The tumour suppressor p53, known as "the guardian of the cell", is involved in antiproliferation by inducing apoptosis of cells harboring uncontrolled, abnormal growth [START_REF] Fu | The Emerging Role of Polo-Like Kinase 1 in Epithelial-Mesenchymal Transition and Tumor Metastasis[END_REF]. For instance, PLK1 has been shown to regulate p53 activity through four differents pathways:

-PLK1 inhibits p53 apoptotic activity as well as p53-dependent transcriptional activation by direct phosphorylation.

-PLK1 phosphorylates MDM2 at Ser260, an E3 ubiquitin ligase for p53, and stimulates MDM2-mediated p53 turnover, thus inhibiting p53 activity.

-Topors is an ubiquitin and SUMO E3 ligase involved in p53 stability. PLK1 has been shown to activate Topors by phosphorylating it at Ser178, leading to an inhibition of Topors-mediated SUMOylation of p53, reducing its stability in association with an increased ubiquitin-mediated degradation of p53.

-PLK1 is also able to phosphorylate G2 and S-phase-expressed 1 protein (GTSE1), leading to its nuclear translocation where it can bind to p53, resulting in p53 shuttling out of the nucleus for degradation.

PLK1 and the tumour-suppressor PTEN

Phosphatase and tensin homologue (PTEN) governs a plethora of processes including survival, proliferation and energy metabolism, mostly by suppressing the PI3K-AKT-mTOR pathway through its lipid phosphatase activity. In addition, it has been shown that PTEN could act in a phosphatase-independent manner, thereby increasing its spectrum of activities. Among with p53, Thesis Adrien FOCA

92

PTEN is one of the best-characterized and commonly misregulated tumour suppressors in cancers [START_REF] Song | The functions and regulation of the PTEN tumour suppressor[END_REF]. In 2014, Choi and colleagues have shown that PLK1 was able to phosphorylate PTEN at the Ser380 in vivo, which was associated with the accumulation of PTEN on the chromatin, thus, increasing mitotic activity [START_REF] Choi | Plk1 protein phosphorylates phosphatase and tensin homolog (PTEN) and regulates its mitotic activity during the cell-cycle[END_REF]. Moreover, Li and colleagues showed that PLK1 enhanced aerobic glycolysis through the inactivation of PTEN and the activation of the PI3K pathway.

Indeed, PLK1 phosphorylates PTEN and Nedd4-1, an E3 ubiquitin ligase of PTEN, resulting in PTEN inactivation and promoting tumorigenesis [START_REF] Li | Plk1 phosphorylation of PTEN causes a tumor-promoting metabolic state[END_REF].

PLK1 interplays with several oncogenes

On the one hand, as discussed before with p53 and PTEN, PLK1 is able to negatively regulate tumour suppressors. On the other hand, PLK1 also activates intensively oncogenes. Both mechanisms are known to be mandatory for cancer initiation. As for the section "3.3.7.1.1. PLK1 inhibits tumour suppressors", PLK1 is found to be associated with several oncogenes but we decided to focus on PLK1 interaction with FoxM1 and the Myc family members, as they are the best documented and characterized regarding involvement in oncogenesis process.

PLK1 and FoxM1: a cell-cycle story

The Forkhead box protein M1 (FoxM1) transcription factor is required for nomal cell proliferation and is found elevated in a wide range of cancers [START_REF] Halasi | FOX(M1) NEWS -it is cancer[END_REF]. Interestingly, FoxM1 plays a role far beyond the cell-cycle progression and is found involved in all major hallmarks of cancer depicted by Weinberg [START_REF] Hanahan | Hallmarks of cancer: the next generation[END_REF]. The transcriptional activity of FoxM1 correlates with its phosphorylation level, which is at a basal level in G1 and fully phosphorylated at the G2/M transition. In this context, PLK1-dependent phosphorylation of FoxM1 is required to fully activate FoxM1 [START_REF] Fu | Plk1-dependent phosphorylation of FoxM1 regulates a transcriptional programme required for mitotic progression[END_REF]. In counterpart, activated FoxM1 is in capacity to increase PLK1 expression, thereby creating a positive loop, amplifying PLK1 levels and FoxM1 activity.

Using mouse neural progenitor cells, the overexpression of FoxM1 accelerated neurosphere formation, while FoxM1 expression increased as cells progressed to tumorigenic progenitors and glioma-like cells. This founding was correlated with PLK1-dependent activation of FoxM1 [START_REF] Joshi | MELK-dependent FOXM1 phosphorylation is essential for proliferation of glioma stem cells[END_REF].

PLK1 and the Myc family

The MYC family contains three members that are described as oncogenes: c-Myc, L-Myc and N-Myc. These three proteins are mainly involved in the cell-cycle regulation albeit a wealth of data has shed new light on the functions of Myc and how their deregulations could contribute to cellular transformation [START_REF] Adhikary | Transcriptional regulation and transformation by Myc proteins[END_REF]. Overexpression of c-Myc is sufficient for the induction of cell-cycle progression while a conditional knockout in mice implies that c-Myc function is mandatory for proliferation in most cell types [START_REF] Trumpp | c-Myc regulates mammalian body size by controlling cell number but not cell size[END_REF]. PLK1 is known to directly phosphorylate c-Myc, leading to its accumulation and in fine to oncogenic transformation, tumor-initiating cell activation and resistance to mTOR-targeted therapy [START_REF] Tan | PDK1 signaling toward PLK1-MYC activation confers oncogenic transformation, tumorinitiating cell activation, and resistance to mTOR-targeted therapy[END_REF]. transcription, resulting in a positive feed-forward that strengthens N-Myc oncogenic programs [START_REF] Xiao | Polo-like Kinase-1 Regulates Myc Stabilization and Activates a Feedforward Circuit Promoting Tumor Cell Survival[END_REF].

Recently, a study using integrated network analysis and machine learning approach unveiled 12 upregulated genes in triple-negative breast cancer, amongst which PLK1 and Myc were found.

Due to aggressiveness of this breast cancer subtype and the lack of approved targeted therapies, the identification of key hub genes and pathways may serve to the development of potential targeted drugs [START_REF] Naorem | Integrated network analysis and machine learning approach for the identification of key genes of triple-negative breast cancer[END_REF].

Interplays between PLK1 and viruses

PLK1, as seen before, is a key protein for the cell-cycle and could be considered as a bona fide oncogene. Frequently, viral infections and oncogenesis affect common cellular mechanisms and pathways, aiming to enhance cell division and escapement from immune system to spread.

Interestingly, studies showed deregulation of PLK1 expression and/or activity during viral infections.

In this part, examples from hepatitis C virus (HCV), human papillomavirus (HPV), influenza A virus and parainfluenza virus 5 (PIV5) are detailed (Figure 36).

Interplays between PLK1 and HCV

HCV is a positive strand RNA virus. It encodes a unique polyprotein, further processed into four structural proteins (Core, E1, E2 and p7) and six non-structural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (Chen et al., 2010). NS5A exists in basal phosphorylated form (p56) and in hyperphosphorylated form (p58) and these post-translational modifications are of importance for HCV replication [START_REF] Ross-Thriepland | Hepatitis C virus NS5A: enigmatic but still promiscuous 10 years on![END_REF]. In cells transfected with PLK1-specific shRNA, HCV replication and NS5A hyperphosphorylation were significantly reduced, indicating that PLK1 kinase activity is required for this process. In adequation with these results, overexpression of PLK1 in the knockdown cells rescued HCV replication. Moreover, evidences indicated that PLK1

immunoprecipitates and colocalizes with NS5A. At last, it has been shown that PLK1 induces both phosphorylated and hyperphosphorylated forms of NS5A, highlighting further the importance of this kinase for HCV replication (Chen et al., 2010).

Interplays between PLK1 and HPV

HPV infection often causes no symptoms and resolve spontaneously, however the infection may persist causing warts and precancerous lesions. Up to 40 types of HPV are typically transmitted through sexual contact but HPV-16 and HPV-18 are involved in nearly all cervical cancers [START_REF] Castle | Impact of human papillomavirus vaccination on the clinical meaning of cervical screening results[END_REF]. The E2 protein of HPV is essential for viral life cycle. Through a yeast two-hybrid screening,

Wang and colleagues have shown that PLK1 interacts and phosphorylates HPV-5 E2 [START_REF] Wang | Interaction between human papillomavirus type 5 E2 and polo-like kinase 1[END_REF]. However, HPV-16 E2 does not interact with PLK1, suggesting a E2 variability between HPV subtypes. Moreover, both HPV-5 E2 and PLK1 are able to interact with Brd4, this latter being known to promote G1 and S phases' progression [START_REF] Mochizuki | The bromodomain protein Brd4 stimulates G1 gene transcription and promotes progression to S phase[END_REF]. During HPV infection, Brd4 acts as a transcriptional cofactor of E2. HPV-5 E2 may form a stable complex with Brd4, preventing its phosphorylation by PLK1, leading in fine to deregulated cellular functions of Brd4.

Interplays between PLK1 and influenza A virus

Influenza is a respiratory disease caused by influenza A and B viruses. Every winter, season epidemics result in high economical losses due to sick days. Currently, vaccines and antiviral drugs are available, but both come with limitations. In recent years, several genome-wide RNAi screenings have unveiled cellular factors required for influenza virus infections in human, in order to establish some of them as drug targets. PLK1 was found to be one of these hits in four independent screens and in 2017, Pohl and colleagues reported that knockdown of PLK1 as well as kinase activity inhibition by four different compounds resulted in a loss of influenza A replication in infected human lung cells. This phenotype was reported to be specific of PLK1 activity with no measurable toxicity and thus established PLK1 as potential drug target [START_REF] Pohl | Identification of Polo-like kinases as potential novel drug targets for influenza A virus[END_REF]. death and cytokine production, thus, authors hypothesized that PIV5 limits its viral gene expression through PLK1-dependent phosphorylation of P, in order to avoid these host effects [START_REF] Sun | PLK1 down-regulates parainfluenza virus 5 gene expression[END_REF]. Interplays between PLK1 and viruses", PLK1 is largely involved in both carcinogenesis process and virus replication, mainly by endorsing proviral roles for this latter. HBV being responsible for an estimated >50% of HCC globally, the community went naturally to study the involvement of PLK1 in this virus-induced oncogenic transformation. In the late 2000s, there was still debate on the exact implication of PLK1 regarding cellular transformation. On the one hand, PLK1 could be a key driver of carcinogenesis, mostly by promoting cell proliferation and genetic instabilities. On the other hand, the overexpression of PLK1 could be just a side effect of other key driver events and reflect solely the high proliferative rate observed in tumours [START_REF] Cholewa | The role of polo-like kinase 1 in carcinogenesis: cause or consequence?[END_REF]. However, rencently, several studies have pointed out PLK1 to be a key driver of hepatocarcinogenesis. In a study from Wang and colleagues aiming at deciphering the key signaling pathways, genes and transcription factors associated with hepatocellular carcinoma, PLK1 was defined as a key gene involved in HCC development [START_REF] Wang | Key signaling pathways, genes and transcription factors associated with hepatocellular carcinoma[END_REF]. Furthermore, in 2017, a paper published in Cancer Cell reported that PLK1 is a key driver of HCC subtype C1, this latter being mainly associated to HBV [START_REF] Chaisaingmongkol | Common Molecular Subtypes Among Asian Hepatocellular Carcinoma and Cholangiocarcinoma[END_REF].

PLK1 and the mechanisms behind HBV-induced cellular transformation

In an in vitro model of murine hepatocyte transformation featuring the overexpression of the HBx protein and selection of polyploidy events, it was shown that PLK1 was the main driver of the process (Studach et al., 2010). Some of the PLK1-mediated oncogenic mechanisms were elucidated.

The group of Dr Andrisani has shown that PLK1 induces the degradation of SUZ12 and ZNF198, two components of the polycomb repressive complex (PRC2) and LSD1-CoREST-HDAC1 complex, which are involved in gene repression. This is associated with the re-expression of stemness genes (e.g., BAMBI, EpCAM, Myc…), which are also found overexpressed in HBV-related HCC (Wang et al., 2011).

Moreover, a link with the long noncoding RNA HOTAIR, which is found overexpressed in many cancers, was established. HOTAIR helds together the two repressive complexes PRC2 and LSD1-

CoREST-HDAC1 and it was shown that PLK1-dependent ubiquitination of SUZ12 and ZNF198 was enhanced by expression of HOTAIR. Clinically, the overexpression of HOTAIR and PLK1 was correlated with aggressiveness of the tumours that are more prone to develop satellite nodules (Zhang et al., 2015).

In addition to the mechanisms described above which linked HBV-induced carcinogenesis and PLK1, mostly by modifying the epigenetic landscape, the L-HBsAg was also unveiled as potent HCC-inducer and related to PLK1. In The Journal of Pathology, Li and colleagues reported that expression of L-HBsAg was a priming event in HCC development by inducing cytokinesis failure and consequent aneuploidy through induction of DNA damage and overexpression of PLK1 in hepatocytes. Interestingly this PLK1 overexpression was associated with G2/M checkpoint overriding, promoting cell-cycle progression and genomic instabilities. Inhibiting PLK1 resulted in G2/M checkpoint restauration and in a decrease of tumour formation incidence, tumour volume and tumour weight in vivo (Li et al., 2018).

In the section "2.3.4. Mechanisms of HBV-induced HCC", several mechanisms involved in HBV-induced oncogenesis where reviewed. HBV acts as a weak oncovirus and cannot lead to one-hit cellular transformation, thus several interplays are more likely coopting to induce oncogenesis.

Moreover, the hepatocellular carcinoma is well known as a multifactorial originating cancer and could not be defined by a single event. In this respect, several studies have identified PLK1 to be part of this HBV-induced transformation and inhbiting PLK1 could be of benefit for anti HBV-related HCC therapy.

PLK1 as a target for anticancer drugs

Due to its large involvement in the cell-cycle progression and in pathways that are related to carcinogenesis, PLK1 has been considered as a druggable target for the development of new therapies in a wide variety of cancers. In this respesct, remarkable efforts from academics and private companies are underway. Two major class of chemical inhibitors of PLK1 are available, either acting in an ATP-competitive manner or by interfering with the PBD to suppress protein-protein interactions. Furthermore, genomic tools such as siRNA targeting PLK1 are under development/investigation. In the part below, the principal anti-PLK1 molecules are overviewed. For a more detailed review, please refer to "Plk1 inhibitors in cancer therapy: From laboratory to clinics" from Gutteridge and colleagues (Gutteridge et al., 2016).

BI-2536 is one of the earliest PLK1 inhibitors and was developed by Boehringer Ingelheim Company. In 2007, Steegmaier and colleagues reported that the ATP-competitive BI-2536 inhibits tumor growth in vitro as well as in vivo [START_REF] Steegmaier | BI 2536, a potent and selective inhibitor of polo-like kinase 1, inhibits tumor growth in vivo[END_REF]. Furthermore, BI-2536 was found to exhibits a relatively safe profile in clinical trials [START_REF] Frost | Phase i study of the Plk1 inhibitor BI 2536 administered intravenously on three consecutive days in advanced solid tumours[END_REF]. However, this molecule is no longer used in monotherapy, mostly due to modest response rates observed in phase 2 trial for the treatment of unresectable exocrine adenocarcinoma of the pancreas [START_REF] Mross | A randomised phase II trial of the Polo-like kinase inhibitor BI 2536 in chemo-naïve patients with unresectable exocrine adenocarcinoma of the pancreas -a study within the Central European Society Anticancer Drug Research (CESAR) collaborative network[END_REF]. BI-2536

showed excellent results when combined to other targeted therapies such as synergistic effects observed with Vincristine in neuroblastoma [START_REF] Czaplinski | Polo-like kinase 1 inhibition sensitizes neuroblastoma cells for vinca alkaloid-induced apoptosis[END_REF] or with Eribulin in rhabdomyosarcoma [START_REF] Stehle | Eribulin synergizes with Polo-like kinase 1 inhibitors to induce apoptosis in rhabdomyosarcoma[END_REF].

Volasertib, known as BI-6727, is a second generation of PLK1 inhibitors and was also developed by Boehringer Ingelheim by tailoring the dihydropteridinone structure of BI-2536 [START_REF] Rudolph | BI 6727, a Polo-like kinase inhibitor with improved pharmacokinetic profile and broad antitumor activity[END_REF]. Volasertib is a potent molecule in inhibiting mitosis with very low IC50 of 0.87μmol/L. First studies using Volasertib were shown to impede proliferation in preclinical models of acute myeloid leukemia (AML) [START_REF] Rudolph | Efficacy and mechanism of action of volasertib, a potent and selective inhibitor of Polo-like kinases, in preclinical models of acute myeloid leukemia[END_REF] and to induce cell mortality in bladder cancer [START_REF] Brassesco | In vitro targeting of Polo-like kinase 1 in bladder carcinoma: comparative effects of four potent inhibitors[END_REF]. In order to improve the efficacy, Volasertib was combined to other therapies and has shown synergystic effects with the MEK inhibitor Trematinib [START_REF] Posch | Combined Inhibition of MEK and Plk1 Has Synergistic Antitumor Activity in NRAS Mutant Melanoma[END_REF] and with histone deacetylase (HDAC) inhibitors in vitro [START_REF] Wissing | Targeting prostate cancer cell lines with polo-like kinase 1 inhibitors as a single agent and in combination with histone deacetylase inhibitors[END_REF]. Finally, Volasertib inhibits the subcutaneous xenograft growth of HCC in nude mice, mostly by inducing apoptosis, thereby providing new therapeutic potential of Volasertib on hepatocellular carcinoma [START_REF] Zheng | Volasertib suppresses the growth of human hepatocellular carcinoma in vitro and in vivo[END_REF].

Like many others targeted therapies, Volasertib may become less effective as cancers develop resistances. In 2015, Wu and colleagues reported that overexpression of the ATP-binding cassette (ABC) drug transporter ABCB1 confers resistance to Volasertib (Wu et al., 2015). One may suggest that the use of ABCB1 modulators combined to Volasertib could prevent this mechanism and delay resistance phenomenon. Regarding clinical trials, several have tested Volasertib, in mono or combined therapies, in recent years. Notably, dose escalations to determine the safety profile of Volasertib were done in solid tumours and this drug was considered being well-tolerated [START_REF] Lin | A phase I study of two dosing schedules of volasertib (BI 6727), an intravenous polo-like kinase inhibitor, in patients with advanced solid malignancies[END_REF]). Volasertib appears to have better response rates in some cancers, thus combination therapy trials are being actively pursued. The most advanced study was a phase III trial of Volasertib plus low dose cytarabine LDAC for the treatment of acute myeloid leukaemia, but unfortunately, did not meet the primary endpoint of objective response. The first results were published in 2016 and the primary analysis showed a higher number of patients responded to volasertib plus LDAC (25.2%) than placebo plus LDAC (16.8%) but the overall result was not statistically significant. The occurrence of fatal infection frequency was considered as the major reason for a negative overall survival trend. This trial is still ongoing and updates will be presented in 2019 (NCT01721876).

The Poloxin is the major representative of PLK1 inhibitors shown to block the PBD-dependent protein-protein interactions [START_REF] Reindl | Inhibition of polo-like kinase 1 by blocking polo-box domain-dependent protein-protein interactions[END_REF]. Poloxin leads to mitotic arrest and apoptosis of several cancer cell lines, mostly through modulating Kizuna, a key substrate of PLK1 at centrosomes.

Furthermore, in xenograft mouse models, Poloxin was shown to suppress tumor growth [START_REF] Yuan | Polo-Box Domain Inhibitor Poloxin Activates the Spindle Assembly Checkpoint and Inhibits Tumor Growth in Vivo[END_REF]. In 2015, in order to improve Poloxin efficacy, Scharow and colleagues reported the identification of Poloxin-2, an optimized analog of its predecessor with improved potency and selectivity [START_REF] Scharow | Optimized Plk1 PBD Inhibitors Based on Poloxin Induce Mitotic Arrest and Apoptosis in Tumor Cells[END_REF]. Further studies are still needed to document the anti-cancer activity of Poloxin-2 in mono or combined therapies as well as to define its toxicity.

Rigosertib, also known as ON 01910 acts as a Ras mimetic and as a non-ATP-competitive small molecule inhibitor of PLK1 and PI3K [START_REF] Gumireddy | ON01910, a non-ATP-competitive small molecule inhibitor of Plk1, is a potent anticancer agent[END_REF]. Rigosertib has been largely trialed in clinic with increased overall response rates in severals cancers (Gutteridge et al., 2016). In some cases, PLK1 inhibitors have been employed successfully to overcome acquired Gemcitabine resistance in certain cancer lines. In this respect, a phase 3 clinical trial was conducted to determined the efficacy of Rigosertib plus Gemcitabine compared to Gemcitabine alone in previously untreated metastatic pancreatic cancer. The combination increased the partial response in patients by 6%, however, due to an overall survival decreased by 3 months compared to Gemcitabine alone, the trial was deemed unsuccessfull (NCT01360853).

An innovative approach to inhibit PLK1 tends to emerge and resides in the genomic knockdown of PLK1 using siRNA. In 2002, Spänkuch-Schmitt and colleagues first demonstrated the potential of inhibiting PLK1 by a siRNA approach in order to induce apoptosis in cancer cells [START_REF] Spänkuch-Schmitt | Effect of RNA silencing of polo-like kinase-1 (PLK1) on apoptosis and spindle formation in human cancer cells[END_REF].

Polo-Like-Kinase 1 Is a Proviral Host Factor for Hepatitis B Virus Replication

Ahmed Diab, 1-3* Adrien Foca, 1,2* Floriane Fusil, 2,4 Thomas Lahlali, 1,2 Pascal Jalaguier, 1,2 Fouzia Amirache, 4 Lia N'Guyen, 1,2 Nathalie Isorce, 1,2 Franc¸ois-Lo€ıc Cosset, 2,4 Fabien Zoulim, 1,2,5,6 Ourania Andrisani, 3 and David Durantel 1,2,6 Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC) and current treatments for chronic hepatitis B and HCC are suboptimal. Herein, we identified cellular serine/threonine Polo-like-kinase 1 (PLK1) as a positive effector of HBV replication. The aim of this study was to demonstrate the proviral role of PLK1 in HBV biosynthe-sis and validate PLK1 inhibition a potential antiviral strategy. To this end, we employed physiologically relevant HBV infection models of primary human hepatocytes (PHHs) and differentiated HepaRG cells in conjunction with pharmacologic PLK1 inhibitors, small interfering RNA (siRNA)mediated knockdown, and overexpression of constitutively active PLK1 (PLK1 CA ). In addition, a humanized liver Fah 2/2 /Rag2 2/2 /Il2rg 2/2 (FRG) mouse model was used to determine the antiviral effect of PLK1 inhibitor BI-2536 on HBV infection in vivo. Finally, in vitro PLK1 kinase assays and site-directed mutagenesis were employed to demonstrate that HBV core protein (HBc) is a PLK1 substrate. We demonstrated that HBV infection activated cellular PLK1 in PHHs and differentiated HepaRG cells. PLK1 inhibition by BI-2536 or siRNA-mediated knockdown sup-pressed HBV DNA biosynthesis, whereas overexpression of PLK1 CA increased it, suggesting that the PLK1 effects on viral biosynthesis are specific and that PLK1 is a proviral cellular factor. Significantly, BI-2536 administration to HBV-infected humanized liver FRG mice strongly inhibited HBV infection, validating PLK1 as an antiviral target in vivo. The proviral action of PLK1 is associated with the biogenesis of the nucleocapsid, as BI-2536 leads to its decreased intracellular formation/ accumulation. In this respect, our studies identified HBc as a PLK1 substrate in vitro, and mapped PLK1 phosphorylation sites on this protein. Conclusion: PLK1 is a proviral host factor that could be envisaged as a target for combined antiviral and antitu-moral strategies against HBV infection and HBV-mediated carcinogenesis. (HEPATOLOGY 2017;66:1750-1765).

cancer is the fifth and seventh most common cancer worldwide according to sex and the third most common in terms of death. With approximately 250 million people chronically infected with HBV, according to the latest World Health Organization estimation, many patients with chronic hepatitis B are expected to progress to HCC, despite existing therapies. (1) Although a prophylactic vaccine is available, new infections still happen because vaccination campaigns are not always well-implemented, and the vaccine is not always 100% protective. (2) Furthermore, children of infected mothers still become chronically infected in endemic areas. (2) Current treatments include use of pegylated interferon-a (IFN-a) and/or antiviral nucleoside analogs; the latter require life-long administration, which can result in viral resistance when drugs with a low genetic barrier are used. (3) New and effective therapies are urgently needed. (3) In particular, drugs that target both viral replication and prevent neoplastic transformation could represent a valuable addition to the therapeutic arsenal.

Replication of HBV within hepatocytes involves both nuclear and cytoplasmic events. (4) After viral entry, nucleocapsids are transported to the nucleus, releasing the viral DNA and core/capsid HBV core protein (HBc) into the nucleoplasm. There, the viral genome, initially a relaxed circular partially doublestranded DNA (rcDNA), is converted into covalently closed circular double-stranded DNA (cccDNA), which is responsible for viral persistence. This nuclear form of the genome serves as a template for viral RNA transcription, including pregenomic RNA (pgRNA). (4) In the cytoplasm, pgRNA interacts with HBV polymerase and is packaged into capsids, where it is reverse-transcribed to generate newly synthesized rcDNA. (4) HBc plays an essential and dynamic role in HBV biosynthesis, including selective binding to pgRNA in association with viral polymerase, encapsidation, and reverse-transcription of pgRNA, transport of mature capsids to the site of viral envelopment for virion generation, and transport to the nucleus for recycling. (5) Although the mechanism by which HBc and capsid assembly regulate viral DNA synthesis is not fully understood, it is well established that phosphorylation of HBc has an important role in various aspects of pgRNA reverse-transcription. (5,6) Kinases that mediate HBc phosphorylations include cyclindependent kinase 2 (CDK2), which phosphorylates serine/proline (SP) sites located at the C-terminus of HBc, including S155, S162, and S170. (7,8) Another study has identified additional serine/threonine (S/T) sites as having a role in regulating all aspects of HBV replication, and serine/arginine protein kinase 1 as another kinase that phosphorylates HBc in vitro. (9) Our earlier studies, aiming at understanding the mechanism by which the HBV X protein (HBx) mediates oncogenic transformation of hepatocytes, (10-12) led us to the study of Polo-like-kinase 1 (PLK1), an S/T kinase involved in cell-cycle regulation. (13,14) In physio-logic conditions, PLK1 is required for checkpoint recovery, mitotic entry, and progression, whereas it is overexpressed in many human cancers, including HBV-mediated liver cancer. (13,14) We have shown that HBx activates PLK1 prematurely, allowing propaga-tion of DNA damage to daughter cells by attenuating both DNA repair and p53 apoptosis. (11,12) In a cellular model of HBx-mediated transformation and in liver tumors of animals modeling HBx-and HBV-mediated hepatocarcinogenesis, expression of PLK1 is elevated. (11,12,15,16) However, whether PLK1 is activated during physiologic HBV infection of normal hepatocytes, and whether it has a role in the establishment and maintenance of infection is unknown. To address this issue, in this study, we used HBV infection models of nontransformed primary human

ARTICLE INFORMATION:

From the 1 hepatocytes (PHHs) and differentiated HepaRG (dHepaRG) cells, a bipotential human dedifferentiated hepatocyte cell line that upon differentiation supports HBV infection. (17,18) In this study, we demonstrate that the cellular PLK1 enzyme acts as a proviral host factor for HBV replication in vitro by favoring nucleocapsid formation and subsequent reverse transcription.

In addition, we identified the viral core protein HBc as a PLK1 substrate in vitro and mapped the sites of PLK1 phosphorylation. Finally, we provide evidence that PLK1 inhibitors serve as a novel class of anti-HBV molecules in vitro and validate the antiviral effect of PLK1 inhibitor BI-2536 in HBV-infected liver humanized mice in vivo.

Materials and Methods

CHEMICALS, ANTIBODIES, AND OTHERS REAGENTS

All chemicals were purchased from Sigma-Aldrich unless specified otherwise. PLK1 inhibitors (BI-2536, BI 6727, MLN 0905, and ON 01910) were from Selleckchem. Polyclonal HBc (19) and commercially available HBc antibodies were used for immunofluorescent staining and immunoblots. Rabbit polyclonal anti-HBc was from Dako (B0586) or Abcam (ab140243) and monoclonal anti-HBc from Abcam (ab8637; Clone C1). PLK1 antibodies used: rabbit polyclonal (ab21738) for detection of phospho-PLK1-S 137 in western blotting and immunofluorescence; rabbit monoclonal (ab115095) for detection of phospho-PLK1-T 210 in western blotting and immunofluorescence, and rabbit polyclonal (ab109777) for detection of PLK1. Small interfering RNA (siRNA; SmartPool or sequence designed) were purchased from Dharmacon and transfected into dHepaRG cells or PHHs using Darmafect-1 reagent according to the manufacturer's instructions. Tenofovir (TFV) and HBV capsid assembly inhibitors Bay41-4109 (20) and AT130 (21) were kindly provided by Gilead Sciences and Novira Pharmaceutics, respectively. IFN-a (i.e., Roferon) was obtained from Hoffmann-La-Roche.

HepaRG CELLS AND PRIMARY HUMAN HEPATOCYTES AND INFECTION BY HBV

Human liver progenitor HepaRG cells (18) were cultured for 2 weeks in complete William's medium (Gibco) supplemented with 10% Fetal Clone II serum (Thermo Scientific), 50 U/mL penicillin/streptoMycin (Gibco), 2 mM glutaMax (Gibco), 5 lg/mL human insulin (Sigma), and 5 3 10 25 M hydrocortisone hemisuccinate (UpJohn, SERB) at 378C in humidified CO 2 (5%) incubators and differentiated into hepatocyte-like cells by way of treatment with 1.8% dimethyl sulfoxide (DMSO; Hybrid-max, Sigma) for 2 weeks as described previously. (22) Engineered Hep-aRG cell lines were also used. The HepaRG-TR-PLK1 CA cell line enabling tetracycline-inducible expression of a constitutively active form of PLK1 (i.e., T210D mutant (23) ) was constructed as described for the HepaRG-TR-HBc cell line. (24) PHHs were obtained from surgical liver resections after informed consent of patients (kindly provided by Prof. Michel Rivoire [Centre L' eon B' erard, Lyon, France]; agree-ment numbers DC-2008-99 andDC-2008-101). PHHs were prepared as described previously (25) and were cultured in complete William's medium supplemented with 1.8% DMSO.

HBV INFECTION AND ANALYSIS OF VIRAL PARAMETERS DURING REPLICATION

dHepaRG cells and PHHs were infected as described previously, (26) with HBV inoculum prepared either from HepG2.2.15 or HepaAD38 cells. Intracellular accumulation of viral RNA and DNA, secretion of hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) were monitored by quantitative polymerase chain reaction (qPCR), reverse transcription qPCR (RT-qPCR), southern blotting, and enzymelinked immunosorbent assay (ELISA), as described. (26) Briefly, HBeAg and HBsAg were quantified in culture medium by way of ELISA, using a chemiluminescence immunoassay kit (Autobio, China) according to the manufacturer's instructions. Total DNA was purified from infected cells using MasterPure Complete DNA Purification Kit (Epicentre). Southern blot analysis of HBV DNA was performed using equal amounts of total DNA, electrophoresed on 1.1% agarose gels, hybridized to HBV DNA radiolabeled with [a-32 P]CTP (22) and analyzed by autoradiography. Total RNA was extracted from infected cells using NucleoSpin RNA (Macherey-Nagel) and transcribed into complementary DNA using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA). Real-time PCR for total HBV DNA and cccDNA was performed using LightCycler 96 (Roche) as described previously. (26,27) :s :s :s

TRANSFECTIONS, PLASMIDS, AND SMALL INTERFERING RNAs

Transient transfections in HEK293T cells were performed by employing Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. HBc-WT (EL43), HBc-3A (EL113), and HBc-3D (EL114) were kindly provided by Dr. Daniel Loeb, derived from EL43 plasmid. (28) The CTD-GST fusion protein plasmids GST-HCTD141 (CTD WT) and GST-HCTD141-AAAAAAA (CTD 7A) (7) were a kind gift from Dr. Jianming Hu. siRNAs (Smart pool grade; Dharmacon) for PLK1, HBV, and hepatitis C virus (HCV) (5-25 nM) were transfected in HBV-infected HepaRG or PHHs using Dharmafect I (Dharmacon) following the manufacturer's protocol.

HUMANIZED FRG MOUSE MODEL AND HBV INFECTION

All animal studies were reviewed and approved by our local ethics committee. Highly immunosuppressed FRG mice deficient in T cells, B cells, and natural killer cells were used (29) and maintained in a pathogenfree facility. High-quality cryopreserved human hepatocytes were purchased (BD, Biosciences) and injected through an intrasplenic route into 2-to 3-month old mice as described previously. (30) Liver humanized FRG mice featuring serum production of human albumin, at least 5 mg/mL, were infected with 200 lL of HBV inoculums (1.10 8 virus genome equivalents [veg] to 1.10 9 veg in phosphate-buffered saline) through an intraperitoneal route. (30) Mice were treated with intraperitoneal injection of BI-2536 (10 mg/kg twice a week) for 1 month. Serum was harvested every week by retro-orbital bleeding and stored at -80°C in aliquots for further antigenemia and viremia analysis. Mice were sacrificed at week 8 postinfection and hepatic tissues were frozen and processed for virologic parameter analyses or fixed in formalin and embedded in paraffin for immune-staining.

CAPSID MIGRATION ASSAY

The intracellular formation/accumulation of HBV nucleocapsids in in vitro infected hepatocytes or in mousederived liver resection was accessed from cell or liver lysate by way of native agarose gel electrophoresis followed by transfer onto the enhanced chemiluminescence membrane and western blot analysis, as described previously. (6,31)

IN VITRO PLK1 KINASE ASSAYS

Assays were performed as described previously (10) using recombinant PLK1 (BPS Bioscience, Protein One). Core protein was immunopurified from the HepaRG-TR-HBc cell line or purchased from Meridian Life Science, Inc. Site-directed mutagenesis of putative PLK1 phosphorylation sites in HBc-WT and HBc-3D was performed employing the Quick-Change Lightning site-directed muta-genesis Kit (Agilent). Point mutations in the GST-CTD-WT and GST-CTD-7A plasmids were introduced following the same procedure. Mutations were confirmed by DNA sequencing. For protein staining, PageBlue Protein Staining Solution (ThermoFisher) was used according to the manufacturer's protocol.

STATISTICAL ANALYSIS

Statistical analysis was performed using two-way analysis of variance, t tests, or nonparametric Mann-Whitney tests using GraphPad Prism software. For all tests, P 0.05 , P 0.01, and P 0.001 were considered statistically significant.

Results

PLK1 IS ACTIVATED BY HBV INFECTION IN NON-DIVIDING/ DIFFERENTIATED HEPATOCYTES

Our earlier studies demonstrated that (1) HBx activates the mitotic S/T kinase PLK1, in a conditional HBx-expressing cell line, (11) (2) PLK1 activation initiates proteasomal degradation of chromatin modifying nuclear proteins SUZ12 and ZNF198, (10) and ( 3) SUZ12 down-regulation in HBV replicating hepatocytes results in expression of hepatic cancer stem cell markers and pluripotency genes. (32) We have also shown activation of PLK1 in HBV-replicating HepAD38 cells, (10) further suggesting a link between HBV infection and PLK1 activation. However, it has yet to be determined whether PLK1 activation occurs in the context of physiologic infection of nondividing, differentiated, and nontransformed hepatocytes. To this end, PHHs and dHepaRG cells were infected with HBV and expression and activation of PLK1 was quantified. Upon infection of dHepaRG cells, PLK1 messenger RNA (mRNA) increased by 15-fold 24 hours postinfection, followed by a constant level of expression of 3

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to 5-fold from 48 to 168 hours postinfection (Fig. 1A). This resulted in a transient increase in PLK1 protein levels (Fig. 1B). More interestingly, an increase in PLK1 phosphorylation on S137 and/or T210, indicative of PLK1 activation, was detected as a function of HBV infection by immunoblotting (Fig. 1B) and immunofluorescence microscopy (Fig. 1C) employing phospho-specific PLK1 antibodies. Remarkably, this activation of PLK1 by HBV infection was also detected by immunoblots of lysates from various preparations of PHHs (representative blots are shown in Fig. 1D).

PLK1 INHIBITORS, INCLUDING BI-2536, SUPPRESS HBV DNA ACCUMULATION IN PERSISTENTLY HBV-INFECTED HEPATOCYTES

To test whether PLK1 activation has a proviral effect, dHepaRG cells were infected with HBV virions and on day 7 postinfection, when infection had reached a plateau in terms of viral replication, cells were treated with various PLK1 inhibitors. Specifically, increasing concentration of adenosine triphosphate (ATP)-competitive (i.e., BI-2536 and BI 6727) or non-ATP-competitive (i.e., MLN 0905 and ON 01910) PLK1 inhibitors were added for 3 days; accumulation of intracellular HBV DNA was measured by way of Southern blotting and qPCR (Supporting Fig. S1A,B). Among all drugs tested, BI-2536 was the most active and selected for further studies, after monitoring batch-to-batch stability (Supporting Fig. S2). HBV-infected dHepaRG treated with BI-2536 on day 7 postinfection exhibited no effect on levels of secreted HBeAg and HBsAg, or intracellular viral RNA (Fig. 2A). Interestingly, BI-2536 significantly inhibited synthesis of total HBV DNA, with an effective concentration at 50% (EC 50 ) of approximately 4 nM (Fig. 2A), in the absence of any toxicity (cytotoxic concentration at 50%) of approximately 10 lM Fig. 2B). Interestingly, BI-2536 reduced the level of capsid (i.e., assembled HBc/core protein) detected by immunofluorescence microscopy (Fig. 2C) with an antibody recognizing HBc assembled into capsid, and not monomeric forms of HBc (Supporting Fig. S3). Finally, BI-2536 was shown to strongly (up to 95%) and dose-dependently reduce the formation/accumulation of HBV nucleocapsids in infected cells (Fig. 2D). Taken together, these results demonstrate that PLK1 inhibition by BI-2536 affects the assembly of HBV capsids, as specific HBc assembly inhibitors (such as BAY41-4109 or AT130) do, thereby inhibiting subsequent rcDNA synthesis. Similar results were also obtained in HBV-infected PHHs (Fig. 3), with an EC 50 at approximately 50 nM. Interestingly, in this model, a trend toward an increase in intracellular viral RNA was observed (Fig. 3A), thus inversely correlat-ing with total DNA decrease. This corroborates the hypothesis that rcDNA synthesis is likely inhibited by an impaired assembly of capsids around pgRNA.

We also analyzed the effect of BI-2536 on the establishment of HBV infection and found that the antiviral effect was even greater with not only an effect on neosynthesis of HBV DNA but also effect on other measured viral parameters (Supporting Fig. S4).

LOSS-OF-FUNCTION OF PLK1 SUPPRESSES HBV DNA ACCUMULATION IN INFECTED HEPATOCYTES

To conclusively establish that PLK1 is a proviral factor, we investigated effect of loss of function of PLK1 on HBV replication by siRNA-mediated knockdown of PLK1 (siPLK1). As controls, we employed siRNAtargeting HBV transcripts (siHBV; positive control), and siRNA against HCV (siHCV) serving as negative control. The effectiveness of PLK1 knockdown by siRNA transfection was determined by quantification of PLK1 mRNA levels as a function of time after transfection (data not shown). Transfection of siPLK1 reduced by nearly 90% endogenous level of PLK1 mRNA at 72 hours after transfection, whereas all nontargeting siRNAs had no effect on PLK1 mRNA levels (Fig. 4A). Trans-fection of increasing amounts of siPLK1, ranging from 5 to 25 nM in HBV-infected dHepaRG cells, resulted in a progressive reduction of 50% to nearly 70% in the accu-mulation of intracellular HBV DNA (Fig. 4A). By con-trast, transfection of 25 nM siHCV had a minimal effect, whereas transfection of 25 nM siHBV reduced HBV DNA by nearly 80% (Fig. 4A). With siPLK1, we again observed a decreased accumulation of assembled HBc within infected cells by way of immunofluores-cence using an antibody recognizing assembled cap-sids (Fig. 4B) and by way of native capsid migration assay (Fig. 2D), thus confirming the potential rela-tionship between proviral action of PLK1, formation of HBV capsids, and decrease in newly synthetized rcDNA. Similar results were obtained in HBV-infected PHH cultures transfected for 72 hours with siRNAs targeting PLK1, HBV, or an irrelevant target (Fig. 4C). Specifically, Southern blot analyses dem-onstrated a 50% reduction in HBV DNA by transfec-tion of siPLK1, a 10% reduction with siHCV, and a 65% reduction with siHBV (Fig. 4D).

• • • • • • • • • • • • • • • • • • •• •• •• •• •• •• •• • •• •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• • •• •• •• • • FIG.
• • • • • • • • • • • • • • • • • •• •• •• •• •• •• •• • •• •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• • •• •• •• • • • • • • • • • • • • • • • • • • • • •• •• •• •• •• •• •• • •• •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• • •• •• •• • • FIG. 3.
Effect of BI-2536 on HBV replication in nondividing and nontransformed PHHs. PHHs were infected with 100 vge/cell of HBV for 4 days followed by treatment with increasing concen-tration of PLK1 inhibitor BI-2536, as indicated, for 3 days. (A) Quantification of secreted HBsAg and HBeAg by ELISA using supernatants of HBV-infected PHHs on day 7 postinfection. Total RNA and DNA extracted from HBV infected cells, day 7 postin-fection, were analyzed by HBV-specific RT-qPCR or qPCR, nor-malized to housekeeping genes, and compared with mock-treated cells. Fold change in expression or secretion quantified relative to untreated controls are expressed as the mean 6 standard error of the mean of at least three independent experiments. (B) Represen-tative Southern blotting (n 5 3). Total DNA extracted from cells on day 7 postinfection was analyzed by way of agarose gel electro-phoresis, transferred onto a nylon membrane, and hybridized to a radioactive HBV probe as described previously. (22) •

• • • • • • • • • • • • • • • • • •• •• •• •• •• •• •• • •• •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• • •• •• •• • • Thesis Adrien FOCA 112 FIG.
• • • • • • • • • • • • • • • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• • •• •• •• • Thesis Adrien FOCA

113

A MODEST GAIN-OF-FUNCTION OF PLK1 IS ASSOCIATED WITH INCREASED HBV REPLICATION

To investigate the effect of gain-of-function of activated PLK1 on HBV replication, we generated an HepaRG cell line expressing a constitutively active form of PLK1 (PLK1 CA ) in a tetracycline inducible manner (i.e., Hep-aRG-TR-PLK1 CA ) (Supporting Fig. S5A). HBVinfected dHepaRG-TR-PLK1 CA cells were induced to express PLK1 CA on day 7 postinfection by addition of increasing amounts of tetracycline (0, 16, 80, and 400 ng/ mL) for 3 days. Quantification of viral DNA and secreted antigens was performed day 10 postinfection. Under these conditions of established infection, a modest expression of PLK1 CA enhanced HBV replication by 1.5-to 2-fold, whereas no effect on HBeAg secretion was observed (Supporting Fig. S5B). This result suggests that active PLK1 enhances the formation of new viral genomes through reverse transcription of pgRNA; that is, it exerts a positive effect on capsid-assisted reverse transcription of pgRNA.

REDUCTION OF HBV VIREMIA IN LIVER-HUMANIZED INFECTED FRG MICE TREATED WITH BI-2536

To demonstrate the effect of BI-2536 on inhibition of HBV replication in vivo, we employed liverhumanized FRG mice (30) infected with HBV (5.10 8 vge/mouse). Once serum viremia reached 7 log 10 vge/ mL, antigenemia 5 log 10 IU/mL for HBsAg, and 2500 net control unit (NCU)/mL for HBeAg at week 4 postinfection, treatment was administered by way of intraperitoneal injection of 10 mg/kg of BI-2536 (or same concentration of DMSO diluted in saline) biweekly for 4 weeks. Serum viremia and antigenemia were monitored at the end of weeks 1, 2, 4, 6, 7, and 8, whereas weeks 6, 7, and 8 were those under treatment. Treated mice exhibited at week 8 a mean reduction of 1.406 log 10 ± 0.26 in viremia (Fig. 5A), whereas a minimal effect was observed on antigenemia (Fig. 5B,C). Intraliver analyses using samples collected at euthanasia confirmed the reduction of total HBV DNA while showing no change in HBV RNA and cccDNA, further confirming the antiviral phenotype of BI-2536 (Fig. 5D). If immunohistochemistry staining with a polyclonal anti-HBc antibody, which recognize all forms of HBc (Supporting Fig. S3), revealed a slight decrease in accumulation of this protein at the end of the treatment period (representative immuno-histochemistry [IHC] shown in Fig. 6A), an analysis of the accumulation of assembled capsid by native cap-sid migration assay confirmed the inhibitory effect of BI-2536 (representative IHC is shown in Fig. 6B,C). This antiviral effect was obtained in the absence of toxicity as measured by monitoring mouse weight, secretion of human albumin in serum, and level of alanine aminotransferase (Supporting Fig. S6). It is worth noting that similar duration of treatment with IFN-a (25 lg/kg twice a week; n = 5 mice treated) or entecavir (2.5 lg/mouse per day in drinking water; n = 3 mice treated) in liver-humanized FRG mice led to a viremia reduction of 0.668 log 10 ± 0.18 or 1.919 log 10 ± 0.23, respectively (Supporting Fig. S7). These results indicate that a mean reduction of 1.406 log 10 ± 0.26 in monotherapy obtained with BI-2536 was relevant and in between performance of IFN-a and entecavir in this model.

• • • • • • • • • • • • • • • • • • •• •• •• •• •• •• •• • •• •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• • •• •• •• • • FIG. 5.
Effect of BI-2536 on HBV replication in liver humanized HBV-infected FRG mice. Eight mice engrafted with human PHHs for 2 months were infected with HBV (5.10e8 vge/mouse). At week 4 postinfection, treatment was initiated as indicated. BI-2536 (10 mg/kg) was injected twice per week intraperitoneally for 4 weeks. Blood was collected at the end of weeks 1, 2, 4, 6, 7, and 8 and viremia (A) and antigenemia (B, C) were monitored by way of qPCR and ELISA. From liver pieces, intrahepatic HBV DNA (D.i), RNA (D.ii), and cccDNA (D.iii) were quantified respectively by qPCR, RT-qPCR, and cccDNA-specific qPCR. (27) •

• • • • • • • • • • • • • • • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• • • • •• •• •• •• •• •• •• •• • •• •• •• •• •• •• •• • • • •• •• •• •• • •• •• •• • • Thesis Adrien FOCA 115 • • • • • • • • • • • • • • • • • • •• •• •• •• •• •• •• • •• •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• • •• •• •• • • FIG.
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116

COMBINATION OF BI-2536 WITH APPROVED OR INVESTIGATIONAL ANTI-HBV DRUGS

Our in vitro and in vivo data demonstrated that PLK1 inhibitors could be used as efficient and safe anti-HBV drugs. PLK1 inhibitor, including BI-2536, seems to target the capsid-associated reverse transcription of pgRNA into rcDNA. Other drugs also target this step of the viral life cycle, including nucleoside/ nucleotide analogs and core inhibitors (also called core protein allosteric modulators). However, all of these drugs have different molecular targets and therefore could be used in combination to enhance the inhibitory phenotype. To establish whether BI-2536 could be used in combination with nucleoside/nucleotide analogs or core protein allosteric modulators without leading to antagonistic effect, we combined these molecules to treat infected dHepaRG cells. A combination of BI-2536 and IFN-a was also included. As expected, only BI-2536 1 IFN-a treatment led to a reduction in HBeAg secretion (Fig. 7A), whereas all other combinations led to a sharp reduction of intracellular HBV DNA (Fig. 7B). No obvious antagonism was evidenced for all combinations tested, thus suggesting that these drugs could be further considered for combination studies.

HBc IS A SUBSTRATE FOR PLK1 IN VITRO

During established infection, inhibition of PLK1 by treatment with BI-2536 suppressed only viral DNA synthesis (Figs. 2A, and3A,B), suggesting that PLK1 may have a role in capsid-associated reverse transcription of pgRNA into rcDNA. Conversely, inhibition of PLK1 could prevent these processes, based on capsid assembly. Because HBc has an essential and dynamic role in this process of capsid assembly, pgRNA encap-sidation, and rcDNA biosynthesis, we investigated whether HBc is a PLK1 phosphorylation substrate.

To determine whether HBc is a direct target of PLK1, we performed in vitro kinase assays with puri-fied recombinant HBc and PLK1 (Fig. 8). We observed in vitro PLK1 phosphorylation of HBc using either recombinant HBc or HBc isolated from HepaRG-TR-HBc cells (Fig. 8A); furthermore, addition of 500nM BI-2536 inhibited this phosphorylation (Fig. 8A, left panel). The Cterminal domain of HBc (CTD), spanning amino acids 143 to 183 (Fig. 8B), contains 8 S/T residues.

We performed in vitro PLK1 kinase assays with GST-CTD fusion peptide isolated from transfected cells and featuring either wild-type or mutated CTD sequences at all S/T sites. This approach was validated by showing that fusion protein with seven (not including T147) or all eight S/T posi-tions mutated exhibited absence of PLK1 phos-phorylation (Fig. 8B). PLK1 requires a priming phosphorylation of its substrate, usually by CDK1 or CDK2 at SP sites. Interestingly, HBc is known to be phosphorylated by CDK2 at three SP phosphorylation sites (Fig. 8B). Accordingly, we tested whether HBc proteins containing S to A or S to D substitutions at all three SP positions (labeled as 3A or 3D) could serve as PLK1 substrates. Interestingly, only the phosphomimetic substitutions-S155D, S162D, and S170D (referred to as 3D)-exhibited phosphorylation by PLK1 (Fig. 8C). Next, we observed that S168A exhibited significantly reduced phosphorylation in the context of the phospho-mimetic substitutions at SP sites, whereas no difference in the phosphorylation by PLK1 was detected by T146A and T147A substitutions (Fig. 8D). Single point mutations at residues 168, 176, and 178, again in the context of the phospho-mimetic substitutions at SP sites, resulted in significant reduction of HBc phosphorylation by PLK1 (Fig. 8E). Therefore, we conclude that S168, S176, and S178 are PLK1 phosphorylation sites in vitro.

Discussion

The hepatitis B virus (HBV) is one of the most important human oncogenic viruses. (1,2) Approximately 250 million individuals worldwide are chronically infected with HBV, having increased risk for developing HCC. (1,2) If clinicians dispose of several anti-HBV drugs, they have limited chemotherapeutic options for HCC. It is thus crucial to identify novel molecules that target both virus biosynthesis and neoplastic transformation. In this study, we provide evidence that PLK1, a kinase already demonstrated to be involved in HCC, could represent a potential target to tackle both HBV replication and liver oncogenesis. Earlier studies demonstrated that HBx activates PLK1 in a hepatocyte model cell line (11) and that PLK1 was activated in the HepAD38 cellular model of HBV replication. (10) However, this cellular model of viral replication does not model physiologic infection. In this study, using physiologically relevant HBV infection models comprising PHHs and HepaRG cells, we show that HBV infection leads to early activation of PLK1. Here, we show that PLK1 phosphorylation occurs upon HBV infection in nondividing and differentiated cells; the mechanism of PLK1 activation during HBV infection is yet to be determined. HBx protein, remains the main candidate for this activation likely mediating PLK1 activation via the

• • • • • • • • • • • • • • • • • • •• •• •• •• •• •• •• • •• •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• • •• •• •• • • FIG. 7.
Combination treatment of BI-2536 and tenofovir, IFN-a, or Bay41-4109. dHepaRG cells were infected with 100 vge/cell of HBV for 7 days, followed by treatment with the indicated concentration of drugs for 3 days. (A) HBeAg secretion in supernatant was monitored by way of ELISA. (B) Intracellular HBV DNA was quantified by way of qPCR.

• • • • • • • • • • • • • • • • • • •• •• •• •• •• •• •• • •• •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• • •• •• •• • • Thesis Adrien FOCA 118 • • • • • • • • • • • • • • • • • • •• •• •• •• •• •• •• • •• •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• • • • •• •• •• •• • •• •• •• • • FIG. 8.
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Thesis Adrien FOCA 119 p38-MAPK. (11,33) Here, we provide evidence for the functional advantage to the viral life cycle of this early activation of PLK1 by HBV infection.

Employing PLK1 pharmacological inhibitors and an RNA interference approach, we have shown that PLK1 enzymatic activity is crucial for maintenance of HBV replication in nondividing hepatocytes. Indeed, BI-2536, an ATP-dependent PLK1 inhibitor, drastically inhibited HBV replication in vitro in both HepaRG cells and PHHs, as well as in vivo in liver humanized FRG mice, without toxicity. Interestingly, the antiviral activity of BI-2536 was potent, with an EC 50 ranging from 5 to 50 nM, depending on the cell type used; such an antiviral efficacy is rarely obtained with host targeting agents and corresponds to the efficacy of direct acting agents including nucleoside analogs or assembly inhibitors. Accordingly, an in vivo antiviral activity was also obtained with BI-2536 in HBV-infected liver humanized FRG mice, and this antiviral activity was comparable to that obtained with entecavir or IFN-a in the same model. The RNA interference (RNAi) approach confirmed the PLK1 specificity, as kinase inhibitors are often nonspecific. The antiviral phenotype obtained by RNAi targeting PLK1 was also very strong in vitro. The use of such an approach in vivo using siRNA that could be specifically targeted to infected hepatocytes could represent an interesting alternative to pharmacological inhibitors, which may be quite toxic when administrated systemically. Recently, it has been shown that RNAi nanoparticles in complex with PLK1 siRNA, administered systemically, efficiently delivers PLK1 siRNA to the liver with no observable toxicity. (34) Finally, we have also shown that BI-2536 could be combined in vitro and presented no antagonism to currently approved (i.e., IFN-a and tenofovir) anti-HBV drugs and Bay41-4109, (20) a representative core inhibitor, the latter being a novel class of inhibitors in current clinical development. (5) The molecular mechanism underlying the proviral role of PLK1 has not been completely determined in this study. Nevertheless, we have shown that a target of PLK1 is the capsid/core protein (HBc), which is responsible for capsid assembly around the pgRNA, and indirectly reverse-transcribed pgRNA, as the latter occurs only within capsids. PLK1-mediated phosphor-ylation requires prior phosphorylated primed sub-strates. Here, we present evidence that HBc is a direct target of PLK1 in vitro, and importantly, efficient phosphorylation of HBc required prior phosphorylation at positions S155/S162/S170, sites shown to be phosphorylated by CDK2. (7,8) Our studies identified S168, S176, and S178 as PLK1 phosphorylation sites in vitro, dependent on prior phosphorylation of S155, S162, and S170. Thus, PLK1-mediated phosphorylation of HBc in a sequential phosphorylation process could directly mediate the dynamic events associated with capsid formation, pgRNA encapsidation, and reverse transcription. Further mechanistic studies and molecular tools (phospho-specific antibodies) are required to fully delineate this mechanism of PLK1 involvement.

Many other viral proteins are directly phosphorylated by PLK1, including the nonstructural 5A protein from HCV, (35) the mumps virus nucleoprotein, (36) and the parainfluenza virus 5 protein. (37) PLK1-mediated phosphorylation of the target can either lead to its degradation or change in function; therefore, PLK1 can either act as a proviral or antiviral factor. For HCV, PLK1 seems to be a proviral factor, as it contributes to the hyperphosphoryaltion of nonstructural 5A, (35) which is a crucial event to switch from a replicative cycle (i.e., amplification of viral RNA) to a productive one (i.e., generation and secretion of novel virions). Nevertheless, the use of anti-PLK1 drugs has only limited value to inhibit HCV replication, because the virus replicates better in vitro in dividing cells, thus precluding nontoxic PLK1 inhibition. However, in the case of HBV, this framework for a potential use of PLK1 targeting drugs does exist. Indeed, HBV only replicates in nondividing and highly differentiated cells. Although we have shown here that an anti-HBV phenotype could be obtained in vivo, more experiments are warranted to further demonstrate the safety of such a host targeting agent-based approach. Many PLK1 pharmacological inhibitors as well as strategies based on PLK1 silencing are in current development for treating several cancers (38) ; a repositioning of such drugs for the treatment of chronic hepatitis B patients could be foreseen. The fact that PLK1 inhibitors could reinforce the inhibition of the neosynthesis of encapsidated rcDNA, as nucleoside analogs already do represent an excellent framework for the potential development of combination therapies. ETV (2.5 g/day) was administrated in drinking water. Blood was taken at the end of weeks 4 and 8 and viremia monitored by qPCR. Polo-Like-Kinase 1: A Key Cellular Target for Anti-HBV Therapy?

M#A M#B M#C M#D M#E M#F M#G M#H M#I m#J M#K 1.
PLK1 is a serine/threonine-protein kinase. The investigators demonstrated that HBV infection transiently elevates PLK1 expression and activates PLK1 ore than 240 million individuals are estimated to be chronically infected with hepatitis B virus (HBV) (1) and face a lifetime risk of developing hepatocellular carcinoma (HCC). Current treatments using nucleoside analogues and interferon are effective in inhibiting HBV replication, but unable to clear virus from all hepatocytes. Thus, an alternative strategy of targeting host factors indispensable for HBV replication began to attract serious thought. In this issue of HEPATOLOGY, Diab et al.

show that Polo-like-kinase 1 (PLK1) fulfils the role of such a host factor and can serve as an anti-HBV target. (2) To mimic the living cell condition for HBV infection, the investigators utilized various models to highlight the role of PLK1 in HBV replication, including nontransformed primary human hepatocytes, differentiated HepaRG cells, and a humanized liver mouse model. kinase activity, which, in turn, phosphorylates hepatitis B core protein (HBc), thereby facilitating capsid formation and DNA synthesis of HBV. HBc has long been known to be essential for multiple steps in HBV biosynthesis, including capsid assembly, encapsidation, and reverse transcription of pregenomic RNA (pgRNA), transport of DNA into the nucleus, virus maturation, and release. (3) HBc performs specific functions by different domains. N-terminal domain is sufficient to form the capsid shell, and the C-terminal domain (CTD) is essential for viral replication. The CTD, covering residues 150-183, of HBc is highly basic and rich in arginine. CTD contains eight potential serine/threonine phosphorylation sites, and some phosphoacceptor residues have been demonstrated to be critical for viral replication. (4) PLK1 is composed of a kinase domain and a Polo-Box domain (PBD). In the current publication, Diab et al. (2) demonstrated that, upon HBV infection, PLK1 could be activated by phosphorylation at S137 and T210, which, in turn, phosphorylates HBc at S168, S176, and S178. How PLK1 phosphorylates HBc remains an interesting question. In general, the PBD of PLK1 can accommodate phosphoserine or phosphothreonine residues to mediate PLK1 interaction with targets. In other words, phosphor-priming by other kinases is required for most PLK1 targets. Several other kinases, including cyclin-dependent kinase 2 (CDK2), protein kinase C, SRPK, serinearginine protein kinase (SRPK) 1, and SRPK2, have previously been known to mediate phosphorylation on HBc. (3) In this study, the investigators showed that mutation of three putative CDK2 phosphorylation sites (S155, S162, and S170) to alanine residues strikingly reduced phosphorylation of HBc by PLK1, implying that HBc may undergo hierarchical phosphorylation. How these sequential phosphorylations proceed is worthy of continued investigation. Interestingly, PLK1 is also involved in hepatitis C virus (HCV) RNA replication as well as virus production by hyperphosphorylation of viral nonstructural protein Abbreviations: CDK2, cyclin-dependent kinase 2; CTD, C-terminal domain; HBc, hepatitis B core protein; HBV, hepatitis B virus; HBx, HBV X protein; HCC, hepatocellular carcinoma; HCV, hepatitis C virus; NS5A, nonstructural protein 5A; PBD, Polo-Box domain; pgRNA, pregenomic RNA; PLK1, Polo-like-kinase 1; rcDNA, relaxed circular DNA; si, small interfering; SRPK, serinearginine protein kinase.

• • • • • • • • • • • • • • • • • • •• •• •• •• •• •• •• • •• •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• FIG. 1.
The role of PLK1 in HBV-mediated HCC. Upon HBV infection, PLK1 is induced and activated by HBx. The phosphorylated PLK1, the active form of PLK1, then phosphorylates HBc to induce HBV replication by facilitating capsid formation and reverse transcription of pgRNA into rcDNA. In addition, the activated PLK1 allows propagation of DNA damage and epigenetic reprogramming, resulting in HBV hepatocarcinogenesis. PLK1 activity can be effectively abolished by PLK1 inhibitor. The boxes colored in blue and orange depict cellular events and viral events, respectively. (5) Intriguingly, like HBV core protein, HCV NS5A can be phosphory-lated by multiple kinases to different degrees of phos-phorylation, suggesting that phosphorylation of viral proteins are under exquisite control, with PLK1 pro-viding the last piece of brick. The important roles of PLK1 in both HBV and HCV replication imply that HBV and HCV may share some common features in the mechanism of viral hepatocarcinogenesis.

• • • • • • • • • • • • • • • • • • •• •• •• •• •• •• •• • •• •• •• •• •• •• •• •• • • • •• •• •• •• •• •• •• 5A (NS5A).
Moreover, the investigators demonstrated that inhibition of PLK1 activity by PLK1-inhibitor BI-2536 or small interfering (si)RNA reduced the formation of HBV capsid and synthesis of rcDNA (relaxed circular DNA). The decreases in rcDNA synthesis may be caused by reduced pgRNA encapsidation, preferential encapsidation of spliced RNAs, and disruption of reverse transcription. Of note, selectivity in RNA encapsidation has been shown to be regulated by the optimum amount of negative charges from phosphoserines located at the CTD of HBc and encapsidated nucleic acids. (6) However, whether PLK1 plays a direct role in RNA encapsidation remains unknown. Further study to delineate the mechanism by which PLK1 affects rcDNA synthesis, for example, pgRNA encapsidation or reverse transcription, will strengthen the key role of PLK1 in regulating HBV replication.

Besides its role in the HBV life cycle, PLK1 is a key player in HBV carcinogenesis. (7) PLK1 plays multiple roles in the cell-cycle and is essential for precisely regu-lating the cell division and maintaining genome stabil-ity in mitosis, spindle assembly, and DNA damage response. Homozygous loss of PLK1 resulted in early embryonic lethality. (8) PLK1 expresses ubiquitously in eukaryotic cells and is highly expressed in most of human cancers, including HCC. (9) PLK1 has been shown to participate in numerous cancer-associated pathways. The role of PLK1 in HBV-related carcino-genesis is focused here. It has been investigated that PLK1 can be activated by HBV X (HBx) protein to allow propagation of DNA damage by simultaneously attenuating the DNA damage checkpoint checkpoint kinase 1 activity and destabilizing DNA repair protein Mre11. (7) In addition, PLK1, together with HOX transcript antisense RNA, induces proteasomal degradation of two transcription repression factors, suppressor of zeste 12 homolog and zinc finger protein 198, to alter the chromatin landscape, leading to epigenetic reprogramming associated with oncogenic transformation. (10) Combined with the current finding by Diab et al., (2) the HBV-infected hepatocyte appears to suffer a double whammy by PLK1 through its enhancing effect on HBV replication and its disruptive effects on cellular DNA repair, resulting in HBV-induced carcinogenesis.

The role of PLK1 in both HBV biology and carcinogenesis makes it an ideal therapeutic target for HBV infection and HBV-induced neoplastic transformation or HCC. PLK1 has been proposed as a therapeutic target for HCC. The recent study demonstrated that Huh-7 cells-transplanted nude mice showed significant tumor regression in siPLK1-treated mice as compared to control mice. (9) Besides, normal human cells are largely unaffected by loss of PLK1, whereas PLK1 depletion promotes apoptosis in cancer cells. These preclinical studies suggest that PLK1 is both a feasible and relevant target for suppressing tumor cell growth. Several PLK1 inhibitors have been tested by clinical trial. However, these small-molecule inhibitors of PLK1 have not achieved a satisfactory therapeutic effect in clinical trials. (11) The monotherapy could be supplemented by combined therapy. The investigators Thesis Adrien FOCA 133 LAI AND SU HEPATOLOGY, December 2017 treated HBV-infected dHepaRG cells with the PLK1-specific inhibitor and approved or investigational anti-HBV drugs, and found that most of these combined treatments had additive or synergistic effects on reducing reverse transcription of pgRNA into rcDNA. It suggests the possibility of combined therapies to combat chronic hepatitis B and HBV/HCVinduced HCC by suppression of viral replication and enhancing the immune response with immunotherapeutic interventions. This study not only revealed the function of HBc phosphorylation, but also suggested a new think-ing on HBV hepatocarcinogenesis. PLK1 could serve as a promising target for HBV/HCVinduced HCC.

Paper in preparation

Expected Submission date: December 2018

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ABSTRACT

A link between HBV and PLK1 has now been clearly evidenced in HBV-driven carcinogenesis, and we have also recently shown that PLK1 is a proviral factor in the early phases of HBV infection. Relative to the latter, we have shown that BI-2536, a chemical PLK1 inhibitor, was very efficient at inhibiting rcDNA neosynthesis, notably by impacting nucleocapsid assembly, via HBc phosphorylation regulation. The benefit of a PLK1 inhibitor could therefore be dual, as it could help inhibiting HBV replication, while preventing/modulating carcinogenesis. Yet, as kinase inhibitors often feature poor selectivity, there is a need for a more specific targeting of PLK1, whether this therapeutic strategy is to be developed in chronic HBV infection.

Here, we analysed in primary human hepatocytes, and in dHepaRG the anti-HBV properties of an LNP-encapsulated PLK1-targeting siRNA (related to TKM-080301, which had shown indications of efficacy in a phase-2 clinical trial NCT01262235 for neuroendocrine tumours and adrenocortical carcinoma). Standard assays were used to monitor the effect of LNP-siPLK1, or controls (LNP-siHBV and LNP-siNon-Targeting), on HBV replication and cell viability.

A dose as low as 100ng/mL LNP-siPLK1 resulted in a >75% decrease in HBV DNA release/viremia, which was comparable to that obtained with LNP-siHBV or 10μM of Tenofovir (TFV), without affecting the cell viability. Interestingly, and in contrast to that obtained with TFV, a strong inhibition of viral RNA and HBe/HBsAg secretions was also observed under LNP-siPLK1 treatment. This correlated with a significant intracellular decrease of vRNA accumulation, which was independent of any change in cccDNA levels, thus indicating a transcriptional or post-transcriptional modulation. Such an effect was not obtained with a biochemical approach of PLK1 inhibition, unveilling an enzymatic-Thesis Adrien FOCA 136 independent role of PLK1. The mechanism behind this phenotype is not fully deciphered, yet LNP-siPLK1 treatment leads to increased amount of vRNA in the nucleus compared to the cytosolic compartment, suggesting an involvement in HBV RNA metabolism.

These results warrant additional investigations in preclinical animal model to further determine the usefulness of such a therapeutic strategy.

INTRODUCTION

Hepatitis B virus (HBV) infection represents the main cause of hepatocellular carcinoma (HCC), which is the 2 nd leading cause of cancer deaths worldwide (WHO data). An estimated 2 billion people have been exposed to HBV and 257 million remain currently chronically infected (CHB) (WHO data; global hepatitis report, 2017). If left untreated, these patients have a 100 fold increased risk of developing HCC compared to the general population [1].

When patients are sucessfully treated, using safe nucleos(t)ide analogs (NUCs), their risk of developing HCC is 2 to 5 times reduced compared to the normal risk [2,3]. The reminiscent risk of HCC development under treatment is likely due to the fact that NUCs do not allow the elimination of an HBV replicative intermediate called cccDNA (i.e., covalently closed circular DNA), which is responsible for viral persistence and rebound when treatment is stopped [4,5]. In addition to the development of drugs that may target cccDNA [4,5], the discovery of host-targeting agents (HTA) acting both on HBV replication and HCC initiation and/or progression could increase the efficacy of the current standards of care (SoC) for curing patients from the infection and associated liver diseases.

We previously identified the serine/threonine polo-like-kinase 1 (PLK1) as such a candidate [6]. PLK1 is known to be overexpressed in many human cancers [7], and is thought to be an important driver of HBV-induced HCC [8][9][10][11]. Moreover, we found that PLK1 is a pro-HBV factor and small molecules inhibiting PLK1 (SM-PLK1i) (e.g., BI-2536) had a negative impact on HBV replication [6]. The inhibition of PLK1 could therefore be of interest for the treatment of CHB patients with potential effects on both virologic and oncologic sides of the disease.

Due to their poor specificity and overall low safety profile, SM-PLK1i cannot be used to treat CHB patients. Interestingly, small interfering RNAs directed against PLK1 were also shown to Thesis Adrien FOCA 138 be efficient at targeting PLK1 oncogenic activity [12]. Chemically modified and injectable versions of PLK1 siRNA were also validated in preclinical cancer animal models [13,14], and one PLK1 siRNA encapsulated in proprietary lipo nanoparticles (LNPs) was clinically trialed in patients with advanced HCC (NCT02191878).

In this study, we found that the use of those LNP-siPLK1 in HBV-infected primary human hepatocytes (PHHs) lead to a decrease in the amount of intracellular HBV RNAs, that subsequently affect the amount of secreted viral particles and HBe/HBs antigens. Knowing that a loss of HBsAg in patients is a clinical end-point goal [3], our finding could have relevant therapeutic implications. Moreover, these data unraveled a novel PLK1 proviral function, which is independent of its kinase activity. Indeed besides being involved in HBV reverse transcription through the regulation of capsid assembly [6], our data suggest that PLK1 is also involved in the regulation of viral RNA accumulation.

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MATERIALS and METHODS

Chemicals, antibodies, and others reagents

PLK1 inhibitor BI-2536 was purchased from Selleckchem. All other chemicals were purchased from Sigma-Aldrich. Rabbit polyclonal anti-HBc antibodies were purchased from Dako (B0586) or Abcam (ab115992) and monoclonal anti-HBc antibodies from Abcam (ab8637; Clone C1). Rabbit polyclonal anti-PLK1 antibodies from Abcam (ab21738) were used for detection of phospho-PLK1-S137 ; rabbit monoclonal anti-PLK1 antibodies from Abcam (ab115095) were used for detection of phospho-PLK1-T210 and rabbit polyclonal anti-PLK1 antibodies from Abcam (ab109777) for detection of total PLK1.

HepaRG and primary human hepatocyte cultures

Human liver progenitor HepaRG cells [15,16] were cultured and differentiated (dHepaRG) as described previously [17]. Primary human hepatocytes (PHH) were freshly prepared from human liver resection obtained from the Centre Léon Bérard (Lyon) with French ministerial authorizations (AC 2013-1871, DC 2013 -1870, AFNOR NF 96 900 sept 2011) as previously described [18].

Production of siRNA-containingliponanoparticles production and use in cell culture

LNP-siPLK1 corresponded to TKM-080301, as described in NCT02191878. Information on the equivalence between molarity of siPLK1 and amount of LNP-siPLK1 in nanogram/mL is given in Table 1. Controls LNP-siRNAs were manufactured the same way. The sequences of siRNA

37,5 500

Corresponding concentrations between naked siPLK1 in nM and LNP siPLK1 in ng/mL

HBV infection and analysis of viral replication

Differentiated HepaRG cells (dHepaRG) and PHHs were infected with HBV genotype D (concentrated from HepAD38 cells supernatants) as described previously [19], [20]. After treatment procedures, levels of intracellular and/or extracellular HBV RNAs and DNAs, HBc, secretion of HBeAg and HBsAg were monitored by quantitative polymerase chain reaction (qPCR), reverse transcription qPCR (RT-qPCR), immunoblot and enzyme-linked immunosorbent assay (ELISA) respectively, as previously described [6,19]. Briefly, HBeAg and HBsAg were quantified in culture medium using a chemiluminescence immunoassay kit according to the manufacturer's instructions. All qPCR analyses were performed with LightCycler™ 96 and 480 systems (Roche), except for cccDNA and pgRNA specific PCRs that were processed under a QuantStudio™ 7 (Applied), using homemade TaqMan assays [19].

Primary human hepatocytes preparation from chimeric FRG mice and use for antiviral

This study was approved by the WuXi IACUC (Institutional Animal Care and Use Committee).

FRG™ (Fah -/-/Rag2 -/-/Il2rg -/-) mice were purchased from Yecuris (Tualatin, OR, USA), engraphted with PHH and infected with 1.10 9 virus genome equivalent (vge) of HBV genotype D. When HBV viremia had reached a maximum (i.e., around 10e9 vge/mL), the liver was digested by in situ collagenase perfusion and recovered hepatocytes seeded in DMEM medium into 24-well plates (2.7.10 5 cells/well). LNP treatments were conducted every 4 days starting at day 0 and BI-2536 treatment was started on day 0 and replenished every tow days. DNA in the culture supernatants was isolated using QIAamp 96 DNA Blood Kit (Qiagen) and quantified by qPCR. HBsAg was quantified using specific ELISA kit (Autobio)

according to the manual.

Separation of cytoplasm and nucleopasm

Extractions of nuclear and cytosolic proteins were performed using NE-PER Nuclear and Cytoplasmic kit (Thermo Scientific), according to the manufacturer's instructions. Sodium orthovanadate, cOmplete™ EDTA-free protease inhibitor cocktail (Roche) and sodium fluoride were added freshly to all lysates. Nuclear and cytosolic RNAs were isolated as described in Weil et al., [21], followed by a purification with TRI reagent (Merck) and precipitation with isopropanol. Purity of nuclear vs cytoplasmic extracts, was assessed by RT-qPCR using primers to amplify the nuclear U6 snRNA (Fw: 5'-CTCGCTTCGGCAGCACATATAC-3' / Rv: 5'-GGAACGCTTCACGAATTTGCGTG-3') and the cytoplasmic Human Tyr-tRNA (Fw: 5'-

CCTTCGATAGCTCAGCTGGTAGAGCGGAGG-3' / Rv: 5'-CGGAATTGAACCAGCGACCTAAGGATGTCC-

3').

Capsid migration assay

The intracellular HBV nucleocapsids were analyzed by agarose electrophoresis in native condition followed by transfer onto an enhanced chemiluminescence membrane (Amersham) and western blot analysis, as described previously [19].

Southern Blotting

Total DNAs extracted with NucleoSpin® 96 Tissue kit (Macherey-Nagel) were pooled, precipitated using 5M NaAc and ethanol and analyzed by Southern blot as previously described [22]. DNAs from HepG2-NTCP cells extracted using a modified Hirt procedure were used as positive controls [23].

Statistical analysis

Statistical analysis was performed using two-way analysis of variance, t tests, or nonparametric Mann-Whitney tests using GraphPad Prism software, version 7.04. For all tests, P ≤ 0.05*, P ≤ 0.01**, and P ≤ 0.001*** were considered statistically significant.

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RESULTS

Efficacy and safety of LNP siPLK1 in primary human hepatocytes

Compared to control LNP siRNAs, LNP siPLK1 was efficient at inhibiting PLK1 expression, with 70% loss of PLK1-mRNA at 13.3ng/mL without toxicty in proliferating HepG2 cells. Of note, LNP siPLK1 induced 50% mortality in proliferating HepG2 cells at 133ng/mL due to active replication and basal involvment of PLK1 in G2/M checkpoint (Fig. Sup. 1). Considering these data, as well as the concentration correspondence table (Table1) and our previous work done using naked siRNA targeting PLK1 [6], we tested the effect of LNP-siPLK1 and controls in HBV-infected primary human hepatocytes with a range of concentration from 100 to 500 ng/mL. We observed a strong reduction in the levels of of PLK1 mRNA (Fig. 1A) and PLK1 phosphoprotein (Fig. 1B). As previously reported, LNP-siHBV led to a reduction of PLK1 mRNA to a level closer to that observed in uninfected cells, thus confirming that HBV increase PLK1 expression in PHH. Confirming also our previously published data obtained with a biochemical approach [6], the inhibition of PLK1 expression was not cytotoxic in PHH (3 different batches tested) even when LNP siPLK1 was administrated 3 times at 500ng/mL (Fig. 1C and1D). Moreover PHH remained functional, as measured by the apolipoprotein B (ApoB) secretion (see Fig. 2C).

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LNP siPLK1 strongly and specifically inhibits the secretion of HBV virions and antigens by infected primary human hepatocytes

To assay LNP siPLK1 antiviral activity, freshly isolated PHH from three different donors were infected with HBV, and at day-4 post-infection (p.i.), when the average HBsAg production reached around 1000 IU/mL, cells were treated three times every three days with increasing LNP siPLK1 doses or control molecules (Fig. 2A). Secretion of viral DNA (i.e., infectious virion)

and RNA (i.e., RNA-containing particles; [24]) were significantly inhibited by LNP siPLK1 (Fig. 2B). The inhibiton was comparable to that observed with LNP siHBV in this model. The lack of dose-dependency of the phenotype suggests that even a weak loss of PLK1 (Fig. 1B) is associated with a strong inhibition of virus progeny. Interestingly, we also observed a 70%

inhibition of viral antigens HBeAg and HBsAg secretion (Fig. 2C), without affecting ApoB secretion, highlighting a specificity of LNP siPLK1 action on those secreted viral proteins. As expected, tenofovir (TFV) did not affect HBV antigens secretion and the inhibition of HBV antigens secretion was stronger with LNP siHBV, as expected due to the direct targeting of HBV RNAs. Such an inhibitory phenotype on viral antigens was not obtained with SM-PLK1i, as previously reported [6]; the decrease of PLK1 protein is more deletarious for the virus than kinase activity inhibition, thus suggesting an enzymatic-independent function of PLK1 towards HBV.

These results were confirmed in the HBV-infected dHepaRG cells with the same experimental setting (Fig. Sup. 2). Moreover, the inhibitory phenotype on virion and HBsAg secretion was also observed by ex vivo treatment with LNP siPLK1 of freshly isolated hepatocytes from HBV-infected liver-humanized FRG mice [25], which replicate HBV at high level (i.e., around 1.10 9 copies/mL at day-26 post-infection), mirroring the highly viremic « immune tolerant » phase in human. In this model, 300ng/mL of LNP siPLK1 led to a Thesis Adrien FOCA 147 decrease of 60% in HBV DNA and 65% in HBsAg, whereas treatment with LNP siHBV was very efficient (Fig.

Sup. 3).

LNP siPLK1 induces a reduction of intracellular viral RNA accumulation without significant modification of cccDNA amount

To further describe the anti-HBV potential of LNP siPLK1 and get insigths on the observed phenotype on secreted HBV antigens, intracellular viral parameters were also analyzed. We or Southern Blot) (Fig. 3). These data suggest that the decrease of viral RNA observed with LNP siPLK1 is a consequence of a transcriptional and/or post-trancriptional event.

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LNP siPLK1 does not inhibit better HBV replication in pre-infection treatment setting

To determine if PLK1 affects HBV infection establishment, we treated PHH twice with LNP siPLK1 or control molecules, prior to HBV infection, as indicated in Fig. 4A. A third treatment was done at the time of HBV inoculation in order to maintain PLK1 protein level low from HBV entry to cccDNA formation. Analyses were done at 5d.p.i. PLK1 mRNA knockdown was still >75% with 100 and 300ng/mL of LNP siPLK1 (Fig. 4B). As seen before LNP siHBV allowed a PLK1 mRNA decrease, in accordance to the level found in uninfected cells. Interestingly a similar phenotype was observed with PreS1 peptide, which very efficiently prevented HBV infection in this setting (as shown in other panels of the Fig. 4). Regarding antiviral phenotypes obtained with LNP siPLK1, they were very similar to that obtained in postinfection treatment setting (compare results of Fig. 2 and4). Importantly, cccDNA establishment was not blocked by either LNP siPLK1 or LNP siHBV, as compared to that observed with PreS1 peptide, which acts a genuine entry inhibitor. The observed trend of a decrease in cccDNA levels observed with LNP siPLK1 and LNP siHBV was not significant compared to controls (LNP si Ctrl and TFV), and could therefore not be taken into account in the other inhibitory phenotypes observed. It is worth mentioning here that PreS1 peptide did not lead to an absence of detection of intracellular HBV DNA in this setting, as compared to other papameters (cccDNA, intracellular RNA, HBeAg, HBsAg), thus confirming that residual HBV DNA from inoculation perturbs analyses as discussed earlier. This is the reason why results obtained from supernatants analyses are more trustful, and without ambiguity demonstrate the overall strong effect of LNP siPLK1 on HBV replication.

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- ------------------------------------------------------------------------------------------------------------------------- Taqman qPCR [19]. P ≤ 0.05*, P ≤ 0.01**, and P ≤ 0.001*** were considered statistically significant.

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PLK1 knockdown results in a partial nuclear vRNA sequestration in HBV-infected PHH

In HBV-infected PHH, activated pPLK1-S137 was mainly localized in the nucleus (Fig. 1D and [6]). As we observed a decrease in HBV RNAs in absence of change in the level of cccDNA under LNP siPLK1 treatment, we wondered if PLK1 could be involved in the modulation of HBV RNA levels. To address this, we infected PHHs, treated them as above and harvested cells three days after the last treatment (Fig. 2A). We performed a cytosolic/nuclear fractionation in order to quantify HBV RNAs in both compartiments. RNA purity of our extracts was checked using nuclear-specific U6 snRNA and cytosolic-specific Tyr-tRNA. LNP siHBV resulted in a decrease of HBV RNAs levels in both compartments, whereas LNP siPLK1 led to a dual phenotype, depending on subcellular compartments. Indeed, in the cytosol, a 50% decrease of HBV RNAs levels was observed, similar to what was observed with whole cell extracts (Fig. 5A andB). By contrast, in the nuclear compartment, a dose-dependent accumulation of HBV RNAs was observed under LNP siPLK1 treatment (Fig. 5C). As the amount of cytosolic HBV RNAs was overall 10 times higher than that observed in the nucleus, this accumulation of HBV RNAs in the nucleus induced by LNP siPLK1 did not modify the overall observed phenotype (i.e., reduction of total RNA accumulation), but certainly accounted for the decrease in viral protein synthesis.

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- ------------------------------------------------------------------------------------------------------------------------- , [21]. RNA were extracted using trizol and reverse transcribed. Home-designed primers were used to amplify the nuclear specific U6 snRNA and the Human Tyr-tRNA for the cytoplasm compartiment and served as purity control as well as housekeeping genes. P ≤ 0.05*, P ≤ 0.01**, and P ≤ 0.001*** were considered statistically significant.

DISCUSSION

Hepatocellular carcinoma ranks 2nd in term of cancer deaths worldwide and is responsible of an estimated 600 000 deaths/year. HCC development is carried out by the interaction of genetic predisposition, environmental factors as well as oncogenic viruses; the latter are involved in 80% of HCC and HBV for itself accounts for 50%. As 257 million people are chronically infected with HBV and only a minority of them are effectively treated, the HBVmediated HCC burden is far from being eradicated. If a better diagnostic of infected patients and more universally use of antiviral therapies are needed, it is also crucial to identify novel molecules that would both target viral replication and HCC occurrence.

Our previous study demonstrated that PLK1 is activated upon HBV infection and acts as a proviral factor. Consequently, we showed that its inhibition by a SM-PLK1i leads to an inhibition of HBV replication. In addition, the group of Dr O. Andrisani have shown in several studies that PLK1, that is induced by HBx, is a driver of murine hepatocytes transformation in vitro and that its inhibition with SM-PLK1i can prevent it [11,26]. Moreover PLK1 being a validated target in oncology, research efforts to identify potent SM-PLK1i have been important as reviewed recently [27]. The possibility to use siRNA to specifically target kinases involved in cancer has finally opened novel perspectives of use. In this line, the clinical trial (NCT02191878) performed with LNP siPLK1 in subjects with advanced HCC was instrumental and led us to consider LNP siPLK1 as a good candidate for early inhibition of HBV replication.

Here we confirmed that PLK1 is crucial for HBV replication in non-dividing hepatocytes by using of these PLK1-targeting-siRNA encapsulated in lipid nanoparticles (Arbutus Bioparma proprietary composition). In particular, we demonstrated that LNP siPLK1 inhibits more efficiently HBV replication than naked siRNA or SM-PLK1i (reported in [6]) in the absence of Thesis Adrien FOCA 158 cell toxicity. Indeed 100 to 300ng/mL of LNP siPLK1 (corresponding to 7.5 to 22.5nM siPLK1) led to a >75% decrease in HBV viremia in vitro. Moreover, in contrast to BI-2536, the SM-PLK1i that was previously profiled in depth in vitro and in liver-humanized FRG mice [6], we found that LNP siPLK1 was also capable to potently inhibit the secretion of HBV antigens in the supernatant of treated cells. This indirectly indicates that a kinase-independent function of PLK1 could account for production and secretion of HBV antigens. To this extent, it was precedently shown by Vitour and colleagues that the polo-box domain of PLK1 could bind to the mitochondria-bound innate immunity adapter MAVS and inhibit its ability to activate the IRF3 and NF-kappaB pathways, without any requirement of the kinase activity [28]. Such an unexpected phetotype on HBV antigens production is particularly relevant, as the next step in the improvement of current HBV therapy in clinic is precisely to be capable to reduced antigenemia in patients to potentially restore immune responses and control infection [3].

SiRNA targeting HBV itself are currently clinically evaluated in this respect (e.g., Phase-2 for ARB-001467; NCT02631096). The co-administration of siRNAs targeting mainly HBV and PLK1, could be an original way to further improve such a therapeutic approach.

The LNP siPLK1-induced inhibition of HBeAg and HBsAg in supernatant correlated with a reduced accumulation of intracellular HBV RNAs in LNP siPLK1 treated cells, in the absence of any change in cccDNA amount. This observation points out toward a role of PLK1 in transcriptional and/or post-transcriptional regulation. In this study, we limited our analysis to the impact of LNP siPLK1 on the nuclear versus cytoplasmic fractionation, and found that HBV RNAs tended to accumulate in the nucleus under LNP siPLK1 treatment, while the total amount of RNA was decreased. Although the precise underlying mechanistic of this transcriptional and/or post-transcriptional regulation (i.e., cccDNA activity, vRNA trafficking and/or fate) is yet to be defined, it is tempting to speculate that the previously described interplay between PLK1 and HBc could also be at work. Indeed, HBV core protein is known to Thesis Adrien FOCA 159 interact with cccDNA [29] and potentially in the regulation of transcription [30]. Moreover, it has been suggested that the HBc CTD could be determinant for HBc-cccDNA interaction [30] and in our precedent study, we have identified 3 PLK1-dependent phosphorylation sites in HBc CTD. Finally, HBc is also known to be an RNA-binding protein physically interacting with vRNAs. Taken together, this provided a nice hypothetical landscape for further mechanistic studies to document LNP siPLK1 mode of action (MoA).

Of course, it would also be important to assay the anti-HBV efficacy and MoA of LNP siPLK1 in a liver humanized mouse model to confirm its potential therapeutic interest on the virology side. Regarding the possibility to test the relevance of using LNP siPLK1 as a double bullet compound, which would be able to both contribute to HBV replication inhibition in "add on therapy" with SoC and prevention of HBV-driven carcinogenesis, we would need a preclinical model able to recapitulate the entire pathogenesis process. A mouse model based on immune-competent mice transduced with AAV-HBV vectors could be of interest, as it features a persistent viral replication [31], in the context of an HBV immune tolerance [32],

and was also reported tu support HCC development [33]. Alternatively other transgenic mouse models featuring the over expression of HBV proteins, HCC development and a demonstrated implication of PLK1 in carcinogenesis could be used to this end. Recently, it was shown that genomic integrity of hepatocytes was disrupted by viral LHBS and that PLK1 acted as a major modulator in LHBS-mediated G2/M checkpoint override. In this model, SM-PLK1i resulted in a G2/M checkpoint restauration and suppression of tumorigenesis [34].

To summarize, in this study we have reported a potent antiviral activity of LNP siPLK1 in HBV-infected PHH, which includes a strong loss of intracellular vRNA accumulation, leading to reduction of HBV antigens secretion. This stands as proof of concept that PLK1 plays other proviral roles in HBV life-cycle that extend beyond it already described involvement in capsid Thesis Adrien FOCA 160 assembly [6] and further validate its potential interest for the anti-HBV arsenal. Moreover as it is now rather well accepted that PLK1 is an important HCC driver, its inhibition could result in a combined effect on the virus and viro-induced carcinogenesis and this warrants further preclinical investigation.

Chapter 3: Discussion and Outlooks

An estimated 15% of cancers worldwide are attributed to viruses. Among them, HBV is a well-known hepatocellular carcinoma-inducer and contributes to 800,000 deaths per year with 40% related to cancers [START_REF] Lozano | Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study[END_REF]. Despite the existence of a prophylactic vaccine and relatively efficient nucleotide analogs, there are still 250 million people being chronically infected by HBV, with a high risk of progression to end-stage liver diseases. Regarding the management of non-resecable HCC, there are only two available targeted options, allowing a survival gain of few months only, highlighting the urgent need of efficient drugs development. Checkpoint inhibitors, which are very efficient for other cancer, are yet to be tested in the case of HCC.

As viral infections and oncogenesis often deregulate the same cellular pathways, aiming to enhance cell division and escape from immune system, the community has gained interest in key actors of these pathways. In this respect, PLK1, regarding its global involvement in the cell-cycle regulation, was extensively studied and proposed as a promising target for drugs development in the oncology field. Focusing on HBV-related HCC, several groups have shown an overexpression of PLK1, linked with bad prognosis for patients with cancer. Morevover, PLK1 was shown to be activated by HBx, a potent HBV transactivator, but the mechanism of PLK1 activation early on after HBV infection need still to be determined (Studach et al., 2009b). However, "the chicken and egg" paradigm is still relevant as we do not know yet if PLK1 acts as a key driver of the carcinogenesis or if the overexpression of PLK1 is a result of the high proliferative rate observed in tumours [START_REF] Cholewa | The role of polo-like kinase 1 in carcinogenesis: cause or consequence?[END_REF]. At last, no one focused on the possible involvement of PLK1 in HBV life cycle early-on after infection in untransformed and quiescent hepatocytes.

Eventually, we have shown the efficacy of targeting PLK1 in vitro and in preclinical mouse model to inhibit HBV infection but the exact mode of action of PLK1 is still elusive and seems to be plurimodal, with an involvement of this kinase at several key steps of the HBV life cycle. In this discussion, all these points are addressed in order to delineate a big picture serving as a clue to answer the question:

Is there a place for PLK1 inhibitors to reinforce the anti-HBV arsenal, and can we hope for a bimodal mode of action by acting also on the prevention of neoplastic events occurence?

How PLK1 is activated upon HBV infection in quiescent and differentiated cells?

The group of Dr Andrisani has shown that PLK1 was activated in 4pX-1 cells expressing the HBx protein after release from double thymidine block. However, this phenomenon occurs after 40 cell generations and seems to be indirectly induced by the polyploidy and is of importance regarding oncogenesis process but may have limited impact on early HBV infection and replication (Studach et al., 2009b). In 2010, using the same model, they demonstrated that HBx mediates activation of PLK1 in the G2 phase of untransformed hepatocytes through the activation of the p38 MAPK pathway (Studach et al., 2010). However, these cells, albeit being untransformed are still undergoing cell division and HBx could be just another cue responsible for PLK1 activation and not playing the driving trigger in this process.

The HepAD38 cell line, which was derived from HepG2 stably transformed with an HBV transgene, is capable to replicate HBV in a tet-off inducible manner. In this model, PLK1 activation, witnessed by the induction of pPLK1T210 in immunofluorescence experiments, was found early-on after HBV replication (Zhang et al., 2015). However, although this model recapitulates some steps of HBV life cycle (except the entry steps), cells are not quiescents and were synchronized in G2/M using nocodazole, a cell-cycle stage where PLK1 is described to be already present. Once again one could not exclude that PLK1 was preactivated, possibly being phosphorylated on its Ser137 (confer section "3.3.2. PLK1 activation: a multi-step and dynamic process"), and HBV in this context might not represent the main inducer of PLK1 activation.

In our hand, using quiescent and untransformed models (e.g., dHepaRG and PHH), where the basal level of PLK1 was neither detectable by western blot nor by immunofluorescence, we observed this early activation of PLK1 upon HBV infection. Indeed, as early as 24h post-infection, we observed a peak in PLK1 mRNA, consistent with an increased amount of pPLK1S137 at D3 and a fully activated pPLK1T210 at D7 post-infection (confer "A. First study: Polo-like-kinase 1 is a proviral host factor for hepatitis B virus replication"). This means that HBV could represent a bona fide trigger of PLK1

activation, yet the underlying mechanism remained elusive. Indeed, HBx still represents the best candidate as it is the first HBV protein being expressed upon infection and may help transactivating PLK1 expression directly. Alternatively, HBx could act by indirectly, by regulating transcriptions factors found to be associated with the PLK1 promoter, such as STAT5 and/or E4BP4, which were described to interplays with HBx [START_REF] Lee | Signal transducers and activators of transcription 5b activation enhances hepatocellular carcinoma aggressiveness through induction of epithelial-mesenchymal transition[END_REF][START_REF] Qiao | SREBP-1a activation by HBx and the effect on hepatitis B virus enhancer II/core promoter[END_REF]. In our hand, the use of a HBxdeficient virus led to a short delay in the PLK1 activation but was not statistically significant (data not shown). However experimental issues due to viral production did not allow to firmly conclude on the involvement of HBx in PLK1 activation early-on after HBV infection.

This induction being observed quickly after the infection, another hypothesis regarding PLK1 activation resides in the incoming viral material from the HBV inoculum. Indeed as PLK1 and HBc are able to interact (confer "First study: Polo-like-kinase 1 is a proviral host factor for hepatitis B virus Thesis Adrien FOCA 166 replication"), one could suggest that the core protein may sequester PLK1, rendering its activation by Aurora A/Bora impossible due to steric hindrance. Finally it would be worth to address the status of protein know to inhbitits PLK1 such as Map205, stabilizing the autoinhibited state of PLK1, or BubR1, a protein shown to physically interacts with PLK1 and inhibiting its phosphorylation during interphasic quiescent cells, under HBV infection [START_REF] Izumi | BubR1 localizes to centrosomes and suppresses centrosome amplification via regulating Plk1 activity in interphase cells[END_REF]. Moreover, HBx was shown to bind BubR1, thereby deregulating the mitotic checkpoint and enhancing the accumulation of chromosomal instability in HBV-associated hepatocarcinogenesis [START_REF] Kim | HBV X protein targets hBubR1, which induces dysregulation of the mitotic checkpoint[END_REF]. Thus, it is reasonable to speculate that HBx could bind to BubR1 early-on after HBV infection, resulting in a release of BubR1-dependent inhibition of PLK1.

All these described mechanisms could either act in a standalone manner or more possibly act together in order to activate PLK1 in a short time after HBV infection of quiescent hepatocytes.

Furthermore, some of them could last long enough to make a bridge between activation of PLK1, which is mandatory for HBV replication, and high level of PLK1 found in HCC.

However, as shown in the section "Second study: Antiviral activity of PLK1-targeting siRNA delivered by lipid nanoparticles in HBV-infected hepatocytes", a knockdown of PLK1 before HBV infection does not lead to a defect in HBV infection establishment, suggesting a minor role of PLK1 in steps from HBV entry to cccDNA formation. Thus the early activation of PLK1 upon infection could be induced by HBV in order to support subsequent steps of the viral life cycle (from cccDNA transcription to rcDNA-containing capsids formation).

Finally, the overexpression of PLK1 at the early steps of HBV infection could be the result of a transient NF-KB activation via the TLR4/MyD88/IκBα axis. Indeed PLK1 was associated with the downregulation of TLR2/TLR4-induced inflammation which is known to be activated by HBsAg [START_REF] Jing | HBsAg/β2GPI activates the NF κB pathway via the TLR4/MyD88/IκBα axis in hepatocellular carcinoma[END_REF]. The initial inoculum we used contains up to 10000 fold more subviral particles than infectious virions and thereby could explain such activation. As HBV is described to be a stealth virus or more possibly is able to shut down very quickly the innate immune response, the activation of PLK1 through this axis could in part explain this phenomenon by attenuating the NFkB pathway via direct inhibition of the IKK complex [START_REF] Higashimoto | Regulation of I(kappa)B kinase complex by phosphorylation of (gamma)-binding domain of I(kappa)B kinase (beta) by Polo-like kinase 1[END_REF]. In a second time, after cccDNA formation and active replication, the virus could deflect PLK1 activity to this advantage as we identified this kinase to be involved in pgRNA encapsidation and reverse-transcritpion (confer "First study: Polo-like-kinase 1 is a proviral host factor for hepatitis B virus replication").

To summarize, two pictures that may explain the PLK1 activation upon HBV infection are proposed here. On the one hand, HBV through direct mechanisms could lead to PLK1 activation in order to support the upcoming replication. On the other hand, PLK1 activation could come from the transient activation of the innate immune response by HBV infection, and PLK1, by downregulating Thesis Adrien FOCA 167 the IKK complex, could be part of the explaination on how HBV succeed in inhibiting the innate immune response (Figure 37).

In accordance with kinase cascades which are often observed (e.g., MAPK, PI3K/Akt/mTOR) to transduced a signal; one could address the question of the PLK1 activation kinetic/dynamic. Are PLK1 activators such as Aurora A found overexpressed/deregulated under HBV infection? Recently the nonsynonymous polymorphism of the AURKA gene Ile31Phe (encoding the kinase Aurora A) was associated with increased risk of occurrence of HBV-related hepatocellular carcinoma in Chinese population [START_REF] Bao | Association between the functional polymorphism Ile31Phe in the AURKA gene and susceptibility of hepatocellular carcinoma in chronic hepatitis B virus carriers[END_REF]. Although the authors have not deciphered the underlying mechanisms, it would be interesting to focus on the role of this polymorphism regarding early HBV infection and study its potential impact on PLK1. This could strengthen the link between the overexpression of PLK1 induced quickly after HBV infection and elevated PLK1 levels found in HBV-related HCC. et al., 2018). NFkB is a well described transcription factor of PLK1, leading to increase its transcription. PLK1 was identified as a gammaBD kinase, which negatively regulates IKK activation [START_REF] Higashimoto | Regulation of I(kappa)B kinase complex by phosphorylation of (gamma)-binding domain of I(kappa)B kinase (beta) by Polo-like kinase 1[END_REF] and could explain how HBV shut down innate immune response quickly after infection.

Thesis Adrien FOCA

168

PLK1 is involved in several steps of HBV life cycle, strengthening the interest of targeting this kinase for antiviral therapies

As discussed before, although PLK1 is found overexpressed and activated early-on after HBV infection, its inhibition before infection does not impede the establishment of HBV cccDNA, implying a minor involvement of PLK1 regarding entry step, rcDNA translocation and cccDNA formation (confer "Second study: Antiviral activity of PLK1-targeting siRNA delivered by lipid nanoparticles in HBV-infected hepatocytes"). From our studies, we identified PLK1 as a proviral factor for HBV and found that the neosynthesis of HBV DNA was the only parameter impacted drastically by small molecules inhibitors of PLK1 (e.g., BI-2536). The critical step for the rcDNA generation being the pgRNA encapsidation and subsequent reverse transcription, we focused our efforts on the HBc-PLK1 interaction. As explained, a dynamic process of phosphorylation of the HBV capsid is mandatory for a proper pgRNA encapsidation and reverse transcription. Moreover, several groups have already identified kinases able to phosphorylate the capsid, such as CDK2 which phosphorylates SP sites located at the C-terminus of HBc, including S155, S162, and S170 (Ludgate et al., 2012) or SRPK1/2 (Daub et al., 2002). PLK1 is known to mostly interact with already phospho-primed peptides and was Thesis Adrien FOCA

169

This antiviral activity using the PLK1 inhibitor BI-2536 is very potent in decreasing HBV DNA with EC 50 ranging from 5 to 50 nM, depending on the cell type used; such an antiviral efficacy is rarely obtained with host targeting agents and corresponds to the efficacy of direct acting agents including nucleoside analogs or assembly inhibitors. Accordingly, an in vivo antiviral activity was also obtained with BI-2536 in HBV-infected liver humanized FRG mice, and this antiviral activity was comparable to that obtained with entecavir or IFN-a in the same model.

However, as kinase inhibitors often demonstrate limits in term of specificity, we decided to knockdown PLK1 using siRNA. Thanks to Arbutus Biopharma, we had acces to an innovative delivery strategy that allow a specific targeting of the hepatocytes; the siRNA-containing lipid nanoparticles (LNP) (Figure 39). Using this technology and siRNA targeting PLK1 which were already clinically trialed for anti-HCC activity (NCT02191878), we knocked-down PLK1 in a context of HBV infection. Surprisingly, knocking-down of PLK1 at a protein level, instead of solely inhibiting its kinase activity, uncovered new phenotypes regarding the anti-HBV activity. As mentioned earlier, the use of the small molecule PLK1 inhibitor BI-2536 resulted in an inhibition of HBV DNA neosynthesis .

The use of LNP siPLK1, which abolish totally the expression of PLK1 protein, resulted in a loss in HBV DNA, but also in HBV RNA and secreted HBe/HBsAg (confer "Second study: Antiviral activity of PLK1-targeting siRNA delivered by lipid nanoparticles in HBV-infected hepatocytes"). This implies that PLK1 may play roles in HBV life cycle in a kinase-independent manner and regulates several steps of the HBV cycle. To this extent, it was precedently shown by Vitour and colleagues that the polo-box domain of PLK1 could bind to the mitochondria-bound innate immunity adapter MAVS and inhibit its ability to activate the IRF3 and NFkB pathways, without any requirement of the kinase activity [START_REF] Vitour | Polo-like kinase 1 (PLK1) regulates interferon (IFN) induction by MAVS[END_REF]. Furthermore, the lack of a functional kinase domain in PLK5 highlights the question of whether other PLKs have kinase-independent roles. Notably, PLK2 leads to modulation of distal dendrite synapses during chronic excitation in a kinase-and PBD-independent manner [START_REF] Evers | Plk2 attachment to NSF induces homeostatic removal of GluA2 during chronic overexcitation[END_REF]. Besides the "classical" functions of PLKs in the regulation of the cell-cycle originally described in Drosophila and yeasts, it is now obvious that these are important regulators of non-proliferative events, such as cell differentiation, and these functions may be independent of the kinase activity.

The mechanisms behind this antiviral activity is not characterized yet, however we saw an accumulation of HBV RNAs in the nucleus of cells treated with LNP siPLK1 treatment, while the total amount of vRNA was decreased, indicating an involvement of PLK1 in cccDNA activity, vRNA trafficking and/or fate. Undergoing experiments attempt to answer this question, mostly by ChIP/RIP analysis to see if activation/repression marks on the cccDNA are altered under PLK1 inhibition. The second point of interest regarding this axis will be to determine if PLK1 is found associated with vRNA, playing a role in their stability/trafficking. It is tempting to speculate that the previously described interplay between PLK1 and HBc could also be at work. Indeed, HBV core protein is known to interact with the HBV cccDNA (Bock et al., 2001) and potentially plays a role in the regulation of its transcription. Moreover, it has been suggested that the HBc CTD could be determinant for HBc-cccDNA interaction (Chong et al., 2017) and in first precedent study, we have identified 3 PLK1dependent phosphorylation sites in HBc CTD. Finally, HBc is also known to be an RNA-binding protein physically interacting with vRNAs. Taken together, this provided a nice hypothetical landscape for further mechanistic studies to document LNP siPLK1 mode of action.

Such an unexpected phetotype on HBV antigens production is particularly relevant, as the next step in the improvement of current HBV therapy in clinic is precisely to be able to reduced antigenemia in patients to potentially restore immune responses and control infection.

Other groups in the HBV field have found that PLK1 plays proviral roles. Recently, the team of Jake Liang from the NIH has performed a genome-wide screen on HepG2-NTCP, targeting 21,500 genes using three unique siRNA sequences per gene in order to identify host factors related to HBV.

In addition, they did a counter-screen with a cell viability assay measuring ATP as an endpoint to assess the effect of knockdown on cytotoxicity. Interestingly they found that siPLK1 led to a loss of secreted HBV antigens (HBV meeting 2018, oral presentation N°167; A genome-wide siRNA screen for host factors involve in hepatitis B virus infection), strengthening our results. Furthermore, they identified HNF4α in the screen to have an impact on HBV infection. This master regulator of hepatocyte diffentiation has been described to bind to the cccDNA, thus, activating its transcription in infected HepG2-NTCP cells. The IL6 inhibitory effect on HBV transcription could be in part mediated by a loss HNF4α binding to the cccDNA and a redistribution of STAT3 binding from the cccDNA to IL6 cellular target genes [START_REF] Palumbo | IL6 Inhibits HBV Transcription by Targeting the Epigenetic Control of the Nuclear cccDNA Minichromosome[END_REF]. HNF4α has also been linked to PLK1 expression. Indeed, under normal conditions, HNF4α tends to inhibit hepatocytes proliferation while PLK1 is known as a potent cell-cycle promoter. In the liver of mice deleted for HNF4α, PLK1 is found upregulated in a 13 fold manner at the genomic level [START_REF] Walesky | Hepatocyte Nuclear Factor 4 alpha Deletion Promotes Diethylnitrosamine-induced Hepatocellular Carcinoma in Mice[END_REF]. However, one may suggest that in the case of an HBV infection, the relocatlization of HNF4α to the cccDNA could allow an inhibition release of the PLK1 transcription, leading to increased expression/activity of PLK1 that is mandatory for a proper HBV replication. Regarding this hypothesis, it would be interesting to perform ChiP experiments to determine whether HNF4α could bind to PLK1 promoter/gene and to see if HBV infection in primary human hepatocytes leads to a relocalization of HNF4α from PLK1 gene to the cccDNA (Figure 40). In non-infected hepatocytes (left), HNF4α acts as a repressor of PLK1 transcription, thereby promoting the quiescence of the hepatocytes and the differentiation [START_REF] Walesky | Hepatocyte Nuclear Factor 4 alpha Deletion Promotes Diethylnitrosamine-induced Hepatocellular Carcinoma in Mice[END_REF]. Under HBV infection (right), HNF4α is found to bind the cccDNA where it promotes the viral transcription [START_REF] Palumbo | IL6 Inhibits HBV Transcription by Targeting the Epigenetic Control of the Nuclear cccDNA Minichromosome[END_REF]. The dashed black line represents the relocalization of HNF4α from genomic DNA to the cccDNA, thus, releasing the inhibition of PLK1. In turn, PLK1 act as a proviral factor either by increasing the cccDNA activity or by promoting stability/trafficking/maturation of the viral RNAs.

Does the PLK1 proviral effect towards HBV is pan-genotypic?

The majority of the experiments were done using HBV genotype D concentrated from HepAD38 cells supernatants. However, recently we have developed news tools allowing the production of the eight HBV genotypes (A-H) either in HepG2 or in HepaRG backbone. As explained in the introduction section, the different HBV genotypes lead to divergences in term of natural history and progression of the disease. This highlights the fact that molecular mechanisms are affected in a different way, depending on the HBV genotype. Actually, the community put efforts on testing potent antiviral molecules in several HBV genotypes. From our side, PLK1 was found overexpressed in all HepG2 cells producing A-H genotypes compared to naive HepG2 and the antiviral effect of LNP siPLK1 was confirmed in genotype C (data not shown). These preliminary results seem to indicate that PLK1 is involved in the replication of all HBV genotypes ans that its inhibition could be of benefit in a pan-genotypic manner. Indeed, the CTD of HBc being extremely well conserved among all the genotypes, one would expect that PLK1 inhbition should at least exhibit the same antiviral phenotype regarding the encapsidation/reverse-transcription of the pgRNA.

PLK1 as a first-choice target for anti-HBV and anti-HCC therapies?

Besides its involvement in the HBV life cycle, PLK1 is a key player in HBV carcinogenesis. PLK1 is expressed ubiquitously in eukaryotic cells and is highly expressed in most of human cancers, including HCC (Mok et al., 2012). PLK1 can be activated by HBV X protein to allow propagation of DNA damage by both attenuating the DNA damage checkpoint Chk1 activity and destabilizing DNA repair protein Mre11 (Studach et al., 2010). Furthermore, PLK1, together with the long non-coding RNA HOTAIR, induces proteasomal degradation of two transcription repression factors, SUZ12 and ZNF198, to alter the chromatin landscape, leading to epigenetic reprogramming associated with oncogenic transformation (Zhang et al., 2015). Finally, L-HBsAg was found to be involved in HCC development through induction of cytokinesis failure, leading to aneuploidy in hepatocytes. This phenotype appears to be mediated via DNA damage and G2/M checkpoint override due to PLK1 overexpression.

However, we do not know yet if the initial overexpression of PLK1 upon HBV infection is related to the PLK1 elevated levels found in HBV-related carcinogenesis (Figure 41). To answer this question it would be of value to follow the PLK1 expression in a long term kinetic of HBV infection.

The main issue is the limits of experimental models. Regarding the in vitro part, PHH cannot support more than three weeks of plating without dedifferentiation and loss of viability. The HepaRG model can last hypothetically for months/years in differentiation state, yet, we saw a decrease in HBV replication after aproximatly a month of infection. Regarding in vivo model, FRG liver-humanized mice tend to develop spontaneously liver tumours of murine origin and thus are not the optimal model to perform a long-term follow up of HBV infection. The solution may arise from the HepaRGproducing all the HBV genotypes in a stable manner. Altough being pauciclonal, these cells lines could theoretically produce virion and all HBV intermediates in a long-lasting way. It would be interesting to monitor the PLK1 expression level in these cells in order to:

-Know if HBV genotypes induce PLK1 expression in a different quantitative manner.

-See if PLK1 overexpression (plasmid transduction of PLK1 constitutively active form) in these cells leads to cellular transformation.

If the two latters points are proven exact, can we imagine different time-courses regarding cellular transformation as we known that genotypes C2 or F1b are more aggressive and lead rapidly to HCC.

The developpement of new innovative tools (e.g., 3D spheroids culture, humanized-miced)

and efforts bring to understand the interface between PLK1 involvement in HBV replication and PLK1

functions in carcinogenesis will eventually converge to the development of new drugs, which will act in a bimodal way, acting on both HBV replication and prevention of the occurrence of neoplasic events. In this respect, inhibiting PLK1 may emerge as a pionner strategy. Furthermore, due to its large involvement in carcinogenesis, several molecules targeting specifically this kinase have been developed and clinically trialed. Thus, a repositioning of drugs (e.g., Volasertib, Rigosertib) could be foreseen to reinforce the anti-HBV/HCC arsenal. At last, direct acting agents such as HBV RNA destabilizers, CpAMs, siHBV, NAs should be combined with host targeting agents like kinase inhibitors to combat in an efficient way HBV and to maintain the hepatocytes landscape in a near normal state to prevent carcinogenesis. Upon HBV infection, PLK1 is overexpress and stay at an elevated level in a long-lasting way. This alters the hepatocytes and microenvironment landscape by promoting proliferation, DNA damages, chromosomic instabilities … and eventually along with the occurrence of satellite events, lead to cellular transformation. In this model PLK1 acts as a key driver of carcinogenesis. Bottom: Initially the situation remain unchanged, PLK1 is overexpressed due to HBV infection. However, once the infection is stably establish, the PLK1 level returns to a basal state untill a driver carcinogenic event occurs such as p53 mutation, wich lead in an indirect manner to PLK1 overexpression. In this situation PLK1 overexpression plays a role of satellite event towards transformation process and is just the results of high-proliferative rate of uncontrolled cells. In both cases it is of importance to note that with our actual technology, we can pretend in best cases to detect tumour approximately 10 years after the emergence of the first transformed cell.

Take-home message

This thesis project aimed at exploring the impact of cellular partners on HBV and has led to the identification of PLK1 as a new proviral factor. Using several approaches such as ATP-competitive inhibitors of PLK1 or innovative lipid nanoparticles system to deliver siRNA, we brought forward the proof-of-concept that PLK1 could be an attractive druggable target in HBV infection. We uncovered partially the mechanisms behind this antiviral effect and found that PLK1, through its kinase activity, is able to phosphorylate the HBV core protein, thus affecting the pgRNA encapsidation and subsequent reverse-transcription. Furthermore, PLK1 seems to be involved in other steps of the HBV life cycle, in a kinase-independent manner albeit the exact mechanism needs still to be determined.

Finally, as PLK1 is found overexpressed in HBV-related HCC, we hypothesized that its inhibition could act in a bimodal way, both acting on HBV replication and preventing the occurrence of neoplastic events.

Place of my project within the research group

At last, my thesis project on PLK1 involvement in the HBV life cycle led the group to explore new opportunities regarding host-virus interactions, with a special focus on cellular kinases. Indeed, interactomes of HBc were done and allowed to identify kinases involved in RNA metabolism (e.g., maturation, splicing, trafficking) which could be of importance for HBV replication. Notably, a patent was established regarding the inhibition of a specific kinase which could serve as a new drug target to reinforce the anti-HBV arsenal. The next step would be to do a kinome between uninfected and infected PHH to decipher the general pattern of kinases being deregulated under HBV infection.

Thus, my work allowed the team initiating a new axis of research around host-virus interactions with a special focus on kinases that may be involved in vRNA metabolism as well as strengthening several links between subgroups.

My contribution outside this project

During my PhD, I was not only focused on my personal project but always get involved in neighboring projects and some examples are presented in Appendix.

Introduction

Hepatitis B virus (HBV) is a major hepatotropic human pathogen, with at least 2 billion people having been exposed to date to the virus according to World Health Organization [START_REF] Revill | New insights into how HBV manipulates the innate immune response to establish acute and persistent infection[END_REF]. HBV exposure leads to chronic infection in 85-95% of infected neonates/ children and 5-15% of infected adults [START_REF] Hadziyannis | Natural history of chronic hepatitis B in Euro-Mediterranean and African countries[END_REF]. Chronic infections are associated with a higher risk of developing liver cancer [START_REF] Revill | New insights into how HBV manipulates the innate immune response to establish acute and persistent infection[END_REF], with 50% of hepatocellular carcinoma (HCC) attributed to HBV. Viral persistence is due to the ability of the virus to escape the host immune system, and due to establishment of an episome in the nucleus of infected cells, known as covalently closed circular double-stranded DNA (cccDNA). This cccDNA resembles the host genome, as it assumes a chromosome-like structure, and serves as main template for viral transcription. However, cccDNA is devoid of telo-meres and replication origins, implying that it can be lost when cells divide; consequently, its reservoir is maintained by recycling of neo-synthesized, relaxedcircular DNA (rcDNA) containing nucleocapsids. For an in-depth discussion of cccDNA biology see recent reviews [START_REF] Allweiss | The role of cccDNA in HBV maintenance[END_REF]Lucifora and Protzer, 2016). CccDNA is a 3.2 kb molecule that expresses at least 6 overlapping RNAs, from 4 open reading frames (ORF), leading to production of 7-8 proteins, including viral polymerase (P), regulatory X protein (HBx), envelop proteins (S, M, and L), HBeAg, and core/capsid (HBc). In this review, we focus our discussion on the function of HBc.

HBc, a small versatile protein of 21 kDa, has been classically viewed as a structural protein that selfassembles to form the viral nucleo-capsid. In turn, the nucleocapsid initially contains the pregenomic RNA (pgRNA), subsequently converted into rcDNA by reverse transcription. The versatile nature of HBc is primarily a consequence of posttrans-lational modi cations occurring at its Cterminal domain (CTD), and thus, this protein directs several viral processes. Accumulated evidence supports that HBc functions extend well beyond this structural role. HBc plays a role in nearly every stage of the HBV life cycle, including: subcellular tra cking and release of HBV genome, RNA metabolism, capsid assembly and transport, and reverse transcription (Fig. 1) (Basagoudanavar et al., 2007;Nassal, 1992;Perlman et al., 2005;Rabe et al., 2003;Zlotnick et al., 2015). Moreover, recent ndings implicate an active role for HBc in epigenetic regulation of the viral and host genomes.

Collectively, this multiplicity of functions elevates HBc as an excellent target for mechanism-based antiviral therapeutics (Durantel and Zoulim, 2016;Zlotnick et al., 2015). This review will highlight how the unique structure of HBc, its capacity to multimerize, as well as its post-translational modi cations, can explain its multiple roles in the HBV life cycle. (Blondot et al., 2016). 2) HBc associates with cccDNA (Guo et al., 2011). 3) HBc modulates host gene expression [START_REF] Xie | Multi-omics analyses reveal metabolic alterations regulated by hepatitis B virus core protein in hepatocellular carcinoma cells[END_REF]. 4) HBc is required for pgRNA encapsidation (Nassal, 1992). 5) HBc is required for reverse transcription (Lewellyn and Loeb, 2011), and 6) persistence of cccDNA involves recycling of nucleocapsids into the nucleus (Nassal, 2015).

Contents lists available at ScienceDirect

1. HBc: from translation to capsid structure and self-assembly

The core protein is encoded by the HBV pgRNA which also encodes the viral polymerase, and serves as template for rcDNA synthesis via reverse-transcription. The viral polymerase acts as a molecular switch to regulate these two otherwise competitive processes allowing the virus to replicate e ciently [START_REF] Ryu | Proximity between the cap and 5' epsilon stemloop structure is critical for the suppression of pgRNA translation by the hepatitis B viral polymerase[END_REF][START_REF] Ryu | Hepatitis B virus polymerase suppresses translation of pregenomic RNA via a mechanism involving its interaction with 5' stem-loop structure[END_REF]. The 21 kDa HBc protein is the building block or capsomer of the HBV genome-con-taining icosahedral capsid. The rst crystal structure, resolved by Wynne and colleagues, revolutionized our understanding of HBc biology [START_REF] Wynne | The crystal structure of the human hepatitis B virus capsid[END_REF]. HBc is a helical protein with ve alpha-helices and lacking β-sheets. Helices 2 and 3, making a long alpha-helical hairpin, are responsible for the HBc folding pattern. Interest-ingly, most nucleocapsid proteins that form icosahedral shells usually fold into barrel structures instead of helical ones, e.g., those seen in the HIV-1 capsid [START_REF] Wynne | The crystal structure of the human hepatitis B virus capsid[END_REF]. A hydrophobic region makes up the base of the protein. HBc assembles rst into dimers via alpha-helical hairpins, which form a four-helix bundle, assuming an inverted T shape. A disul de bridge between the two Cys-61 residues, stabilizes the association, although this cystine formation is not necessary for dimer assembly (Nassal, 1992;Zheng et al., 1992;[START_REF] Zlotnick | Dimorphism of hepatitis B virus capsids is strongly in uenced by the C-ter-minus of the capsid protein[END_REF]. Residues Tyr-132, Arg-127, Pro-129, and Ile-139 appear to be involved in dimer formation [START_REF] Wynne | The crystal structure of the human hepatitis B virus capsid[END_REF]. Following dimer formation, trimers of dimers assemble into hexamers, eventually forming icosahedral capsid particles [START_REF] Birnbaum | Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein[END_REF]. Multimerization of 120 HBc dimers, i.e., 240 capsomers, leads to production of a 22 nm, icosahedral capsid particle, without need of other viral proteins [START_REF] Birnbaum | Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein[END_REF]. The dimers form the icosahedral capsid with a triangulation number T = 4. Of note, a small fraction of capsids consist of only 90 dimers with a triangulation number T = 3 [START_REF] Crowther | Three-dimensional structure of hepatitis B virus core particles determined by electron cryomicroscopy[END_REF]. However, it is unknown whether virions comprised of smaller capsids are infectious or merely represent dead-end products caused by aberrant assembly. Focusing on the HBc primary sequence, the full-length protein consists of 183 amino acids with distinct N-and C-terminal domains connected by a hinge region (Nassal, 1992). The N-terminal domain of HBc, spanning amino acid residues 1-140, contains the capsid assembly domain, as truncated HBc 1-140 is su cient for self-assembly [START_REF] Birnbaum | Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein[END_REF]. On the other hand, the CTD of HBc, spanning amino acids 141-183, is dispensable for in vitro viral assembly. Notably, capsids lacking the last 34 amino acids maintain assembly capacity in vitro, but do not support genome replication in cells (Nassal, 1992), suggesting a role for the CTD in this process. Of the 34 amino acid residues comprising the CTD, 14 are arginines, and at least seven other residues are either serines or threonines, putative phospho-acceptor sites.

Studies to understand the role of HBc CTD in HBV replication have led to the proposal that electrostatic interactions between the highly basic CTD in assembled capsids and the viral RNA/DNA regulate re-verse transcription of HBV DNA [START_REF] Newman | Testing an electrostatic interaction hypothesis of hepatitis B virus capsid stability by using an in vitro capsid disassembly/reassembly system[END_REF]. Charge-driven destabilization of capsids, by either rcDNA maturation and/or changes in post-translational modi cations of CTD, for example by phosphorylation, could explain capsid disassembly at the nuclear pores (Basagoudanavar et al., 2007;Perlman et al., 2005;[START_REF] Rabe | Nuclear entry of hepatitis B virus capsids involves disintegration to protein dimers followed by nuclear reassociation to capsids[END_REF]. In addition, results of a study by Lewellyn and Loeb favor an hypothesis that CTD could serve as a nucleic acid chaperone domain, i.e., CTD could mediate changes in nucleic acid structure hence enabling pregenomic RNA (pgRNA) reverse transcription into rcDNA (Lewellyn and Loeb, 2011). These two models are not necessarily mutually ex-clusive, since the function of HBc as chaperone may be dependent on the overall charge of its CTD.

A recent study employing cryo-EM scanning has determined the highresolution 3D structure of assembled HBV capsids (Yu et al., Thesis Adrien FOCA 203 2013). It was proposed that HBc CTD shuttles between the interior and exterior of capsids, as evidenced by the presence of CTDs either on the exterior of some capsids or RNA-bound in the interior of other capsids [START_REF] Yu | 3.5Å cryoEM structure of hepatitis B virus core assembled from full-length core protein[END_REF]. This model is attractive as it provides a mechanism by which HBc can be accessible for interaction with host proteins involved in intracellular tra cking, capsid maturation, or capsid disassembly. In this model, the positively charged CTD interacts transiently with the negatively charged Asp2 and Glu43 that line the local three-fold channel, presumably allowing movement of CTD in and out of capsids [START_REF] Yu | 3.5Å cryoEM structure of hepatitis B virus core assembled from full-length core protein[END_REF]. In congruence with the cryo-EM study, biochemical binding assays demonstrate a nucleic acid sensitive and transient exposure of the CTD tail [START_REF] Chen | A kinase Chaperones Hepatitis b virus capsid assembly and captures capsid dynamics in vitro[END_REF], further con rming the presence of CTD on the exterior of capsids. Speci cally, RNA-lled capsids failed to bind serine-arginine protein kinase (SRPK), a host chaperone that binds full length HBc capsids, but not HBc 1-149 capsids [START_REF] Chen | A kinase Chaperones Hepatitis b virus capsid assembly and captures capsid dynamics in vitro[END_REF], con rming the dynamic exposure of CTDs is mediated by CTDnucleic acid interactions. These results also agree with biochemical studies showing the CTD of assembled capsids is not only sensitive to proteases, but also accessible to host proteins [START_REF] Chen | A kinase Chaperones Hepatitis b virus capsid assembly and captures capsid dynamics in vitro[END_REF][START_REF] Gallina | A recombinant hepatitis B core antigen polypeptide with the protamine-like domain de-leted selfassembles into capsid particles but fails to bind nucleic acids[END_REF]. The exact mechanism of this dynamic CTD shuttling remains unknown, although a role of phosphorylation has been suggested by site directed mutagenesis of HBc [START_REF] Selzer | Hepatitis B virus core protein phosphorylation sites a ect capsid stability and transient exposure of the Cterminal domain[END_REF]. However, further detailed analyses of the e ect of CTD phosphorylations and dephosphorylations in CTD shuttling are required.

HBc, a nucleic acid chaperone

Nucleic acid chaperones are proteins that catalyze structural rearrangement of genetic material into a functional conformation [START_REF] Semrad | Proteins with RNA chaperone activity: a world of diverse proteins with a common task-impediment of RNA misfolding[END_REF]. The binding of nucleic acid chaperones to their targets is characteristically weak and transient, lacks sequence speci city, and does not require ATP hydrolysis [START_REF] Semrad | Proteins with RNA chaperone activity: a world of diverse proteins with a common task-impediment of RNA misfolding[END_REF]). An array of viral proteins displays nucleic acid chaperone activity. For example, nucleocapsid proteins of HIV [START_REF] Levin | Nucleic acid chaperone activity of HIV-1 nucleocapsid protein: critical role in reverse transcription and molecular mechanism[END_REF], dengue virus [START_REF] Pong | RNA binding property and RNA chaperone activity of dengue virus core protein and other viral RNAinteracting proteins[END_REF], and hepatitis delta virus [START_REF] Huang | Identi cation and characterization of the RNA chaperone activity of hepatitis delta antigen peptides[END_REF]) are described as nucleic acid chaperones. The CTD of HBc contains an arginine-rich domain resembling histone tails with more than 50% of the residues being arginine (Lewellyn and Loeb, 2011). The CTD binds to nucleic acids in vivo due to its positive charge (Nassal, 1992). In addition to its characteristic positive charge, CTD is also structurally disordered [START_REF] Yu | 3.5Å cryoEM structure of hepatitis B virus core assembled from full-length core protein[END_REF], another key feature of nucleic acid chaperones [START_REF] Chu | Nucleic acid chaperone activity associated with the arginine-rich domain of human hepatitis B virus core protein[END_REF]. The localization of CTD to the interior of capsids [START_REF] Zlotnick | Localization of the C terminus of the assembly domain of hepatitis B virus capsid protein: implications for morphogenesis and organization of encapsidated RNA[END_REF] and its ability to bind nucleic acids in vivo support the model that CTD works as a nucleic acid chaperone. This chaperone activity was recently con rmed in vitro using recombinant full length HBc, as well as CTD synthetic peptides [START_REF] Chu | Nucleic acid chaperone activity associated with the arginine-rich domain of human hepatitis B virus core protein[END_REF]. HBc CTD facilitated annealing and unwinding of DNA and enhanced hammerhead ribozyme cleavage in vitro, rendering HBc CTD a bona fide nucleic acid chaperone [START_REF] Chu | Nucleic acid chaperone activity associated with the arginine-rich domain of human hepatitis B virus core protein[END_REF]. However, the high degree of functional complexity associated with the CTD, makes it challenging to demonstrate the nucleic acid chaperone activity of HBc in vivo, without disturbing other HBc functions.

HBc localization: a dynamic process involving active-transport

Reports of subcellular localization of HBc in vivo demonstrated both cytoplasmic as well as nuclear distribution. Histologic analysis of tissues from HBV-infected patients indicated a distribution of HBc/capsids in both cytosolic and nuclear compartments. Sharma et al., as well as Akiba et al., described a predominant nuclear distribution (Akiba et al., 1987;[START_REF] Sharma | Immunohistochemistry for core and surface antigens in chronic hepatitis[END_REF] while others observed mainly a cytosolic core localization [START_REF] Michalak | Crystalline aggregates of hepatitis B core particles in cytoplasm of hepatocytes[END_REF]Petit and Pillot, 1985). In vitro analysis has shown HBc subcellular localization is in fact cell-cycle dependent [START_REF] Yeh | Cell-cycle regulation of nuclear localization of hepatitis B virus core protein[END_REF]. The amount of core protein in the nucleus increases in G1 phase, diminishes in S phase, and increases again to detectable levels in non-proliferating cells [START_REF] Yeh | Cell-cycle regulation of nuclear localization of hepatitis B virus core protein[END_REF].

HBc shuttles between cytoplasm and nucleus rapidly and continuously (Li et al., 2010). These latter points could explain di erences described above. Although HBc is 21 kDa, suggesting passive transport to and from the nucleus, this trapping, as exists for human T-lymphotropicvirus-1 (HTLV-1) [START_REF] Go | Host factors exploited by retroviruses[END_REF] and human papillomaviruses [START_REF] Aydin | Large scale RNAi reveals the requirement of nuclear envelope breakdown for nuclear im-port of human papillomaviruses[END_REF], would be fairly ine cient due to the quasiquiescent state of hepatocytes. Given that HBc is a phosphorylation target of well-characterized cell-cycle regulated kinases, such as cyclindependent kinase 2 (CDK2) (Ludgate et al., 2012) and polo-like kinase 1 (PLK1) (Diab et al., 2017), it is highly plausible that such phosphorylation may contribute to HBc/capsid localization. Investigating this question in a physiologically relevant infection model will most certainly expand our understanding of the dynamics of HBc subcellular localization. Furthermore, it remains unresolved whether HBc enters the nuclear pore complex (NPC) as a dimer or as a mature capsid. Thus, signals modulating HBc import and export from the nucleus are probably distinct from those that guide transport of mature capsids. An early in vitro model for capsid transport by Kann and colleagues suggested mature capsid disassembles into HBc dimers at the cytosolic side of the NPC, releasing the rcDNA-pol complex [START_REF] Kann | In vitro model for the nuclear transport of the hepadnavirus genome[END_REF]. Subsequently, HBc dimers bind importin-β and are transported through a NPC. At this point, HBc dimers dissociate from transport factors and could remain free in the nucleus, eventually playing a role as transcription factors, as described below, or reassemble as empty-capsid forms. On the other hand, as viral polymerase does not contain an importin-β binding (IBB) domain, formation of importin-α/β is required to import the genome complex [START_REF] Kann | In vitro model for the nuclear transport of the hepadnavirus genome[END_REF]. Once at the nuclear side, the rcDNA-pol complex dissociates from importin α/β. Since the viral genome comprises pathogen-associated molecular patterns (PAMPs), it could trigger sensing by cytosolic pattern recognition receptors (PRRs) [START_REF] Paludan | Immune Sensing of DNA[END_REF]. This model of genomic nuclear import, showing a release of viral rcDNA in the cytosol could explain the weak innate immunity activation upon HBV infection [START_REF] Faure-Dupuy | Interplay between the hepatitis B virus and innate immunity: from an understanding to the development of therapeutic concepts[END_REF]Lucifora et al., 2014;[START_REF] Vanwolleghem | Re-evaluation of hepatitis B virus clinical phases by systems biology identi es unappreciated roles for the innate immune response and B cells[END_REF]. However, this model is based on Woodchuck Hepatitis Virus where capsids were denatured using urea treatment in order to release the rcDNA-pol complex [START_REF] Kann | In vitro model for the nuclear transport of the hepadnavirus genome[END_REF]. Under such conditions, changes in conformation of the viral polymerase could enable its interaction with importin-α.

A more recent alternative model (see Blondot et al., 2016 for in-depth review), suggests a direct interaction between mature capsids and an essential component of the NPC's basket, the nucleoporin 153 (Nup153).

It is known that importin-α interacts directly with Nup153, promoting importin-α/β-mediated nuclear import [START_REF] Ogawa | The interaction between importinalpha and Nup153 promotes importin-alpha/beta-mediated nuclear import[END_REF]. Of note, it was shown earlier that mature capsids are transported to nuclear pores by an importin α/β complex [START_REF] Kann | Intracellular transport of hepatitis B virus[END_REF]. The new model provides two options for mature capsids going through nuclear pores: rst, direct binding to Nup153 at the basket of the NPC (cargo size 36 nM) or via coating by importin-α/β and binding to Nup153 (cargo size 38 nM). According to this model, the HBc CTD is not ne-cessary to anchor the capsid at the nuclear basket since importin α/β is required for capsid transport. While both models intend to explain the mechanism of capsid transport through nuclear pores, neither reveals the trigger of such transport. Studies of the CTD tail revealed a role in directing HBc subcellular localization (Blondot et al., 2016;Li et al., 2010). Several attempts have been made to map the nuclear localization sequence (NLS) to the arginine repeats of CTD. However, the results are con icting, probably due to the redundancy of arginine repeats within the CTD [START_REF] Liao | Phosphorylation and nuclear localization of the hepatitis B virus core protein: signi cance of serine in the three repeated SPRRR motifs[END_REF]Rabe et al., 2003). Recently, an exhaustive mutagenesis analysis of the CTD localized the NLS sequence to the 1st and 3rd arginine repeats, while the 2nd and 4th repeats contained the cytoplasmic retention signals (CRS) (Fig. 2) (Li et al., 2010). The presence of more than one NLS in a protein is known to maximize import e ciency [START_REF] Mears | Identi cation of nuclear and nucleolar localization signals in the herpes simplex virus regulatory protein ICP27[END_REF]. Other viral proteins, such as ICP27, of herpes simplex virus type 1 (HSV-1) [START_REF] Mears | Identi cation of nuclear and nucleolar localization signals in the herpes simplex virus regulatory protein ICP27[END_REF] and NS1 of In uenza (Melen (Li et al., 2010(Li et al., ). et al., 2012)), also contain multiple NLS sites. However, one must also consider the accessibility of these sequences in view of the dynamic structural changes of assembled HBc in viral capsids. The dynamic positioning of HBc CTD in assembled capsids makes it quite challenging to de ne clearly the di erent signals regulating HBc transport. Nuclear export of HBc resembles ICP27 of HSV-1 and EBNA1 of Epstein Barr virus (EBV), in that they all bind to the RNA export factor TAP, and all contain the characteristic arginine rich domains (Fig. 2) [START_REF] Yang | Nuclear export of human hepatitis B virus core protein and pregenomic RNA depends on the cellular NXF1-p15 machinery[END_REF]. Since subcellular localization of ICP27 and EBNA1 are regulated by arginine methylation [START_REF] Souki | Arginine methylation of the ICP27 RGG box regulates ICP27 export and is required for e cient herpes simplex virus 1 replication[END_REF], it is reasonable to envisage a similar mechanism driving HBc localization. However, it is still unknown if the arginine repeats of CTD are methylated, an hypothesis worthy of future investigation. Of note, arginine methyltransferase 5 (PRMT5) was reported as a negative e ector of HBV replication and transcription via epigenetic repression of cccDNA transcription and interference with pregenomic RNA encapsidation [START_REF] Zhang | PRMT5 restricts hepatitis B virus replication through epigenetic repression of covalently closed circular DNA transcription and interference with pregenomic RNA encapsidation[END_REF]. Interestingly, PRMT5 was shown to bind HBc, and HBc-de cient virus (HBV-ΔHBc) had less PRMT5 bound cccDNA when compared to the wildtype HBV [START_REF] Zhang | PRMT5 restricts hepatitis B virus replication through epigenetic repression of covalently closed circular DNA transcription and interference with pregenomic RNA encapsidation[END_REF]. The contribution of HBc CTD to capsid localization is only begin-ning to be resolved. The ambiguity is based on the long-standing notion that the CTD localizes to the interior of capsids based on cryo-EM studies [START_REF] Zlotnick | Localization of the C terminus of the assembly domain of hepatitis B virus capsid protein: implications for morphogenesis and organization of encapsidated RNA[END_REF], and has a well-documented role in pgRNA packaging (Nassal, 1992). However, as previously discussed, more recent studies suggest a dynamic model in which HBc CTD shuttles between the interior and exterior of the capsid [START_REF] Chen | A kinase Chaperones Hepatitis b virus capsid assembly and captures capsid dynamics in vitro[END_REF]Li et al., 2010;[START_REF] Selzer | Hepatitis B virus core protein phosphorylation sites a ect capsid stability and transient exposure of the Cterminal domain[END_REF]. Transport of cargo through the nuclear envelope is often facilitated by regulatory phosphorylation/dephosphorylation of speci c residues [START_REF] Nardozzi | Phosphorylation meets nuclear import: a review[END_REF]. Earlier studies reported phosphorylated HBc was only observed in the cytoplasm, based on subcellular fractionation of in vivo 32 P-radiolabed cell lysates [START_REF] Yeh | Cell-cycle regulation of nuclear localization of hepatitis B virus core protein[END_REF]. However, the molecular mechanism regulating subcellular localization of monomeric/dimeric vs. assembled HBc in capsids remains ambiguous. The potential contribution of HBc phosphorylation to the nuclear import of capsids comes from known import mechanisms for other viral proteins, including EBNA-1 [START_REF] Kitamura | Nuclear import of Epstein-Barr virus nuclear antigen 1 mediated by NPI-1 (Importin alpha5) is up-and down-regulated by phosphorylation of the nuclear localization signal for which Lys379 and Arg380 are essential[END_REF]) and SV40 T-antigen [START_REF] Hubner | The protein kinase CK2 site (Ser111/112) enhances recognition of the simian virus 40 large T-antigen nuclear localization se-quence by importin[END_REF]. Speci cally, phosphorylation of EBNA-1 increases its afnity for the import adaptor importin-α5 which mediates the nuclear transport via interaction with importin-β [START_REF] Kitamura | Nuclear import of Epstein-Barr virus nuclear antigen 1 mediated by NPI-1 (Importin alpha5) is up-and down-regulated by phosphorylation of the nuclear localization signal for which Lys379 and Arg380 are essential[END_REF]. SV-40 large T antigen has the best characterized NLS, and phosphorylation upstream of the classic NLS enhances nuclear import [START_REF] Hubner | The protein kinase CK2 site (Ser111/112) enhances recognition of the simian virus 40 large T-antigen nuclear localization se-quence by importin[END_REF]. Thus, the phosphorylation status of HBc CTD could facilitate interaction with nuclear pore complex components. Indeed, in pulldown experiments using HBc CTD-GST fusion proteins, serine to alanine substitution at the three serine/proline (S/P) sites (Fig. 3) showed increased binding to importin-α [START_REF] Liu | Regulation of multiple stages of hepadnavirus replication by the carboxyl-terminal domain of viral core protein in trans[END_REF], suggesting phosphorylation impedes tra cking of HBc to the nucleus. While the in vivo relevance of these observations remains to be determined, other groups have reported that HBc phosphorylated in vitro by PKC in assembled capsids, preferentially bound importin-β [START_REF] Kann | Phosphorylationdependent binding of hepatitis B virus core particles to the nuclear pore complex[END_REF]. However, the phosphorylation sites of HBc required for interaction with importin-β have not been mapped [START_REF] Kann | Phosphorylationdependent binding of hepatitis B virus core particles to the nuclear pore complex[END_REF]. Furthermore, it is unresolved whether protein kinase C (PKC) is the in vivo kinase that phosphorylates HBc CTD (Daub et al., 2002). Thus, further studies are necessary to address this issue, employing well-characterized and functionally documented mutants of HBc. Alternatively, as shown by Schmitz et al., capsid maturation could be a driving force of HBV nuclear import [START_REF] Schmitz | Nucleoporin 153 arrests the nuclear import of hepatitis B virus capsids in the nuclear basket[END_REF]. At the nuclear pore, HBc capsids dissociate into dimers followed by entry into the nucleus [START_REF] Rabe | Nuclear entry of hepatitis B virus capsids involves disintegration to protein dimers followed by nuclear reassociation to capsids[END_REF]. Mature capsids directly interact with Nup153, an essential component of the nuclear basket that facilitates nuclear import via importin-β [START_REF] Schmitz | Nucleoporin 153 arrests the nuclear import of hepatitis B virus capsids in the nuclear basket[END_REF]. Interaction of mature capsids with Nup153 resulted in capsid disintegration followed by release of viral genomes into the nucleus [START_REF] Schmitz | Nucleoporin 153 arrests the nuclear import of hepatitis B virus capsids in the nuclear basket[END_REF]. Interestingly, capsids with immature genomes, i.e., RNA-lled capsids, accumulate at the nuclear pore. Recently, it was reported that serine to glutamate substitutions at the 3 S/P sites of HBc CTD (Fig. 3) reduced CTD exposure to the exterior of capsids suggesting a role for these phosphorylations in directing CTD exposure-inside or outside the capsid [START_REF] Selzer | Hepatitis B virus core protein phosphorylation sites a ect capsid stability and transient exposure of the Cterminal domain[END_REF]. However, the mechanism responsible for distinguishing mature capsids vs. RNAlled capsids remains unclear (Hu and Liu, 2017). Phosphorylation of HBc, shuttling of the HBc CTD to the interior vs. exterior of capsids, and maturation of the genome, may regulate exposure of di erent localization signals to host transport machinery. Such a multilayer and dynamic regulation network can best explain the di culty in deciphering the mechanism of HBc subcellular localization during the HBV life cycle. To understand these important dynamic regulatory networks, it is essential to gain better understanding of HBc structure, HBc interacting partners, and HBc posttranslational modi cations.

HBc phosphorylation

Since the HBV genome does not encode a protein kinase, endogenous/cellular kinases must mediate post-translational HBc modi cations [START_REF] Lanford | Expression of hepatitis B virus core and precore antigens in insect cells and characterization of a core-associated kinase activity[END_REF]. PKC [START_REF] Kann | E ect of core protein phosphorylation by protein kinase C on encapsidation of RNA within core particles of hepatitis B virus[END_REF][START_REF] Wittkop | Inhibition of protein kinase C phosphorylation of hepatitis B virus capsids inhibits virion formation and causes intracellular capsid accumulation[END_REF], serine arginine protein kinase 1 (SRPK1) (Daub Diab et al., 2017Diab et al., et al., 2002) ) and CDK2 (Ludgate et al., 2012) have been suggested as endogenous kinases that associate with or are packaged within viral capsids. The robust phosphorylation of HBc has been demonstrated in vitro by phospho-proteomic approaches [START_REF] Chen | A kinase Chaperones Hepatitis b virus capsid assembly and captures capsid dynamics in vitro[END_REF], and in vivo by labeling with 32 P-orthophosphate (Jung et al., 2014). The HBc CTD contains multiple serine/threonine phospho-acceptor sites which are remarkably well-conserved among related viruses (Jung et al., 2014). Phospho-proteomic analyses of HBc have identi ed at least seven conserved serine and threonine sites that are phosphorylated in vivo [START_REF] Chen | A kinase Chaperones Hepatitis b virus capsid assembly and captures capsid dynamics in vitro[END_REF]. Notably, proline-directed serine (S/P) sites at positions 155, 162 and 170, are highly conserved and several S/P kinases have been reported to phosphorylate these sites (Fig. 3) (Daub et al., 2002;Ludgate et al., 2012). Other putative kinases, including: SRPKs, Glyceraldehyde-3-phosphate dehydrogenase protein kinase (GAPDprotein kinase), PKC, and an unknown kinase of 46 kDa, have also been reported (Daub et al., 2002;[START_REF] Duclos-Vallee | Phosphorylation of the hepatitis B virus core protein by glyceraldehyde-3-phosphate dehydrogenase protein kinase activity[END_REF][START_REF] Kann | Phosphorylationdependent binding of hepatitis B virus core particles to the nuclear pore complex[END_REF]Ludgate et al., 2012) (Table 1). Yet none of these studies have mapped phosphorylation to speci c sites, with the exception of SRPK1 and SRPK2 at the 3 S/P sites (Daub et al., 2002). However, the exact contribution of the SRPK-mediated phosphorylation to viral replication is enigmatic, because, although overexpression of SRPK1 or SRPK2 impaired HBV replication, this e ect was independent of kinase activity [START_REF] Zheng | Suppression of hepatitis B virus replication by SRPK1 and SRPK2 via a pathway independent of the phosphorylation of the viral core protein[END_REF]. These results support that SRPK-mediated phosphorylation of HBc observed in vitro is due to promiscuous substrate speci city of these kinases (Ludgate et al., 2012), and that SRPKs exert other functions a ecting viral replication. For example, SRPKs could act as non-canonical chaperones in capsid assembly [START_REF] Chen | A kinase Chaperones Hepatitis b virus capsid assembly and captures capsid dynamics in vitro[END_REF].

Regarding PKC-mediated HBc phosphorylation, inactivation of PKC led to defective capsid envelopment, but had no e ect on genome maturation [START_REF] Wittkop | Inhibition of protein kinase C phosphorylation of hepatitis B virus capsids inhibits virion formation and causes intracellular capsid accumulation[END_REF]. There is, however, lack of consensus regarding PKC being the candidate host kinase since speci c inhibition of PKC had no e ect on CTD phosphorylation (Daub et al., 2002;Ludgate et al., 2012). CDK2 was shown to phosphorylate the core protein of HBV both in vitro and in vivo (Ludgate et al., 2012). Intriguingly, CDK2 inhibitors had no observable e ect on viral replication (Ludgate et al., 2012). The absence of such e ect could be due to lack of speci city of the chemical inhibitors [START_REF] Sakurikar | Critical reanalysis of the methods that discriminate the activity of CDK2 from CDK1[END_REF] or, more likely, due to redundancy among host kinases in phosphorylating HBc. Knockdown cell lines for CDK2 or other kinases via expression of shRNAs could elucidate their contribution to HBc phosphorylation and viral replication.

Although the kinases that phosphorylate HBc are still to be determined, phosphorylations of HBc do regulate its function at many levels [START_REF] Gazina | Core protein phosphorylation modulates pregenomic RNA encapsidation to di erent extents in human and duck hepatitis B viruses[END_REF]Jung et al., 2014;[START_REF] Kann | Phosphorylationdependent binding of hepatitis B virus core particles to the nuclear pore complex[END_REF][START_REF] Ludgate | Phosphorylation state-dependent interactions of hepadnavirus core protein with host factors[END_REF]. Speci cally, phosphorylation and de-phosphorylation of the HBc protein is required for capsid maturation as demonstrated by mass spectrometric analyses and site-directed mutagenesis studies of HBc (Basagoudanavar et al., 2007;Perlman et al., 2005). Phosphorylation of di erent serine residues of HBc CTD regulates reverse transcription, pgRNA encapsidation, DNA synthesis, subcellular localization, and virion secretion (Basagoudanavar et al., 2007;[START_REF] Gazina | Core protein phosphorylation modulates pregenomic RNA encapsidation to di erent extents in human and duck hepatitis B viruses[END_REF][START_REF] Lan | Roles of the three major phosphorylation sites of hepatitis B virus core protein in viral replication[END_REF]Perlman et al., 2005). HBc phosphorylation is also required for the chaperone activity of CTD [START_REF] Chu | Nucleic acid chaperone activity associated with the arginine-rich domain of human hepatitis B virus core protein[END_REF]. It was also reported that the HBc phosphorylation status in uences capsid stability [START_REF] Ludgate | Cell-free hepatitis B virus capsid assembly dependent on the core protein C-Terminal domain and regulated by phosphorylation[END_REF], since capsids with glutamate phospho-mimetic substitutions at the 3 S/P sites (S/P to E/P) were more stable than wild type capsids. It is likely that during various stages in the life cycle of HBV or as a function of the metabolic and proliferative status of the infected hepatocyte, distinct kinases target HBc phosphorylation, perhaps in an overlapping manner. The identi cation of those kinases, and the mechanisms by which they regulate HBV replication, is of great signi cance and will provide valuable targets for mechanism-based therapeutics.

To this end, our recent study has identi ed the mitotic PLK1 as another cellular kinase that phosphorylates HBc in vitro (Diab et al., 2017). PLK1 substrates are usually primed by phosphorylation at S/P directed sites mediated by CDKs, including CDK1 or 2 (Elia et al., 2003).

Recombinant HBc, as well as immuno-puri ed HBc from trans-fected mammalian cells serves as robust PLK1 substrate in vitro (Diab et al., 2017). We have mapped the phospho-acceptor sites to S168, S176, and S178 (Fig. 3) (Diab et al., 2017), all of which have been identi ed as in vivo phospho-acceptor sites by an unknown kinase (Jung et al., 2014).

Remarkably, inhibition of PLK1 by speci c chemical in-hibitors or siRNA-mediated knockdown of PLK1 inhibited viral re-plication, suggesting HBc phosphorylation by PLK1 is functionally im-portant in virus biosynthesis (Diab et al., 2017). Signi cantly, we made the interesting observation, in accordance with the known substrate preference of PLK1, that alanine substitutions at the 3 S/P sites (S/P to A/P) resulted in loss of PLK1 phosphorylation in vitro. By contrast, PLK1 phosphorylation was robustly recovered in phospho-mimetic mutants, containing serine to aspartic acid substitutions at all three S/P sites (Diab et al., 2017). This suggests that CDK2 functions as the priming kinase for HBc, mediating the phosphorylation of the S/P sites which in turn enables the subsequent phosphorylation by PLK1 (Elia et al., 2003).

Based on our results that PLK1 is a positive e ector of HBV replication, a mechanistic understanding of the role of PLK1 phos-phorylation of HBc in HBV replication has promising therapeutic po-tential. So far, PLK1 is the sole host-kinase whose chemical inhibition results in impaired HBV replication in vivo (i.e., in liver humanized mouse model) at concentrations comparable to other known anti-HBV agents (Diab et al., 2017). Finally, based on this dependence of PLK1 phosphorylation of HBc on its prior phosphorylation by CDK2, we reason combination treatment with inhibitors for CDK2 and PLK1 ki-nases will be e ective in suppressing these modi cations and thus ef-fectively inhibit virus biosynthesis. This hypothesis is particularly at-tractive since safety of PLK1 [START_REF] Gjertsen | Discovery and development of the Polo-like kinase inhibitor volasertib in cancer therapy[END_REF] and CDK2 [START_REF] Asghar | The history and future of targeting cyclin-dependent kinases in cancer therapy[END_REF] inhibitors has been established in clinical trials for di erent types of human cancer.

HBc and interaction with host factors

HBc modulates nearly every stage of the HBV life cycle. With such extensive and intricate functions, HBc has evolved to modulate several host-protein functions. For example, HBc dimers interact with HSP90, and HSP90 facilitates formation of HBV capsids both in vitro and in vivo [START_REF] Shim | Heat shock protein 90 facilitates formation of the HBV capsid via interacting with the HBV core protein dimers[END_REF]. On the other hand, the host HSP40 protein also binds, but de-stabilizes HBc by accelerating its degradation [START_REF] Sohn | Negative regulation of hepatitis B virus replication by cellular Hsp40/DnaJ proteins through destabilization of viral core and X proteins[END_REF]. The binding of HBc to other cellular factors, such as NIRF, an E3 ubiquitin ligase, is implicated in HBc degradation [START_REF] Qian | NIRF, a novel ubiquitin ligase, interacts with hepatitis B virus core protein and promotes its degradation[END_REF]. Thus, dynamic interactions of HBc with host chaperones regulate both HBc stability and capsid assembly [START_REF] Qian | NIRF, a novel ubiquitin ligase, interacts with hepatitis B virus core protein and promotes its degradation[END_REF][START_REF] Shim | Heat shock protein 90 facilitates formation of the HBV capsid via interacting with the HBV core protein dimers[END_REF][START_REF] Sohn | Negative regulation of hepatitis B virus replication by cellular Hsp40/DnaJ proteins through destabilization of viral core and X proteins[END_REF].

Viruses hijack host RNA processing machinery [START_REF] Salvetti | Viruses and the nucleolus: the fatal attraction[END_REF] and HBV is no exception. HBc utilizes the nuclear export factor 1 (NXF1/p15) and transcription export (TREX) machinery for nuclear export as a ribonucleoprotein complex along with the viral pgRNA [START_REF] Yang | Nuclear export of human hepatitis B virus core protein and pregenomic RNA depends on the cellular NXF1-p15 machinery[END_REF]; however, TREX-mediated export of HBc and pgRNA are independent of each other [START_REF] Yang | Nuclear export of human hepatitis B virus core protein and pregenomic RNA depends on the cellular NXF1-p15 machinery[END_REF]. Other viruses that hijack the NXF1/p15 pathway for mRNA processing and export include EBV [START_REF] Juillard | Epstein-Barr virus protein EB2 contains an N-terminal transferable nuclear export signal that promotes nucleocytoplasmic export by directly binding TAP/NXF1[END_REF] and HSV-1 [START_REF] Johnson | The cellular RNA export receptor TAP/ NXF1 is required for ICP27-mediated export of herpes simplex virus 1 RNA, but the TREX complex adaptor protein Aly/REF appears to be dispensable[END_REF]. Interestingly, depletion of di erent TREX components has no apparent e ect on accumulation of viral DNA, suggesting redundancy among host factors utilized by the virus. Another class of host proteins commonly targeted by viruses is serine, arginine rich-proteins [START_REF] Howard | The RNAissance family: SR proteins as multifaceted regulators of gene expression[END_REF] which, along with the kinases (SRPKs) that phosphorylate them, play central roles in alternative RNA splicing [START_REF] Howard | The RNAissance family: SR proteins as multifaceted regulators of gene expression[END_REF]. Intriguingly, HBc CTD resembles serine, arginine-rich proteins and it is not surprising that SRPKs associate with and potentially phosphorylate HBc (Chen et al., 2011;Daub et al., 2002;[START_REF] Zheng | Suppression of hepatitis B virus replication by SRPK1 and SRPK2 via a pathway independent of the phosphorylation of the viral core protein[END_REF]. Interestingly, mass spectrometry analyses of HBc interacting proteins has identi ed SRPK1 as well as several known SRPK1 interacting partners, including: SRSF1, SRSF9, DHX9, CDKN2A, DDX21, and DDX50 (Diab et al. unpublished data). The functional signi cance of these HBc interactions in the viral life cycle is unknown and worthy of investigation. We speculate that HBV usurps the host RNA-processing machinery for production and processing of its own viral RNA. It is also likely that such interactions may allow HBc to interfere with host gene expression. This has been evident in the case of E1E4 protein of the human papilloma type 1 virus (HPV1), which not only binds to SRPK1, but also inhibits phosphorylation of its host serine arginine substrates [START_REF] Prescott | Human papillomavirus type 1 E1ˆE4 protein is a potent inhibitor of the serine-argi-nine (SR) protein kinase SRPK1 and inhibits phosphorylation of host SR proteins and of the viral transcription and replication regulator E2[END_REF]. A yeast two-hybrid screen of a human liver cDNA library identi ed the human protein GIPC1 as another HBc interaction partner [START_REF] Razanskas | Interaction of hepatitis B virus core protein with human GIPC1[END_REF], but the signi cance of such interaction remains to be determined. GIPC1 contains a PDZ domain, a protein-protein interaction domain (reviewed in [START_REF] Hung | PDZ domains: structural modules for protein complex assembly[END_REF]. PDZ-containing host proteins are commonly targeted by viral proteins; examples include the Tax protein of the T-cell leukemia virus type 1 [START_REF] Rousset | The C-terminus of the HTLV-1 Tax oncoprotein mediates interaction with the PDZ domain of cellular pro-teins[END_REF] and the E6 protein of the human papilloma virus type 18 [START_REF] Favre-Bonvin | Human papillomavirus type 18 E6 protein binds the cellular PDZ protein TIP-2/GIPC, which is involved in transforming growth factor beta signaling and triggers its degradation by the proteasome[END_REF].

It is reasonable to speculate that HBc exerts its various roles by interacting with speci c host factors at di erent stages of the viral life cycle in a phosphorylation dependent manner. A better dissection of host factors that interact with HBc could facilitate development of new antiviral strategies or help elucidate core assembly modulators (CAMs).

HBc as a regulator of transcription

In chronically infected patients, HBc is detected in both cytoplasm and nucleus of hepatocytes (Chu and Liaw, 1987). In vitro models of HBV infection also show nuclear as well as cytoplasmic localization of HBc. Nuclear localization of HBc, in biopsies from HBV infected pa-tients, has been linked to high viremia and de cient immune response (Chu and Liaw, 1987;[START_REF] Nguyen | The role of quantitative hepatitis B serology in the natural history and management of chronic hepatitis B[END_REF]. These results prompted investigations of the nuclear function of HBc, in relation to virus biosynthesis and in uence upon host gene expression.

In the nucleus of infected cells, the viral cccDNA assumes chro-matinlike structure in association with histone and non-histone pro-teins, along with viral HBx and HBc proteins (Bock et al., 2001;Lucifora and Protzer, 2016). Association of HBc with the cccDNA mini-chromosome was reported to alter nucleosome spacing (Bock et al., 2001). Furthermore, it was reported that HBc preferentially associated with CpG island 2 of the HBV genome that overlaps enhancers (Enh) I and II involved in viral transcription; HBc binding to CpG island 2 positively correlated with the rcDNA/cccDNA ratio and serum levels of HBV DNA in infected patients (Guo et al., 2011). HBc may also act as transcriptional activator of the HBV pre-Core promoter by enhancing binding of NF-kB upstream of viral Enh II [START_REF] Kwon | Hepatitis B viral core protein activates the hepatitis B viral enhancer II/pregenomic promoter through the nuclear factor kappaB binding site[END_REF]. However, how HBc modulates the function of the cccDNA mini-chromosome remains to be determined. Since HBc is also essential for viral DNA replication, a de nitive genetic approach involving mutagenesis or depletion of HBc is not feasible as a means to delineate its role in cccDNA mini-chromosome functions. Therefore, novel approaches are required to target nuclear HBc, in uencing its capacity to associate with the cccDNA mini-chromosome, without a ecting HBc cytoplasmic functions.

Regarding e ects of HBc on host gene expression, HBc enhanced cAMP-Response-Element (CRE)-mediated transcription via the CRE/ CREB/CBP pathway (Xiang et al., 2015) in a manner similar, but weaker to previously reported HBx-mediated CRE activation [START_REF] Andrisani | CREB-mediated transcriptional control[END_REF][START_REF] Williams | The hepatitis B virus X protein targets the basic region-leucine zipper domain of CREB[END_REF]. HBc may also func-tion as a transcriptional repressor of host genes. For example, binding of HBc to E2F1 reduced the DNA-binding ability of E2F1 at the p53 pro-moter, thereby inhibiting p53 transcription (Kwon and Rho, 2003). In hepatoma cell lines expressing the core protein, HBc blocked the human death receptor 5 (DR5) by repressing its promoter, and inhibition of DR5 desensitized hepatocytes to TRAIL-induced apoptosis [START_REF] Du | Hepatitis B virus core protein inhibits TRAIL-induced apoptosis of hepatocytes by blocking DR5 expression[END_REF]. These results suggest a role for HBc in development of chronic infection by preventing hepatocyte death via blocking DR5 expression. The gene regulatory function of HBc may have evolved as a means whereby the virus may evade host immunity. HBc has been shown to downregulate IFN-induced host antiviral responses in hepatoma cell lines, by interacting with the promoter of MxA, an INFinducible gene widely implicated in antiviral response (Fernandez et al., 2003). However, these conclusions must be further validated by determining in vivo association of HBc with the MxA promoter, in a physiological cellular context of HBV infection. Interestingly, MxA has been shown to inhibit HBV replication by direct interaction with HBc, preventing capsid formation (Li et al., 2012), and providing yet another example of antagonism between viral and host proteins. Ectopic expression of HBc in HepG2 cells and primary hepatocyte cultures sensitized cells to TNF-α induced apoptosis, by disrupting the interaction between mitogenactivated protein kinase kinase 7 (MKK7) and receptor of activated protein kinase C 1 (RACK1) [START_REF] Jia | Hepatitis B virus core protein sensitizes hepatocytes to tumor necrosis factor-induced apoptosis by sup-pression of the phosphorylation of mitogen-activated protein kinase kinase 7[END_REF]. This suggests a direct role of HBc in driving liver pathogenesis in chronically infected patients. These results require validation in a rigorous experimental model. In continuing this e ort to understand the e ect of HBc on host gene expression, recent studies have generated the genome-wide pro le of HBc in HBV-infected hepatocytes, employing chromatin immunoprecipitation microarray studies (ChIP-on-chip). These studies revealed that promoters of nearly 3100 host genes exhibited increased binding to HBc (Guo et al., 2012). However, further studies are necessary to understand the functional signi cance of HBc binding to the host genome in a ecting HBV-mediated disease pathogenesis. Toward this goal, Lucifora et al. recently demonstrated interaction of nuclear deaminases APOBEC3A and APOBEC3B with HBc-bound cccDNA in host nucleus, leading to cccDNA deamination and degradation (Lucifora et al., 2014). Whether HBc recruits these nuclear deaminases to host genes is unknown and of great importance, given the role of APOBEC proteins as cancer driver genes in humans [START_REF] Harris | Molecular mechanism and clinical impact of APOBEC3B-catalyzed mutagenesis in breast cancer[END_REF][START_REF] Morganella | The topography of mutational processes in breast cancer genomes[END_REF][START_REF] Nik-Zainal | Mutational processes molding the genomes of 21 breast cancers[END_REF]. Muli-omics analyses of HBc transfected cells linked HBc over expression up-regulated glycolysis and amino acid metabolism, providing further insights into the role of HBc in the molecular pathogenesis of HBV-mediated tumorigenesis (Xie et al., 2017).

HBc as a target for clinical intervention against chronic HBV infection

Due to the multiple functions of HBc in the HBV life cycle, this protein has become a preferential target for developing novel, directacting agents against HBV replication. Molecules targeting capsid assembly have been known for some time with the discovery BAY41-4109, a heteroaryldihydropyrimidine derivative (Deres et al., 2003), and AT130, a phenylpropenamide derivative [START_REF] Delaney | Phenylpropenamide derivatives AT-61 and AT-130 inhibit replication of wild-type and lamivudine-resistant strains of hepatitis B virus in vitro[END_REF]. This class of inhibitors were subsequently abbreviated core assembly/ allosteric modulators (CAMs), due to their mode of action, which is mainly based on allosteric modulation (Zlotnick et al., 2015). Interestingly, these molecules were shown to be active against nucleoside analogue resistant strains, which render them very attractive when viral resistance was a genuine clinical challenge [START_REF] Billioud | The main hepatitis B virus (HBV) mutants resistant to nucleoside analogs are susceptible in vitro to nonnucleoside inhibitors of HBV replication[END_REF][START_REF] Delaney | Phenylpropenamide derivatives AT-61 and AT-130 inhibit replication of wild-type and lamivudine-resistant strains of hepatitis B virus in vitro[END_REF]Wang et al., 2012). Moreover, CAMs are active against most HBV genotypes [START_REF] Berke | Antiviral pro ling of the capsid assembly modulator BAY41-4109 on full-length HBV genotype A-H clinical isolates and core site-directed mutants in vitro[END_REF]. Surprisingly, CAM therapy has not reached the clinic -probably due to safety or pharmacological issues. Since this early time, active research on structural and biological features of the HBc protein has led to the discovery of many other CAMs. Currently there are 3 types of CAMs according to their di erent molecular architectures, including heteroaryldihydropyrimidine, phenylpropenamide, and sulfamoylbenzamides derivatives (Fig. 4) (Zlotnick et al., 2015). These CAMs are thought to inhibit nucleocapsid-associated reverse transcription of pgRNA into rcDNA by preventing encapsidation of pgRNA, by accelerating formation of HBV nucleic-acid-free, yet normal-in-shape capsids, or by promoting formation of abnormal capsids. This de nes two classes of CAMs based on these in vitro/in tubo structural criteria. In addition to their role in nucleocapsid formation and resulting conversion of pgRNA into rcDNA, CAMs have other modes of action. CAMs have been shown to prevent, in a post-entry manner, formation of cccDNA [START_REF] Berke | Antiviral pro ling of the capsid assembly modulator BAY41-4109 on full-length HBV genotype A-H clinical isolates and core site-directed mutants in vitro[END_REF]. This could be due to retention of rcDNA at the nuclear basket; in this respect, CAMs could bind to nucleocapsid and stabilize the structure, thus preventing its disassembly. CAMs also prevent the release from hepatocytes of RNA-containing and genome-free viral particles [START_REF] Lam | Inhibition of hepatitis B virus replication by the HBV core inhibitor NVR 3-778[END_REF]AASLD abstract). Very recently it has been reported that speci c CAMs prevent release of the HBe antigen (Lahlali et al., HBV meeting 2017). HBeAg, a secreted antigen sharing primary structure with HBc, has potential immune-modulatory functions. An active and fast inhibition of HBeAg secretion by CAMs could promote HBeAg seroconversion, an immunological process that occurs during natural history of HBV infection, and considered an important clinical end-point during therapy. However, bearing in mind some of the other functions described in this review (i.e. regulation of host gene expression, modulation of host and viral RNA metabolism, etc.), one might anticipate CAMs could play a role in HBV replication inhibition and HBV-induced pathogenesis. To date several CAMs are undergoing clinical trials: NVR 3-778 (NCT02112799; Novira Therapeutics), ABI-H0731 (NCT02908191; Assembly Biosceinces), JNJ-56136379 (NCT02662712; Janssen), and GLS-4 (trial with China-CFDA; Morphothiadine Mesilate, HEC Pharm), and many others are the focus of pre-clinical evaluations.

Unanswered questions and future directions

As discussed in this review, HBc is involved in various aspects of HBV life cycle: formation of the viral capsid, pgRNA encapsidation followed by its reverse transcription, and as a component of the viral minichromosome, contributing to its chromatin structure. The unique structure of HBc CTD and its modi cations, mainly phosphorylation, are essential for viral biosynthesis. Accordingly, HBc appears to be an ideal target for mechanism-based therapeutics. Yet many questions remain unanswered when it comes to HBc biology. A de nitive answer to the identity of the kinases responsible of HBc phosphorylation in vivo is yet to be determined. Additionally, systematic investigation of HBchost interactions, in the context of physiologic infection as well as well as details of its molecular properties, will expand our knowledge of how HBc is regulated by post-translational modi cations, as well as how HBc regulates host functions. Moreover, given the unique structure of the CTD, the seemingly promiscuous a nity of HBc for RNA and nucleic acids in general, [START_REF] Birnbaum | Hepatitis B virus nucleocapsid assembly: primary structure requirements in the core protein[END_REF]Nassal, 1992), suggests potential interactions between HBc and host RNA metabolism. This corroborates our recent ndings of long-noncoding (lnc) RNAs NEAT1 and NEAT2 as HBc interacting partners in cellular models of HBV replication and infection (Diab et al., HBV meeting 2017). Ribonucleoprotein immunoprecipitation (RIP) of HBc con rmed HBc associates with NEAT1 and NEAT2, known sca olds of ribonucleoprotein complexes that constitute nuclear speckles (Hutchinson et al., 2007). Employing a nity puri cation of HBc, combined with mass spectrometry-based proteomics, we identi ed nuclear speckle proteins heterogeneous nuclear ribonucleoprotein M (HNRNPM), splicing factor proline/glutamine-rich (SFPQ), and non-POU domain containing protein (NONO) as HBc interacting partners. Thus, we propose that HBc, as an RNA-interacting protein, associates and regulates host RNAs and speci cally lncRNAs. Accumulating evidence supports a regulatory role of lncRNAs in cellular gene expression (Hutchinson et al., 2007). Whether HBc/lncRNA interactions also exerts a role in hepatocyte physiology and virus biosynthesis is presently unknown [START_REF] Zhang | RNA helicase DEAD box protein 5 regulates Polycomb repressive complex 2/Hox transcript antisense intergenic RNA function in hepatitis B virus in-fection and hepatocarcinogenesis[END_REF] and these topics merit further investigation. ABSTRACT The assembly of hepatitis B virus (HBV) core protein (HBc) into capsids represents a critical step of viral replication. HBc has multiple functions during the HBV life cycle, which makes it an attractive target for antiviral therapies. Capsid as-sembly modulators (CAMs) induce the formation of empty capsid or aberrant capsid devoid of pregenomic RNA (pgRNA) and finally block relaxed circular DNA neosyn-thesis and virion progeny. In this study, the novel CAMs JNJ-827 and JNJ-890 were found to be potent inhibitors of HBV replication with respective half-maximal effec-tive concentrations of 4.7 and 66 nM, respectively, in HepG2.117 cells. Antiviral pro-filing in differentiated HepaRG (dHepaRG) cells and primary human hepatocytes re-vealed that these compounds efficiently inhibited HBV replication, as well as de novo establishment of covalently closed circular DNA (cccDNA). In addition to these two known effects of CAMs, we observed for the first time that a CAM, here JNJ-827, when added postinfection for a short-term period, significantly reduced hepatitis B e antigen (HBeAg) secretion without affecting the levels of cccDNA amount, transcription, and hepatitis B surface antigen (HBsAg) secretion. This inhibitory activity resulted from a direct effect of JNJ-827 on HBeAg biogenesis. In a long-term treat-ment condition using persistently infected dHepaRG cells, JNJ-827 and JNJ-890 reduced HBsAg concomitantly with a decrease in viral total RNA and pgRNA levels. Altogether, these data demonstrate that some CAMs could interfere with multiple functions of HBc in the viral life cycle. KEYWORDS hepatitis B virus, core/capsid/HBc protein, nucleocapsid assembly, capsid assembly modulators, mechanism of action, HBV, antiviral, capsid inhibitors hronic hepatitis B (CHB) infections caused by the hepatitis B virus (HBV), a member of the Hepadnaviridae family (1), remain a major public health problem worldwide. According to the World Health Organization, over 250 million people are chronic carriers of the virus (Fact Sheet 204). These patients have a higher risk of developing severe liver diseases, such as decompensated cirrhosis and hepatocellular carcinoma (HCC); the latter being the third leading cause of mortality worldwide (2,3). HBV is a noncytopathic DNA virus that specifically enters hepatocytes through the sodium taurocholate cotransporting polypeptide (NTCP) receptor to replicate and produce virion progeny (4). After delivery to the cytosol, the viral nucleocapsid is translocated to the nuclear pores for disassembly and release of the relaxed circular DNA (rcDNA) (5). Within the nucleus, the rcDNA is converted to a covalently closed circular DNA (cccDNA), which is "chromatinized" to form a long-lived life viral minichromosome that serves as the main template for all viral RNA transcript (pre-C, pregenomic [pg], pre-S, S, and X) synthesis. From these transcripts, seven proteins are translated: HBeAg (hepatitis B e antigen; secreted dimer protein); HBV Pol (viral polymerase); HBc/core (capsid protein); the large (which contains PreS1, PreS2, and S domains), medium (which contains PreS2 and S domains), and small surface envelope glycoproteins; and HBx (transcriptional transactivator) (1,6). The pgRNA is encapsidated in a nucleocapsid and converted into rcDNA by the reverse transcriptase activity of the HBV polymerase, which represents the main replication step. The polymerase-bound pgRNA (Pol-pgRNA) serves as a specific intermediate for the association with several dimers of HBc (nucleation step) from which the fully matured nucleocapsid will arise containing rcDNA (7)(8)(9). Mature nucleocapsids containing rcDNA are then either used to produce progeny virions, following envelopment in HBV-envelope-protein-containing cellular membranes, or are redirected to the nucleus to amplify or maintain the cccDNA pool (this process is called "recycling") (1,10). The secretion of HBV RNA-containing virion-like particles was also recently reported both in vitro and in patient serum. These types of particles, which were shown to be produced in even higher quantity during nucleos(t)ide analogue (NA) treatment, have been very recently described as replication deficient (11). Importantly, the levels of HBV RNA virion-like particles could fluctuate during the natural history of HBV infections and be used as a biomarker of response to CHB treatments (12,13). Currently approved CHB treatments are limited to pegylated interferon alpha (Peg-IFN-a) and NAs. A 48-week Peg-IFN-a treatment leads to complete viral suppression (i.e., undetectable HBV DNA in blood) in approximately 25% of patients but is associ-ated with significant side effects (14,15). NA administration (e.g., tenofovir or entecavir) is well tolerated and induces a strong viral suppression in the majority of patients, but NAs usually have to be taken lifelong to prevent relapses (14,15). Despite viral suppression, a functional cure (i.e., the loss of serum HBsAg [hepatitis B surface antigen] with or without seroconversion) is achieved in only 10% of treated patients after a 5-year follow up (14,15). Therefore, the identification of new targets and development of new antiviral strategies are needed. The core/HBc protein of HBV has recently reemerged as a promising antiviral target due to its multiple functions in the HBV life cycle (16). Indeed, besides its roles in capsid assembly and viral replication in the cytoplasm, nuclear HBc has been reported to modulate cccDNA transcription or posttranscriptional events, as well as host gene expression (17)(18)(19)(20)(21)(22)(23). It is also worth noting that HBc contains the major epitopes recognized by T cells during an HBV-targeted host immune response, thus further emphasizing the importance of this viral protein (1). Molecules targeting HBc, now generically named capsid assembly modulators (CAMs) or core protein allosteric mod-ulators, are being developed as novel direct-acting antivirals. CAMs either induce the formation of morphologically intact empty capsids, referred to as class I mechanism of action (MoA) compounds (e.g., phenylpropenamide derivates such as AT130 [24,25] and sulfamoylbenzamides derivates [26]) or the formation of aberrant empty structures, referred to as class II MoA compounds (e.g., heteroarypyrimidines [HAPs] such as Bayer41-4109 [here abbreviated BAY41] [27,28]). Altogether, they mainly act by preventing and/or blocking the encapsidation of the Pol-pgRNA complex and thus its reverse transcription into rcDNA (29,30). HAPs were the first CAMs demonstrating potent antiviral activity in vitro (31) and in vivo in both HBV transgenic mice (32) and HBVinfected chimeric mice (33). Importantly, CAMs efficiently inhibit replication of HBV mutants resistant to NAs (30,34) and are active against multiple HBV genotypes [START_REF] Chen | Polo-like kinase 1 is involved in hepatitis C virus replica-tion by hyperphosphorylating NS5A[END_REF]. Moreover, CAMs inhibit the production of extracellular RNA-containing particles (36) and prevent de novo formation of cccDNA by acting at a postentry step (37). The inhibition of the establishment of HBV infection could be due to an accelerated breakdown of capsid in cytoplasm before rcDNA could be delivered to nucleus and converted into cccDNA or to a stabilization of the capsid structure, which would prevent uncoating at the nuclear pore (38,39). In addition, it was suggested that CAMs could interfere with the nuclear functions of HBc, including the regulation of cccDNA transcription (40) and host gene expression (19). To date, several CAMs are in preclinical evaluations or have entered clinical trials, including GLS-4 (trial with China-CFDA; morphothiadine mesilate, HEC Pharm), NVR 3-778 (NCT02112799; Novira Therapeutics, now part of Janssen), JNJ-56136379 (NCT02662712; Janssen), ABI-H0731 (NCT02908191; Assembly Biosciences), and RO7049389 (NCT02952924; Hoffmann-La Roche). The aim of this study was to determine whether CAM antiviral effects can extend beyond their two well-described MoA compounds in vitro, which are the inhibition of HBV replication and de novo cccDNA formation. To answer this question, two novel CAMs, JNJ-827 and JNJ-890, were profiled in vitro. Altogether, our data demonstrate that some CAMs could interfere with multiple functions of HBc during the viral life cycle.

C

RESULTS

In tubo and in cellulo effects of JNJ-827 and JNJ-890 on capsid assembly. CAMs were reported to induce either the formation of morphologically intact HBV capsids (class I MoA compounds) (41) or the formation of pleiomorphic/aberrant structures (class II MoA compounds) (28). In this study, two novel CAMs, JNJ-61030827 and JNJ-54138890 (Fig. 1A), abbreviated here as JNJ-827 and JNJ-890, were tested for their anti-HBV activity and cytotoxicity. Before testing them for their antiviral effect in an infectious model, electron microscopy (EM) and size exclusion chromatography (SEC) analyses were performed to categorize JNJ-827 and JNJ-890 as having a class I or II MoA compounds. The compounds were incubated together with the recombinant HBV core assembly domain (amino acids 1 to 149) for 24 h in the presence of 150 mM NaCl before visualization by EM. Images were compared to those obtained in our previous study (37) with the phenylpropenamide AT130 ( 25) and the heteroaryldihydropyrimidine Bayer-41-4109 (here abbreviated as BAY41) (31) compounds with class I and class II MoA compounds, respectively. EM images showed the formation of morphologically intact capsids in the case of JNJ-827 or aberrant structures after treatment with JNJ-890 (Fig. 1B). SEC analysis confirmed these observations (Fig. 1C). The control sample without compound (dimethyl sulfoxide [DMSO]) showed, as expected, two absorbance peaks, one corresponding to capsid and the other to HBc dimers. Control class I MoA compound AT130 increased the formation of capsid as expected, whereas control class II MoA compound BAY41 altered the retention time, indicating the formation of aberrant structures. With JNJ-827, a unique peak was observed at the elution volume corresponding to morphologically intact capsids, whereas a peak was observed at the elution volume corresponding to aberrant structures for the sample treated with JNJ-890. Two inactive compounds (inactive Cp#1 and Cp#2) did not change the SEC profile. Altogether, these studies indicate that JNJ-827 is a class I CAM and that JNJ-890 is a class II CAM.

To study the effect of both CAMs on capsid assembly in cellulo, differentiated HepaRG (dHepaRG) cells were infected with HBV and then treated from day 7 postin-fection for a total of 7 days, during which the compound was refreshed three times. Intracellular capsid formation was visualized by a native agarose gel migration assay. A cell lysate from HepG2.2.15 cells was used as a positive control. As expected, capsid formation was not affected with tenofovir (TDF), whereas there was no band migrating at that same height with BAY41 and JNJ-890 (Fig. 1D). Capsid formation was increased by 2-fold upon AT130 treatment, whereas JNJ-827, which was classified as class I, induced a 2-fold decrease in capsid formation (Fig. 1E).

In vivo studies investigating the subcellular distribution of HBc have reported both cytoplasmic and nuclear localization (42,43). Here, we wanted to know whether CAM treatment could affect HBc subnuclear localization. To address this question, the cellular distribution of HBc was investigated by immunostaining using an antibody, which solely recognizes HBc as part of a capsid and not its monomeric or dimeric form (44). Assembled HBc was mainly found in the nucleus of nontreated HBV-infected dHepaRG (see Fig. S1 in the supplemental material). In untreated (NT) and in TDF-and AT130-treated cells, HBc staining was diffuse in the nucleus, whereas the BAY41 and JNJ-890 treatments induced HBc clustering (see Fig. S1 in the supplemental material). Interestingly, these HBc clusters colocalized with large nuclear bodies containing the promyelocytic leukemia (PML) protein and the proteasomal 20S subunit, two proteins associated with sites of protein modification/degradation [START_REF] Korba | Hepatocellular carcinoma in woodchuck hepatitis virus-infected woodchucks: presence of viral DNA in tumor tissue from chronic carriers and animals serologically recovered from acute infections[END_REF]. An intermediate phenotype was obtained with JNJ-827. Altogether, the data showed that CAM treatment can affect HBc nuclear localization and capsid formation in infected hepatocytes. 1). Moreover, we observed in infected dHepaRG cells a dose-dependent reduc-tion in the amount of intracellular HBV DNA, without cytotoxicity and cell dedifferen-tiation, as measured by the cellular secretion of the apolipoprotein B (ApoB) (see Fig. S2 in the supplemental material). JNJ-827, JNJ-890, and AT130 did not show cytotoxicity in HepG2 cells at any of the concentrations tested (>25, >100, and >50 μM, respec-tively), whereas the 50% cytotoxic concentration (CC 50 ) of BAY41 was 33.9 μM (Table 1). The selectivity index (SI) values (i.e., the CC 50 /iEC 50 value) for JNJ-827 and JNJ-890 in HepG2 cells were >5,319 and >1,515, respectively (Table 1). For the remainder of the mechanistic studies, CAMs were used at a single, noncytotoxic concentration of 10 μM. Ex vivo effect of JNJ-827 and JNJ-890 in infected PHHs. Primary human hepa-tocytes (PHHs) represent the golden-standard in terms of evaluating drug metabolism and hepatoxicity. Moreover, the susceptibility of the cell culture model to HBV infection makes it ideal for studying viral replication ex vivo (46). Three different batches of freshly isolated PHHs were infected with HBV and treated three times with JNJ-827, JNJ-890, control CAMs, and TDF from day 5 postinfection for a total of 7 days. JNJ-827 and JNJ-890 reduced total intracellular HBV DNA by 80 and 60%, respectively, whereas only weak decreases were observed with BAY41 and AT130, and 90% intracellular HBV DNA reduction was obtained with TDF (Fig. 2A). Interestingly, JNJ-827 and JNJ-890 induced also a weak but significant reduction in intracellular total HBV RNA levels, whereas JNJ-890 was the sole compound able to significantly reduce PreC/pg-RNA by 2-fold (Fig. 2A). All treatments had no effect on the established pool of cccDNA in this short treatment duration setting. At the extracellular level, TDF, as already reported for lamuvidine (36), induced a >90% reduction of secreted HBV DNA, along with an accumulation of secreted HBV RNA (Fig. 2B). Among all the CAMs tested, JNJ-827 was the only compound able to strongly decrease both secreted HBV RNA and DNA levels. Moreover, JNJ-827 was the sole CAM observed to affect HBeAg secretion, whereas none of the molecules affected HBsAg secretion (Fig. 2B). The EC 50 of JNJ-827 regarding HBeAg secretion inhibition was around 5 μM (see Fig. S3 in the supplemental material). None of the molecules tested were toxic at the concentrations tested, as indicated by the unchanged levels of HBsAg and ApoB in supernatants (Fig. 2B). Note that results obtained with the three individual batches of PHHs, for cccDNA as well as for secreted viral and ApoB parameters, are also presented as absolute quantifications in Fig. S4 in the supplemental material. Altogether, these results identified JNJ-827 as having the most pronounced effects among the CAMs tested. Moreover, JNJ-827 and JNJ-890 featured interesting novel properties that were further investigated in the rest of the study. Effect of JNJ-827 and JNJ-890 on the formation of cccDNA in a de novo infection setting.

It was recently described that CAMs could also prevent cccDNA formation, thus defining a secondary MoA compound of these molecules based on a postentry mechanism (37).

To evaluate JNJ-827 and JNJ-890 with respect to this MoA compound and compare with control CAMs or the PreS1 peptide (i.e., an entry inhibitor), which is known to inhibit HBV entry by interfering with hNTCP binding (47), compounds were added 1 day before and during viral inoculation of PHHs or dHepaRG cells. This is in contrast to what was done in previous experiments (Fig. 2) that aimed at evaluating the postinfection action of these molecules. TDF was added 24 h postinfection and used here as a technical control for cccDNA quantification, since NAs do not alter the cccDNA level. cccDNA quantification was performed at day 7 postin-fection. None of the drugs were toxic in this setting at the concentration tested, as shown by an absence of variation in ApoB secretion (Fig. 3B andD). As expected, the PreS1 peptide prevented viral entry thus preventing cccDNA establishment and subsequent synthesis of viral intermediates (Fig. 3A). Interestingly, JNJ-827 treatment also reduced cccDNA formation by >90%, resulting in a concomitant decrease of intracel-lular HBV DNA and RNA levels (Fig. 3A), as well as HBeAg and HBsAg secretion (Fig. 3B). JNJ-890 and BAY41 were found to be far less potent than JNJ-827, whereas AT130 was similarly inefficient as TDF at preventing cccDNA establishment. The EC 50 s of JNJ-827 for cccDNA establishment in PHH were between 19 and 61 nM and around 100 nM for HBV total DNA, HBeAg, and HBsAg (see Fig. S5 in the supplemental material). Pheno-typically, JNJ-827 and the PreS1 peptide behaved the same, indicating that a bona fide entry or a postentry inhibition could be equal in terms of antiviral effect.

In the dHepaRG model, all CAMs were found to be efficient to inhibit cccDNA establishment (Fig. 3C), replication intermediates (Fig. 3C), and antigen production (Fig. 3D), suggesting that the compounds might be differentially metabolized in both cell types. Note that results obtained with the three individual batches of PHHs and three independent HepaRG differentiations, regarding cccDNA as well as secreted HBe/HBsAg and ApoB parameters, are also presented in absolute quantification in Fig. S6 and S7 in the supplemental material. Long-term treatment with JNJ-827 and JNJ-890 affects viral RNA accumulation. Shortterm postinfection treatment with JNJ-827 and JNJ-890 in PHHs suggested a modest effect on viral RNA accumulation (Fig. 2A). It was previously speculated that CAM treatment could impact cccDNA transcription and/or posttranscriptional events (40). To further analyze this potential MoA of JNJ-827 and JNJ-890 compared to other CAMs, we made use of dHepaRG that can be maintained for weeks or months in culture in contrast to PHHs, which quickly dedifferentiate when cultured ex vivo. dHepaRG cells were infected with HBV and then treated from day 7 postinfection either for a total of 7 days (total of 3 administrations) or 1 month (total of 15 administrations). Under shortterm conditions using dHepaRG, all CAMs induced a strong reduction of intra-cellular HBV DNA accumulation (Fig. 4A), which is in line with the observed, though less pronounced, effects in PHHs, most likely due to different metabolization properties of each cell type (compare Fig. 4A with Fig. 2A). This inhibition of nucleocapsid assembly was translated into a strong inhibition of secreted HBV DNA and RNA, yet without any effect on HBsAg and ApoB, secretions (Fig. 4B). Regarding the effect on intracellular RNA accumulation, only JNJ-890 led to a weak reduction in dHepaRG (Fig. 4A). It is also worth noting that a significant reduction in HBeAg secretion was observed with both JNJ-827 and JNJ-890, in the absence of any correlative inhibition of HBsAg (Fig. 4B). Such an inhibition was also observed in the PHH model for JNJ-827 (Fig. 2B), suggesting a peculiar feature of JNJ-827 and JNJ-890 with respect to HBeAg biogenesis and secretion.

In the long-term setting, JNJ-827 and JNJ-890 were able to reduce both intracellular total HBV RNA and PreC/pgRNA levels (Fig. 4C), as well as antigens and HBV DNA/RNA secretions (Fig. 4D). Intracellular total HBV RNA and PreC/pgRNA levels were also impacted by BAY41 and AT130, but to a lesser extent. All CAMs induced a strong inhibition of secreted HBV DNA and RNA, whereas TDF gave rise to an expected profile, i.e., decreasing DNA and increasing RNA secretion. JNJ-890 induced a weak but significant reduction of ApoB secretion (Fig. 4D). Altogether, these data suggest that JNJ-827 and JNJ-890 could reduce HBV RNA synthesis/stability in a long-term treatment context and that this reduction can be associated with a decrease in HBsAg production. Unfortunately, in dHepaRG the amount of cccDNA is very low (<0.05 copy/cell), making a reliable quantification of this parameter not possible. Note that results obtained with the three independent HepaRG differentiations in short-and long-term treatment conditions, regarding secreted HBe/HBsAg and ApoB parameters, are also presented in absolute quantification in Fig. S8 and S9 in the supplemental material. Effect of JNJ-827 and JNJ-890 on HBeAg biogenesis and/or secretion. In both PHH and dHepaRG cells and a short-term treatment setting, we observed a reduction in HBeAg secretion with JNJ-827 without intracellular total HBV RNA decrease, leading us to speculate that this CAM has putative effects on HBeAg biogenesis/secretion. To specifically address this question, a HepaRG cell line expressing HBeAg alone in a tetracycline-dependent manner was used (HepaRG-TR-HBe). First, we confirmed that HBeAg secretion increased with the tetracycline amount (see Fig. S10A in the supplemental material). We choose to use 1 μg/ml of tetracycline, for which the HBeAg secretion level (about 150 NCU/ml) was comparable to what we obtained after an infection of dHepaRG cells. By native agarose gel migration assay using also a HepaRG-TR-HBc cell line as a positive control, we confirmed that the HepaRG-TR-HBe cells do not express HBc and as such do not produce capsid (see Fig. S10B in the supplemental material). HepaRG-TR-HBe cells were induced to secrete HBeAg for 1 day and then treated for 5 days (total of two treatments). We observed a reduction in HBeAg secretion of >90% with JNJ-827 (Fig. 5A). The same observation was made with other CAMs tested, but to a lower extent. As expected, TDF had no effect on HBeAg secretion. Note that intracellular quantification of p22/pre-HBeAg by enzyme-linked immunosor-bent assay (ELISA) led to the same ranking of compounds, although with a lower level of inhibition (Fig. 5B). To further extend the analysis to the primary level of p22/pre-HBe/HBeAg expres-sion, supernatants were also submitted to denaturing Western blotting after trichloro-acetic acid precipitation to confirm that the observed effect was genuinely due to an inhibition of HBeAg secretion and not to a problem of detection of the protein by ELISA. At the intracellular level, p22/pre-HBeAg amounts were only affected by BAY41 treatment, whereas other CAMs only reduced extracellular accumulation (Fig. 5Ci, ii, and iii). Altogether, these data suggest that all CAMs tested, except for BAY41, do not tend to prevent the primary expression of p22/pre-HBeAg, while some of them can also potentially interfere with HBeAg biogenesis/secretion. In this respect, JNJ-827 was the most potent in this assay (i.e., use of HepaRG-TR-HBe), and this potency can translate into an inhibition of HBeAg secretion in HBV-infected PHHs (Fig. 2), suggesting that this molecule has a particular feature.

DISCUSSION

Treatment of CHB is limited to the use of NAs and Peg-IFN-a. Unfortunately, these therapeutic options lead to a functional cure (i.e., HBsAg loss with or without HBsAg to HBsAb seroconversion) in only 10% of cases after a 5-year follow-up (14,15). Therefore, there is a need for new therapeutic options. The core/HBc protein plays multiple roles during the HBV life cycle (e.g., forms nucleo-capsid, interacts with cccDNA and human host genes, and may be involved in the regulation of their transcription) (1,17,19,20). Interfering with the multiple functions of HBc using a direct antiviral agent represents a promising strategy to disrupt HBV replication at several steps in the viral life cycle. Currently, several CAMs are being evaluated in clinical trials, and many others are in preclinical development (additional information is available [http://www.hepb.org/treatmentand-management/drug-watch/]). JNJ-827 and JNJ-890 are two novel CAMs. SEC and EM studies revealed that JNJ-827 possesses a class I MoA compound, whereas JNJ-890 possesses a class II MoA compound. However, and in contrast to the hallmark of a class I MoA compound, JNJ-827 was capable of reducing the amount of intracellular capsid by 2-fold, as visualized on native agarose gel. This discrepancy might be explained by the much higher potency of JNJ-827 compared to the other class I CAM in the panel, AT130 (217-fold less active than JNJ-827 in HepG2.117). For the JNJ-890 and BAY41, the iEC 50s were similar.

In the present study, CAMs were mostly used at a high concentration (10 μM) to highlight novel antiviral effects and MoA. In HBV-infected PHHs we confirmed that JNJ-827 and JNJ-890 possess the two described antiviral effects of CAMs: inhibition of HBV replication in already established infections (primary mechanism) and inhibition of cccDNA de novo formation (secondary mechanism) (37, 38). JNJ-827 was the most potent CAM tested in stably HBV-transformed HepG2 cell lines, with an EC 50 in the single-digit nanomolar range. Like the PreS1 peptide entry inhibitor, JNJ-827 inhibited cccDNA formation with an EC 50 around 50 nM. BAY41 and JNJ-890 were similarly efficient in this model. The improved efficacy of JNJ-827 at inhibiting HBV infection by a postentry mechanism was likely due the higher potency of JNJ-827 (iEC 50 at 4.7 nM), as reflected by the lack of inhibition with AT130 (iEC 50 at 1,020 nM). It is worth noting that all CAMs, including AT130, were similarly efficient at preventing HBV infection in the dHepaRG model. This can either point to the difference in metabolic properties of these cells compared to PHH or the lower infection rate in dHepaRG cells compared to PHHs at the same multiplicity of infection (as an indication, HBsAg production ranges between 50 and 100 IU/ml in dHepaRG versus 1,000 to 5,000 IU/ml in PHHs when a multiplicity of infection of 100 genome equivalent of HBV/cell is used). The concentra-tion necessary to achieve a 50% reduction of the formation of cccDNA (EC 50 for cccDNA formation inhibition) was 10 times higher than that necessary to achieve the main inhibitory action (i.e., inhibition of HBV DNA neosynthesis). It remains to be determined whether this inhibition of the establishment of HBV infection is due to an accelerated breakdown of capsid in cytoplasm before rcDNA could be delivered to nucleus and converted into cccDNA (38,39) or to a prevention of disassembly at the nuclear pore. In the first case, it would be interesting to determine whether rcDNA could serve as a pathogen-associated molecular pattern for innate sensing by the dsDNA sensing machinery.

In postinfection and short-term treatment conditions, among all CAMs tested, JNJ-827 was the most potent at inhibiting intracellular HBV replication (i.e., neosynthe-sized rcDNA) in PHHs. Very importantly, as reported both for NVR- 3-1983 (36) in vitro and NVR-3-778 (48) in vivo, we found that, in PHHs, JNJ-827 strongly reduced both extracellular HBV DNA and RNA levels, whereas TDF only decreased extracellular HBV DNA levels (and increased the HBV RNA level), and other CAMs tested were inefficient. This is due to the fact that JNJ-827, in contrast to NAs, blocks the encapsidation of pgRNA, and therefore these results in a reduction of encapsidated DNA and RNA in the cell culture supernatant (11)(12)(13)49). The function of secreted HBV-RNA virion-like particles in HBV life cycle is unclear. HBV RNA virion-like particles were recently reported to be mainly replicationdeficient 11), but they could have immunological functions. Therefore, the inhibition of their release into the blood of CHB patients could be beneficial. Nuclear HBc was suggested to regulate HBV transcription by interacting with cccDNA (17,22). Interestingly, we found, like a very recently published study (50), that CAM treatment induces the relocalization of nuclear HBc to subnuclear sites staining positive for the 20S proteasomal subunit and PML bodies, which are the sites of protein posttranslational modification, activation, sequestration, or degradation [START_REF] Korba | Hepatocellular carcinoma in woodchuck hepatitis virus-infected woodchucks: presence of viral DNA in tumor tissue from chronic carriers and animals serologically recovered from acute infections[END_REF]. Here, we found that BAY41 treatment led to the formation of large HBc aggregates, whereas AT130 treatment had no effect or only a slight effect on nuclear HBc distribution. These observations correlate with the MoA of the compounds and suggest that CAMs could differentially disrupt nuclear functions of HBc. In this respect, in both dHepaRG and PHH cells, JNJ-890 was the sole CAM capable of decreasing intracellular total HBV RNA and PreC/pgRNA levels after 1 week of treatment. However, after 1 month of treatment of dHepaRG cells, all CAMs tended to decrease total HBV RNA levels, as well as HBeAg and HBsAg secretion, with JNJ-827 and JNJ-890 being again the most potent compounds tested. Even if we cannot exclude an effect on cccDNA amount after a long-term treatment (we failed to reliably detect cccDNA in this experimental context by qPCRbased methods), our observations suggest that HBc could be a positive regulator of cccDNA transcription or/and posttranscriptional events and suggest also that (i) CAMs interfere with the nuclear function of core in vitro and that (ii) compound class is not necessarily an indicator of the effect of CAM on HBc nuclear functions; compound potency could also be involved.

HBeAg is a secreted antigen sharing primary structure with HBc. The intracellular precursor of HBe (called p22 or pre-HBe) features 10 additional amino acids at the Nterminal extremity compared to HBc and needs to be processed by both N-and Cterminal maturation to give rise to secreted HBeAg (51, 52). Secreted HBeAg is in fact a dimer of p17 polypeptide. One possible function of secreted HBeAg could be to transiently suppress the immune response against HBV. This could be done by negatively regulating the TLR2 signaling pathway (51, 52) and/or by modulating the immune response against HBc (53, 54). HBeAg seroconversion is an immunological process that occurs during the natural history of HBV infections due to the occurrence of stop or PreC/core promoter mutations in the cccDNA integration events and can be increased upon treatment with Peg-IFN and NAs. It is associated with higher immune activity and is considered an important clinical endpoint during therapy (53,54). In this study, we observed a strong and fast inhibition of HBeAg biogenesis/secretion by CAMs in the HepaRG-TR-HBe model, more particularly with JNJ-827, which was the only compound to recapitulate this phenotype in PHHs with an EC 50 of around 5 μM. The inhibition is unlikely due to an inhibition of the precursor (p22/pre-HBe) synthesis, since Western blot analyses under denaturing conditions did not reveal any differences, but is likely mediated by a direct interaction of these compounds with the intracellular precursor of HBeAg, leading to an impaired processing and dimerization (i.e., biogenesis of the antigen), as well as to an impaired secretion. Although HBc and HBe monomer share very similar primary structures, the quaternary dimeric form of HBeAg is drastically different from HBc (55). This is a consequence of an intramolecular disulfide bridge between the cysteine 61 (common to HBeAg and HBc) and cysteine 7 (specific to HBeAg) that is specifically added in the lumen of the endoplasmic reticulum, in which HBeAg is translocated during translation (56). HBeAg dimers with the C7-C61 disulfide bridge do not assemble in capsid-like structures, while the reduced form does in the cytoplasm (56, 57). CAMs target HBc by binding to a hydrophobic pocket at the dimerdimer interface (58-60). To explain how some CAMs target HBeAg, we can hypothesize that they could force HBeAg dimer-dimer assembly, leading to HBeAg aggregation, as observed for HBc, and could promote a fast degradation.

To summarize, CAMs possess two well-characterized mechanisms of action. They accelerate capsid assembly, which results in an inhibition of HBV replication (primary MoA) and prevent de novo formation of cccDNA by acting at a postentry step (secondary MoA). Here, we demonstrated that treatment with some CAMs can extend beyond these two mechanisms. Indeed, some CAMs can directly reduce secreted HBeAg and act also either at transcriptional or posttranscriptional levels, leading to a reduction in both total HBV RNA and PreC/pgRNA accumulation, as well as HBsAg production. These additional effects with some CAMs require further investigation and may lead to the development of second-generation compounds. Due to their multiple and additional effects compared to NAs, one could expect that some CAMs could lead to higher rates of functional cure in CHB patients, and clinical evaluation of this type of multifunctional CAMs is eagerly awaited.

MATERIALS AND METHODS

Compounds. BAY41, AT130, JNJ-827, and JNJ-890 were provided by Janssen. JNJ-827 and JNJ-890 (see structure in Fig. 1A) were prepared according to methods described in the patent applications WO2014184350 and WO2013102655, respectively. The PreS1 peptide (myristoylated amino acids 2 to 48 from the N-terminal part of PreS1) was synthesized by GenScript, and TDF was obtained from Gilead Sciences (Foster City, CA). All compounds had an high-pressure liquid chromatography-certified purity of >95%.

Antiviral assay using HepG2.2.15 cells. Determinations of the JNJ-827 and JNJ-890 EC50 and EC90 values were performed as previously described [START_REF] Chen | Polo-like kinase 1 is involved in hepatitis C virus replica-tion by hyperphosphorylating NS5A[END_REF]. Cytotoxicity assay using HepG2 cells. Determinations of the JNJ-827 and JNJ-890 CC 50 and CC 90 values were performed as previously described [START_REF] Chen | Polo-like kinase 1 is involved in hepatitis C virus replica-tion by hyperphosphorylating NS5A[END_REF].

EM and SEC studies. Determination of JNJ-827 and JNJ-890 effects on capsid formation in tubo were performed as previously described [START_REF] Chen | Polo-like kinase 1 is involved in hepatitis C virus replica-tion by hyperphosphorylating NS5A[END_REF]. Culture of HepaRG cells and primary human hepatocytes and HBV infection. Human liver progenitor HepaRG cells [START_REF] Devwecan | [END_REF] peptide (used at 100 nM), were evaluated for their effect on the establishment of HBV infections or on established HBV infections. Under the preinfection condition, CAMs and PreS1 peptide were added 1 day before and during infection. TDF was added twice postinfection. Cells and supernatants were harvested at day 6 postinfection. Under the postinfection condition, PHHs or dHepaRG cells were infected by HBV for, respectively, 5 or 7 days and then treated three times at days 5, 7, and 9 (PHHs) or days 7, 9, and 11 (dHepaRG cells) postinfection. Cells and supernatants were harvested at day 12 or 14 postinfection (i.e., 7 days posttreatment).

Analysis of viral parameters. From cell culture supernatants, secreted HBe and HBs antigens were quantified by ELISA, using a chemiluminescence immunoassay kit (Autobio, China) according to manufacturer's instructions. Extracellular viral DNA and RNA were extracted from cell culture supernatant using MagMAx kit (Thermo Scientific) and MagNAPure (Roche), respectively, according to the manufacturer's protocols and treated either with DNase I or RNase A. Intracellular total HBV DNA or RNA were extracted from infected cells using, respectively, NucleoSpin 96 Tissue or NucleoSpin 96 RNA kits (Macherey-Nagel). RNAs were transcribed into cDNA using the SuperScript III reverse transcriptase (Invitrogen). Real-time PCR for total intracellular HBV DNA and RNA was performed using a LightCycler 480 (Roche) and normalized to PRP, a housekeeping gene, with the following primers: 5 -GCTGACGCAACCCCCACT-3 (HBV-sense), 5 -AGGAGTTCCGCAGTATGG-3 (HBVantisense), 5 -TGCTGGGAAGTGCCATGAG-3 (PRP-sense), and 5 -CGGTGCA TGTTTTCACGATAGTA-3 (PRP-antisense). An HBV standard was used for quantification of extracellular viral DNA and RNA. For cccDNA quantification, total DNA was digested by using Plasmid-Safe DNase (New England BioLabs) f or6h at 37°C and then subjected to qPCR using a LightCycler 480 Probes master kit (Roche) with a home-made probe-primer mix. cccDNA was normalized to 13-globin quantification. For PreC/pgRNA quantification, qPCR was performed using TaqMan Fast Advanced master mix (Life Technologies) using a home-made probe primer mix. PreC/pgRNA levels were normalized to GUSB using a commercial probe primer mix (Life Technologies, catalog no. 4448491; GUSB: Hs99999908_m1). ELISA. Apolipoprotein B (ApoB) was quantified in cell culture supernatants using a total human apolipoprotein B ELISA (Alerchek, Inc.) according to the manufacturer's recommendations.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at https://doi.org/10.1128/AAC .00835-18.

SUPPLEMENTAL FILE 1, PDF file, 1.3 MB. which represent, if left untreated, a high risk of developing liver cirrhosis and hepatocellular carcinoma [START_REF] Arzumanyan | Pathogenic mechanisms in HBV-and HCV-associated hepatocellular carcinoma[END_REF]El-Serag, 2012). Hepatitis B virus (HBV), a DNA virus belonging to the Hepadnaviridae family, is highly endemic in some area of the globe where parenteral transmission was until recently possible. The natural history of HBV infection is quite complex with distinct physiopathology and therapeutic stages. The natural course of HBV infection consists of four phases: i) the previously called "immune-tolerance" phase (IT), now referred as "low-inflammatory" phase, during which the virus replicates at very high levels without immune control and apparent liver damage, ii) a chronic-active-hepatitis (CAH) phase, iii) an inactive-carrier phase, and iv) potential reactivation episodes (Busch and Thimme, 2015;[START_REF] Fattovich | Natural history of chronic hepatitis B: special emphasis on disease progression and prognostic factors[END_REF][START_REF] Mcmahon | Natural history of chronic hepatitis B[END_REF].

To establish and maintain a chronic infection, HBV has evolved various strategies to evade the host innate and adaptive immune responses [START_REF] Ait-Goughoulte | Innate antiviral immune responses to hepatitis B virus[END_REF][START_REF] Bertoletti | Innate and adaptive immune responses in chronic hepatitis B virus infections: towards restoration of immune control of viral infection[END_REF][START_REF] Dandri | New insight in the pathobiology of hepatitis B virus infection[END_REF][START_REF] Ferrari | HBV and the immune response[END_REF][START_REF] Tan | Immune Response in Hepatitis B Virus Infection[END_REF]. Several HBV proteins, including those expressed within the infected hepatocytes (e.g. HBx, HBV polymerase) or the HBV antigens released in the bloodstream (i.e. HBsAg and HBeAg), have been shown or proposed to bear immune-modulatory functions within infected hepatocytes or the liver micro-environment, with subsequently a lack of immune cell crosstalk and antigen presentation, as well as a general HBV-specific T cell exhaustion [START_REF] Ait-Goughoulte | Innate antiviral immune responses to hepatitis B virus[END_REF][START_REF] Bertoletti | Innate and adaptive immune responses in chronic hepatitis B virus infections: towards restoration of immune control of viral infection[END_REF][START_REF] Dandri | New insight in the pathobiology of hepatitis B virus infection[END_REF][START_REF] Ferrari | HBV and the immune response[END_REF][START_REF] Tan | Immune Response in Hepatitis B Virus Infection[END_REF]. To restore/favour an immune control of the virus, virus-mediated inhibitory mechanisms would have to be defeated/unlocked in an interventional therapeutic perspective. To this end, a better knowledge of the underlying molecular mechanisms responsible for the blockade of host immune responses is crucial before envisaging such strategies.

The interferon response is a central component of host antiviral strategies (de Weerd and Nguyen, 2012;[START_REF] Randall | Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures[END_REF][START_REF] Wang | The yin and yang of viruses and interferons[END_REF]. This response, which involves the production of a wide range of IFNs (type-I, II and III), may occur within the infected cells or in the microenvironment by the activation of professional innate immune cells (i.e. IPC; IFN producing cells such as pDC or mDC2-BDCA3+). This response is crucial to hold the early spreading of viruses, to reduce the intensity of their replication, to finely their transcription (Fernandez et al., 2003;[START_REF] Rosmorduc | Inhibition of interferon-inducible MxA protein expression by hepatitis B virus capsid protein[END_REF][START_REF] Whitten | Identification of the hepatitis B virus factor that inhibits expression of the beta interferon gene[END_REF]. However, detailed mechanisms about this inhibition remained until now largely unknown.

The aim of this work was to identify the HBV-virion associated factor responsible for the early inhibition of IFN response we evidenced in a previous work {Luangsay, 2015 #95}, and to get more insights on the molecular underlying inhibitory mechanism. In this study, HBc was identified as the major viral determinant responsible for this inhibition. This is the first demonstration that a viral structural protein of HBV virions is able to modulate the host innate immune response by epigenetic regulation of type-I IFN gene expression, shortly after viral entry/penetration.

Results

Virion-associated HBc is the viral determinant involved in the early inhibition of hepatocyte IFN response

We recently reported that HBV could actively and promptly, i.e. within minutes/hours after the onset of infection, and in the absence of de novo viral synthesis, inhibit PRR agonistmediated IFN response in both differentiated HepaRG (dHepaRG) cells and primary human hepatocytes (Luangsay et al., 2015b). We extended these results by very early kinetic studies (Figures 1A and1B). Again, a strong and early inhibition of a dsRNA-mediated IFN response (i.e. prototypic type-I (IFN-α), type-III (IL-29) IFN, as well as OAS-1 as a prototypic ISG) in HBV-infected PHH stimulated with poly(I:C) was observed as early as 2h post-HBV exposure, whereas a delayed inhibition, starting at 8h p.i., was observed for IL-6 (i.e. prototypic pro-inflammatory gene). Interestingly, we showed that a HBV virion/inoculumassociated factor was responsible for this very early inhibition, as the inhibitory phenotype was still maintained with UV-inactivated virus in HBV-infected dHepaRG (Figures 1B). Since HBsAg and HBeAg, the two secreted viral antigens present in the HBV inoculum, were previously ruled out as early negative regulator of IFN response (Luangsay et al., 2015b), we reasoned that HBc, the major protein composing viral nucleocapsid (240 units/capsid) within infectious particles, could be potentially responsible for this inhibition. stimulated with tetracycline (10 μg/ml) for 24h were extracted and HBcAg were evaluated by ELISA (A) and western blot (B) using homemade anti-HBc antibody, anti-PARP (nucleus marker), and anti-tubulin (cytoplasmic marker) antibodies. (C) Differentiated HepaRG-TR-HBc cells were treated or not for 72h with Bayer41 (1 μM) or AT130 (3 μM) prior to be stimulated with tetracycline for 24h (2μg/ml) and then treated with Poly (I:C) ligand for 4h. Expression of indicated genes was analyzed by RT-qPCR and compared to the nontetracycline stimulated HepaRG-TR-HBc cells normalized to 100%. Results in panels A and C are represented as the mean ± SEM of 3 independent experiments.

To further investigate whether the nuclear localization of HBc could significantly impact on the hepatocyte innate immune responses in vitro, HepaRG-TR-HBc cells were treated, prior to tetracycline induction, with two different capsid assembly inhibitors, an heteroaryldihydropyrimidine (i.e. BAY41-4109) or a phenylpropenamide derivative (i.e. AT-130), which both impair nucleocapsid assembly (Deres et al., 2003;Feld et al., 2007). These two treatments, which could prevent both HBc trafficking and translocation into the nucleus (Figure 2A and 2B), reverted the phenotype of HBc-mediated inhibition of expression of IFN-6, IL-29, OAS-1 genes (Figure 2C). These results suggest a correlation between the nuclear localization of HBc and the inhibition of IFN and ISG gene expressions upon engagement of TLR3 or RIGI/MDA5 signaling pathway.

Upon HBV infection, HBc can rapidly interact with hepatocyte innate gene promoters to control IFN response

In conditions of overexpression of HBc in hepatoma cells, it has been suggested that HBc could interact with host promoters to regulate gene expression. For instance, IFN-α and MX-A promoters were shown to be repressed by HBc [START_REF] Rosmorduc | Inhibition of interferon-inducible MxA protein expression by hepatitis B virus capsid protein[END_REF][START_REF] Whitten | Identification of the hepatitis B virus factor that inhibits expression of the beta interferon gene[END_REF], whereas HBc was shown to directly bind to ISRE-containing DNA in EMSA experiments (Fernandez et al., 2003). More recently, using liver biopsies from HBV-infected patients and chromatin immunoprecipitation (ChIP) microarrays, it was also shown that HBc could bind in vivo to a broad spectrum of CpG rich human gene promoters and potentially modulate their expression, even though functional analyses to validate the results were only performed in vitro with few genes (Guo et al., 2012).

To determine whether HBc could interact with our studied gene promoters, we performed ChIP-qPCR experiments on tet-induced dHepaRG-TR-HBc cells using an anti-HBc Ab (and control Ab) and primers specific for IFN-α, IL-29, OAS-1 and IL-6 promoters, or control gene promoters (i.e. HKG, GAPDH, or DHFR). HBc was found to interact with IFN-α, IL-29 and OAS-1 promoters, but not with IL-6 one, as early as 4 hours post-induction (Figure 3A).

Interestingly, the blockade of the nuclear localization of HBc by capsid assembly inhibitor treatments that prevent HBc entry into the nucleus resulted in a marked decrease of the interaction between HBc and gene promoters in HBc-expressing cells (Figure 3B). ChIP analysis was also performed in HBV-infected PHH. The binding of HBc to IFN-α, IL-29 and OAS-1 promoters was evidenced, whereas no binding to IL6 one at any of the time points was observed (Figure 3C). These results are consistent with the observation that IFNα, IL-29 and OAS-1 gene expressions were inhibited by HBc, while the expression of IL6 was not (Figure 1A). Of note, the binding of HBc was long lasting, as at day-12 pi, HBc could be still "ChlPed" on promoters. Importantly, no binding of HBc on IL-6 promoter was again observed, thus confirming that HBc did not regulate IL-6 transcription in our experimental conditions. We previously showed a correlation between HBc nuclear localization and gene expression modulation. To support this observation, we used short-term and non-cytotoxic treatment with nocodazole, which inhibits trafficking and translocation of HBc to nucleus [START_REF] Rabe | Lipid-mediated introduction of hepatitis B virus capsids into nonsusceptible cells allows highly efficient replication and facilitates the study of early infection events[END_REF], and in turn prevents the binding of HBc to target promoters evidenced by ChIP-qPCR experiment (Figure 3D). Altogether, our results suggest that in the very early phase of infection, HBc from "incoming" virions could, after trafficking through the cytosol and translocation into the nucleus, negatively modulate the IFN response by binding to the promoters of some innate genes and, thereby, interfering with their expression.

HBc induces the recruitment on target genes of EZH2, a histone methyl-transferase involved in the establishment of repressive epigenetic marks

Several viruses were shown to manipulate the epigenetic state of the host genome notably by modifying the histone methylation status [START_REF] Berger | The complex language of chromatin regulation during transcription[END_REF][START_REF] Ferrari | Reorganization of the host epigenome by a viral oncogene[END_REF][START_REF] Fonseca | Adenovirus evasion of interferon-mediated innate immunity by direct antagonism of a cellular histone posttranslational modification[END_REF][START_REF] Niller | Epigenetic dysregulation of the host cell genome in Epstein-Barr virus-associated neoplasia[END_REF]. We postulated that HBc-mediated repression of innate genes could be due to its binding to targeted promoters and further epigenetic modulations. To demonstrate this, we performed ChIP studies on HBc-expressing cells using antibodies directed either against hyper-methylated or acetylated histone H3 or against EZH2, a methyl-transferase involved in histone H3 methylation at position K27 [START_REF] Czermin | Drosophila enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites[END_REF][START_REF] Wu | Histone methyltransferase G9a contributes to H3K27 methylation in vivo[END_REF]. Results showed significant enrichment of the repressive H3K27me3 mark on IFN-α, IL-29 and OAS-1 promoters, as early as 4h post-HBc expression (Figure 4A).

Mirroring the pattern of HBc expression after tet-induction and defining a dose response, the enrichment of this repressive mark further increased at 24h and 48h post-induction, while an active mark (H3K27Ac) remained low throughout the kinetic. Confirming this result, we also found EZH2, 252 which contributes to the establishment of the H3K27me3 modification, on HBc-regulated promoters (Figure 4A). ChIP experiments were also performed on HBV-infected PHH at 24h post-infection and showed a concomitant occupation of IFN-α, IL-29, and OAS-1 promoters by HBc, EZH2, and K27me3 histone H3, while no active marks (H3K27Ac and H3K4me3) were found (Figure 4B).

expressing HepaRG cells (Figure S2) and in liver biopsies of chronically HBV infected patients during the immune-tolerance phase (Akiba et al., 1987;[START_REF] Chevallier-Queyron | Immunohistochemistry as a tool for chronic hepatitis diagnosis[END_REF]Chu and Liaw, 1987). Then, ChIP experiments were performed in the liver of these chronically HBV-infected mice to measure HBc binding to innate gene promoters, as well as the association of EZH2 and related epigenetic marks (Figure 6). Results showed that these highly HBV replicative mice featured concomitant similar enrichment to IFN-related gene promoters for HBc, EZH2 and the repressive H3K27me3 mark, whereas active marks (H3K27Ac and H3K4me3) were not changed. In addition, in these mice the epigenetic profile was associated with a low expression of innate genes (Figure S6A and S6C).

Moreover, on the IL-6 promoter, neither HBc, nor EZH2 and the associated repressive mark H3K27me3 were found enriched, whereas positive marks (H3K27Ac and H3K4me3) were found with a low level of enrichment, thus confirming the absence of HBc effect on IL-6 promoter. Interestingly, in mice with low viremia and antigenemia (Mice 323,324,374,408 and 413; Figure S5B), HBc was not found associated to IFN-α, IL-29, and OAS-1 promoters (Figure S6D), and IFN-α and IL-6 genes were found significantly up-regulated (Figure S6B), which nicely correlated with the low replicative status. Thesis Adrien FOCA

257

Capsid assembly inhibitors increase the anti-HBV effect of an IFN-response inducer

As HBc interfere with the dsRNA-mediated induction of IFN response, we wondered whether administration of capsid assembly inhibitors, interfering with HBc localization in infected hepatocyte prior to dsRNA receptor stimulation, could potentiate IFN-mediated antiviral effect. To this end, we used the active enantiomer of BAY41-4109 (Deres et al., 2003), which is capable to block trafficking of HBc to nucleus in our models (Figure S7) and has an efficient concentration 50% (EC50) of around 320 nM (see panel CII of Figure 7).

Persistently HBV-infected dHepaRG cells were treated twice (at day-7 and 10 p.i.) with various concentrations (between 0 to 5 μM) of the active enantiomer of BAY41-4109, then co-treated once (day-13) with the same concentrations of BAY41-4109 and suboptimal antiviral concentrations (0, 0.33, 1, and 3 μg/mL; see Figure S8 for the data on antiviral effect) of a TLR3 ligand (i.e. poly (I:C)-LMH). While the effect of combined treatments on HBeAg secretion was minor (Figure 7A), the inhibition of the intracellular HBV DNA accumulation by poly(I:C)-LMW was potentiated by increasing amounts of the capsid assembly inhibitor (Figure 7B and Ci); and vice versa, the inhibition of the intracellular HBV DNA accumulation by BAY41-4109 was also potentiated by increasing amount of poly(I:C)-LMW, with an EC50 for the former going from 0.321 nM to 0.078 nM (Figure 7Cii). The removal of HBc from the nucleus by specific inhibitors could therefore represent an interesting strategy to restore IFN responsiveness of infected hepatocytes. the same data as in (B) were re-analyzed in order to standardized in a reciprocal manner to 100% for the no drug condition of a given drug while the other is varying in concentration. This could enable the visualization of EC50 that were calculated using the "polynomial equation of individual curves" for each experiment. The precise EC50 is given at the top of the graph.

Thesis Adrien FOCA 260 HBc can also inhibit dsRNA-mediated IFN response in non-parenchymal cells exposed to HBV So far we have shown that HBc is capable to inhibit IFN response in HBV-infected hepatocytes (i.e. liver parenchymal cells). But other cells can be exposed to the virus in the liver, including liver-resident macrophages (i.e. Kupffer cells) and liver endothelial sinusoidal cells (LSEC), the two main scavenger cell types that are also involved in foreign antigen presentation and have innate immune functions in the liver [START_REF] Crispe | Liver Antigen-Presenting Cells[END_REF][START_REF] Knolle | Staying local -antigen presentation in the liver[END_REF]. Moreover, both cell types could also contribute to the active delivery of HBV (or related virus) to hepatocytes by transcytosis mechanisms (Breiner et al., 2001b) (Esser et al., unpublished data), thus suggesting that HBV could penetrate into and deliver HBc to the nucleus of these cells, without replicating in them. To check whether HBV and HBc could also inhibit dsRNA-mediated IFN response in these cell types, we used purified KC from human liver resections, as previously reported [START_REF] Zannetti | Characterization of the Inflammasome in Human Kupffer Cells in Response to Synthetic Agonists and Pathogens[END_REF], and an immortalized LSEC cell line we previously generated (called iLSEC here) [START_REF] Parent | An immortalized human liver endothelial sinusoidal cell line for the study of the pathobiology of the liver endothelium[END_REF], exposed them for 24h to 1000 vge/cell of HBV or 500 pg/cell (i.e. equivalent of 1000 vge/cell) of recombinant HBc, then stimulated them with poly(I:C) for 4h. An immunostaining with an anti-HBc at 24h post exposition to HBV (1000 vge/cell) is shown to evidence viral penetration into iLSEC cells (Figure 8A); auto fluorescence of KC did not allow to generate such data in this cell type. A significant reduction of the induction of IFN-α, OAS-1, and IL-6 was observed in both cell types with HBV, whereas HBc was only able to inhibit induction of IFN-α and OAS-1 (Figure 8B andC), which is consistent with results obtained in hepatocytes (Figure 1). ChIP experiments were also performed on HBV-exposed iLSEC and KC to measure HBc binding to innate gene promoters. As previously shown to prevent cGAS DNA sensing just after entry of virions within cells, and in the absence of viral replication and protein neo-synthesis (Wu et al., 2015).

In the nucleus of newly infected cells, we have shown that HBc binds to promoters of IFN response genes, and somehow induce the recruitment of the histone methyl-transferase EZH2, which is in turn responsible for the establishment of the H3K27me3 repressive mark, leading to the impairment of their inducibility upon dsRNA sensors stimulation. The binding of HBc to IFN response gene promoters was also found in liver-humanized HBV-infected FRG mice, thus suggesting that this early-established mechanism, set with incoming HBc, would also at work in persistent infections. The accumulation of HBc within nucleus is possible throughout the life cycle of the virus thanks to the recycling of neo-synthesized nucleocapsid to nucleus in infected cells. In chronically infected patients, HBc was also found in the nucleus of hepatocytes (Akiba et al., 1987;[START_REF] Chevallier-Queyron | Immunohistochemistry as a tool for chronic hepatitis diagnosis[END_REF]Chu and Liaw, 1987;[START_REF] Nakayama | Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly[END_REF][START_REF] Ng | Dynamic protein methylation in chromatin biology[END_REF], in particular in patients who are in the high-replicative, low-inflammatory phase. Here, we show immune-staining data that further support the idea of a correlation between high viremia and nuclear localization of HBc in the liver of patients. The correlation between the nuclear localization of HBc and the low-inflammatory phase might be explained, at least in part, by the inhibitory mechanism we described here.

As we have shown that the virus does not need to replicate to deliver HBc to the nucleus of hepatocytes, HBc could be delivered in other cell types to set similar inhibitory effect. Indeed in the immune tolerance phase, a high amount of infectious particles, containing HBc, circulates in the blood of patients (between 10 7 and 10 10 HBV-genome-copies/mL) (Seeger et al., 2015), and this concentration may be even higher in the liver microenvironment, thus potentially out numbering liver cells. It has been suggested that HBV could penetrate into LSEC (liver sinusoidal endothelial cells) (Breiner et al., 2001a), Kupffer cells [START_REF] Esser | Hepatitis B virus hijacks neutral lipid transport to target the liver and infect hepatocytes[END_REF], and pDC [START_REF] Vincent | Hepatitis B virus impairs TLR9 expression and function in plasmacytoid dendritic cells[END_REF], which are three important cell types to mount an efficient innate response. Our data suggest that indeed HBc could be delivered to the nucleus of primary KC and immortalized LSEC, and lead to impairment of IFN gene expression. Therefore, the new concept of IFN response inhibition we described for hepatocytes may also be valid for many other non-parenchymal and/or liver resident immune cells.

Finally, we have shown that prevention of the migration of HBc to the nucleus with Thesis Adrien FOCA 265 nocodazole or CpAMs, restore the IFN response to dsRNA ligands. Does this mean that core assembly modulators could be used in vivo to restore immune function in infected hepatocytes and help reinforced immune response against the virus? As HBc possesses both nuclear localization (NLS) and export (NES) sequences (Li et al., 2010), as it has been proposed that the pool of nuclear cccDNA could be constantly replenished by NCs recycling (Seeger et al., 2015), and despite its long-half life in the nucleus (our data not shown), it is tempting to speculate that a long-term treatment with core inhibitors could lead to such a purge. To substantiate this hypothesis, we have finally shown in this study that a capsid assembly inhibitor, leading to nuclear HBc removal (or aggregation), could potentiate the anti-HBV effect of a TLR3-L.

Many core/capsid inhibitors, also called CpAM (core protein allosteric inhibitor) (Zlotnick et al., 2015), are under current development. Selected on their "canonical functions", i.e. inhibition of the packaging of HBV pregenomic RNA and subsequent inhibition of capsid reverse transcription, CpAMs could also have additional antiviral effect due to their impact on HBc nuclear function, and should therefore be also selected on this criteria. Our results suggest that CpAMs could be favorably combined to immune stimulators to obtain a greater anti-HBV effect. Among immune-stimulator in current clinical evaluation is the TLR7 agonist GS-9620, which has shown very interesting antiviral effect, leading in particular to the degradation of cccDNA in animal models [START_REF] Lanford | GS-9620, an oral agonist of Toll-like receptor-7, induces prolonged suppression of hepatitis B virus in chronically infected chimpanzees[END_REF][START_REF] Menne | Sustained efficacy and seroconversion with the Toll-like receptor 7 agonist GS-9620 in the Woodchuck model of chronic hepatitis B[END_REF]. As TLR7 is not expressed in infected human hepatocytes (Luangsay et al., 2015a), and TLR7-L likely acts via pDC-expressed IFN-a, it would be of interest to develop PRR agonist that could both stimulate innate cells and induce expression of IFN-α and related ISGs within infected hepatocytes. In this respect, our study makes a case for a combinatory approach with CpAMs and PRR agonists in order to progress toward the rupture of tolerance to HBV. relative mRNA expression was analyzed with q-base software (Biogazelle, Belgium) using the comparative cycle threshold (2 -titiCt ) method with 2 housekeeping genes (RPLP0 and α-Actin). The relative HBV mRNA level was quantified using the same primer pairs used for the HBV PCR quantification. The sequences of primer pairs were listed on Table 1. Cytoplasmic Extraction Reagents (Thermo Scientific) for nuclear and cytosolic proteins separation. All lysis buffers were complemented with inhibitor cocktail of proteases (Complete EDTA-free tablets, Roche). Extracted proteins were separated electrophoretically on a 10% SDS-PAGE gel and "semi-dry" transferred onto hybond-ECL membranes (Amersham, biosciences). Membranes were blocked with 5% non-fat milk in PBS 0,1% Tween for 1 hr. at RT. Polyclonal rabbit anti-HBc (Petit and Pillot, 1985), antiactin (13E5, Cell Signaling), anti-tubulin (MAB3408, Millipore) and anti-PARP (Zymed, Invitrogen) antibodies were applied overnight at 4°C under gentle agitation. After washing, HRP conjugated secondary antibodies from Sigma Blots were applied. After extensive washing, blots were revealed using the SuperSignal West Pico Chemiluminescent substrate (Thermo Scientific).

Material and Methods

Table 1. Primer pairs for RT-qPCR

GENE

ELISA. Commercial immunoassay kits for HBs antigen (Autobio Diagnostics Co., China), HBe/HBc antigens (Autobio Diagnostics Co., China), and IL-6 and IP-10 cytokine (R&D system) detections were used following manufacturer's instructions. We have identified an epigenetic mechanism hijacked by HBV and linked to poor prognosis HBV-associated liver cancer 1 , involving the chromatin modifying PRC2 complex, the RNA helicase DDX5, and the lncRNA HOTAIR To effect targeted delivery of these anti-miRs to infected hepatocytes, we determined the expression of the folate receptor in HBV replicating cells (HepAD38 cells and PHHs). Interestingly, HBV infection increases expression of the folate

Faculté

  Techniques des Activités Physiques et Sportives Observatoire des Sciences de l'Univers de Lyon Polytech Lyon Ecole Supérieure de Chimie Physique Electronique Institut Universitaire de Technologie de Lyon 1 Ecole Supérieure du Professorat et de l'Education Institut de Science Financière et d'Assurances Directeur : M. F. DE MARCHI Directeur : M. le Professeur F. THEVENARD Directeur : Mme C. FELIX Directeur : M. Hassan HAMMOURI Directeur : M. le Professeur S. AKKOUCHE Directeur : M. le Professeur G. TOMANOV Directeur : M. le Professeur H. BEN HADID Directeur : M. le Professeur J-C PLENET Directeur : M. Y.VANPOULLE Directeur : M. B. GUIDERDONI Directeur : M. le Professeur E.PERRIN Directeur : M. G. PIGNAULT Directeur : M. le Professeur C. VITON Directeur : M. le Professeur A. MOUGNIOTTE Directeur : M. N. LEBOISNE « La science est le calcul fait au plus juste de ce qu'on croyait savoir, et qu'on ne sait pas. » Joseph Michel Antoine Servan מי שאינו מעלה את הידע שלו מפחית אותו Proverbe hébreu; מוּד לְ תַּ Thesis research realised by Adrien Foca within the Centre de Recherche en Cancérologie de Lyon (CRCL), UMR INSERM 1052, CNRS 5286.

Figure 1 : 25 Figure 2 : 25 Figure 3 : 27 Figure 4 : 27 Figure 5 : 31 Figure 6 :Figure 7 : 33 Figure 8 :Figure 9 : 34 Figure 10 :Figure 11 :Figure 12 :Figure 13 :Figure 14 :Figure 15 :Figure 16 :Figure 17 :Figure 18 :Figure 19 :Figure 20 :Figure 21 :Figure 22 :Figure 23 :Figure 24 :Figure 25 :Figure 26 :Figure 27 :Figure 28 :Figure 29 :Figure 30 :Figure 31 :Figure 32 :Figure 33 :Figure 34 :Figure 35 :Figure 36 :Figure 37 :Figure 38 :Figure 39 :Figure 40 :Figure 41 :

 125225327427531673389341011121314151617181920212223242526272829303132333435363738394041 Figure 1: HBV complete virions and subviral particles....................................................…...................25

"Figure 1 :

 1 Figure 1: HBV complete virions and subviral particles. Original electron microscopy pictures taken by Dane and colleagues. Extracted from The Lancet, April 4, 1970, p695.

Figure 2 :

 2 Figure 2: Milestones in HBV research. From first historical description to future goals. Principal facts depicted in three axes: "Description/Characterization", "Therapeutic development" and "Cure".

Figure 3 :

 3 Figure 3: Phylogenic tree of Hepadnaviridae. Avihepadnaviruses (top) and Orthohepadnaviruses (bottom) with prototype members. Created with itol.embl tool.

Figure 4 :

 4 Figure 4: Phylogenetic tree of the genus Orthohepadnavirus, focusing on "great apes". Complete genomes of HBV genotypes A (X02763), B (D00330), C (M12906), D (J02203), E (X75657), F (X69798), G (AF160501) and H (AY090454), and isolates found in chimpanzee (D00220), orangutan (AF193864) and gibbon (U46935), were aligned using Clustal W. Scale bar represents substitutions per site. Adapted from ICTV Ninth Report, 2011.

Figure 5 :

 5 Global distribution of HBV genotypes. Boxe sizes are proportional to genotypic population found in areas. Created using WHO 2017 HBV report data. Figure 6: Hepatitis B virus infection prevalence worldwide (seroprevalence of HBsAg). Based on WHO 2017 HBV report data.

Figure 7 :

 7 Figure 7: Cryo-EM of HBV particles. A: Dane particles (D), 22nm filamentous (F) and spherical (S) subviral particles. B and C: Dane particles harbouring compact and gapped morphology. D: particles with mixed morphology. Areas consistent with the gapped morphology are delineated in white. Scale bar for panels B, C and D equals 20nm. Adapted from Seitz S et al., EMBO J, 2007.

Figure 8 :Figure 9 :

 89 Figure 8: Various HBV particles found in patient serum.

Figure 10 :

 10 Figure 10: Schematic representations of HBV envelope proteins. A: Principal features of S, M and L HBsAg. B: Topology of HBV envelope proteins as processed in the ER. Light blue: S domain. Middle blue: PreS2 domain. Dark blue: PreS1 domain. N-ter are positioned at left. Principal post-translational modifications are shown in orange hexagons. N: N-glycans. O: O-glycans. M: myristic acid.

Figure 11 :

 11 Figure 11: Schematic representation of the different domains of the viral polymerase. The catalytic site YMDD within de C subdomain is a mutational hot point and is critical for resistance to NAs.

Figure 12 :

 12 Figure 12: HBx as the center of a hub in cellular pathways involved in oncogenic process. Extracted from Kong F. et al., Onco. letters, 2017.

Figure 13 :

 13 Figure 13: Schematic representation of HBSP synthesis from pgRNA splicing. 1.3kb intron is removed from pgRNA by splicing, giving rise to SP1 RNA. This latter is translated in the 12.4kDa HBSP protein. The first 46aa (dark grey) are common with the polymerase as HBSP shares the same AUG. The following 47aa (orange) comes from the unique rearrangement of the SP1 RNA sequence.

  Figure 15.

Figure 14 :

 14 Figure 14: Representation of the viral polymerase and pgRNA interaction, leading to its encapsidation. Dashed red circle: terminal protein (TP) of the polymerase. Black dashed circle: reverse transcriptase (RT) domain of the polymerase. A(n): poly-A tail. Red line: 4nt primer synthetized by the TP, initiating the reverse transcription of the pgRNA.

Figure 15 :

 15 Figure 15: Simplified HBV replication cycle. Please refer to the section: "1.4.5. HBV replication strategy and spreading" for details. Extracted from Shih C et al., Trends in Microbiology, 2018.

Figure 16 :

 16 Figure 16: Natural history of hepatitis B. Evolution of seric markers over the course of the disease. Witness of HBV replication rate, inflammation and immunologic status.

Figure 17 :

 17 Figure 17: Pathology of HBV infection. Progression of HBV infection through different clinical states. % represents the progression from one stage to another per year.

Figure 18 :

 18 Figure 18: Global incidence of hepatocellular carcinoma. Incidence means number of cases for 100,000 individuals.

Figure 19 :

 19 Figure 19: Estimated age-standardised incidence and mortality of liver cancer worldwide Male/Female. Cases per 100,000. Globocan 2012.

Figure 20 :

 20 Figure 20: Viral mutation pattern in HBV infected patients over a longitudinal period. Among all the patients with HCC, 65% exhibits BCP/PC and 1938/2051 mutations (panel A, blue). 25% of HCC presents the A2051C mutant with or without other mutations in BCP or precore regions (panel B, yellow + light and dark green). Dx means diagnosis. Extracted from Hayashi S et al., Hepatology, 2018.

Figure 21 : 2 . 3 . 3 . 5 .

 212335 Figure 21: World maps presenting repartition of aflatoxin, HBV and HCC globally. Incidence means number of cases for 100,000 individuals.

Figure 22 :

 22 Figure 22: Infection of a hepatocyte by dslDNA virion. Once in the nucleus, the HBV dslDNA can be repaired into cccDNA or integrate the host genome at double-strand DNA breaks through NHEJ. cccDNA originating from HBV dslDNA has the same fate as cccDNA from rcDNA and permits establishment of long-lasting infection and fully functional viral replication cycle. Adapted from Shih C et al., Trends in Microbiology, 2018.

Figure 23 :

 23 Figure 23: Mapping of HBV integration sites. Each arrow represents a HBV breakpoint from one clinical sample. Boxes represent exons. Extracted from Sung WK et al., Nature Genetics, 2012.

Figure 24 : 2 . 3 . 4 . 1 . 2 .

 2423412 Figure 24: Visualization of breakpoints in HBV genome. The frequency of integration breakpoints at different loci in the HBV genome is shown as a blue histogram, highlighting the 1,800bp region, where the HBx gene is located. Extracted from Sung WK et al., Nature Genetics, 2012.

Figure 25 :

 25 Figure 25: Barcelona Clinic Liver Cancer (BCLC) staging and treatment strategy. The BCLC system establishes a prognosis in accordance with the five stages that are linked to first-line treatment recommendation. ECOG PS=Eastern Cooperative Oncology Group Performance Status. *Patients with end-stage cirrhosis due to heavily impaired liver function. †Currently, Sorafenib followed by Regorafenib has been shown to be effective. Lenvatinib has been shown to be non-inferior to Sorafenib, but no second-line option after Lenvatinib has been explored. Extracted from Forner A et al., The Lancet, 2018.

Figure 26 :

 26 Figure 26: Laparoscopic resection for HCC located on superficial liver areas. This approach exhibits high survival for superficial early stage tumours. Extracted from Galle PR et al., J Hepatol., 2018.

Figure 27 :

 27 Figure 27: Presentation of commonly used percutaneous ablation techniques for HCC. Adapted from Galle PR et al., J Hepatol., 2018.

  , MET, and AXL, which are implicated in the progression of HCC and the development of Sorafenib resistance. The first data from a clinical trial phase 3 were published in July 2018 in the New England Journal of Medicine. The aim was to evaluate Cabozantinib as compared with placebo in previously treated patients with advanced HCC. 707 patients were involved in this trial and among the treated arm, the median overall survival was 10.2 months with Cabozantinib versus 8.0 months with placebo. As mentioned, eligible patients have previously received at least Sorafenib as first-line therapy and were still under disease progression, rendering Cabozantinib a promising drug for advanced, Sorafenib-resistant HCC (Abou-Alfa et al., 2018; NCT01908426).

Figure 28 :

 28 Figure 28: Schematic illustration of production of HBV-TCR redirected T cells.The primary T cells are electroporated with mRNA encoding HBV-specific TCR to transiently redirect their specificity towards HBV. The redirected T cells can be re-infused into the patient, whereby they will recognize and lyse HCC cells expressing HBV antigen within 3-4 days. Extracted fromKoh S et al., Diseases, 2016.

Figure 30 :

 30 Figure 30: Structural domains of human PLKs.The Ser/Thr kinase activity is hold in the kinase domain. PLK5 is described as a pseudokinase as it lacks important residues to fulfil the enzymatic activity. The D-box of PLK1 is important for its degradation through the APC/c complex in anaphase. Degradation of PLK4 is mediated by autophosphorylation of the SCF SBD (S phase kinase-associated protein 1 (SKP1)-cullin-F-box Slimb-binding domain). The SAS-6 binding domain is not found in human PLKs but is important for recruitment of SAS-6 and subsequent appropriate centriole assembly in the C.elegans PLK1 homolog's ZYG-1. PDB means Polo box domain and is involved in interaction with phosphopeptides. Extracted from S.Zitouni et al., Nature Reviews, 2014.

Figure 31 :

 31 Figure 31: Structural organization of human PLK1. Model representation (Top) and quaternary structure (bottom) based on PDB accession identifications 2OU7 for the kinase domain (blue) and 1UMW for the Polo-box domain (green). Thr210 and Ser137 are important for PLK1 activation (orange). A groove between PB1 and PB2 allows the recognition of peptides (purple) containing phosphorylated Ser and Thr residues. IDL = inter-domain linker. Adapted from S. Zitouni et al., Nature Reviews, 2014.

Figure 32 : 5 )

 325 Figure 32: Schematic representation of multileveled regulation of PLK1. 1) PLK1 is autoinhibited by the PBD and the IDL. Map205 stabilizes this resting state. CDK1 can phosphorylate Map205 at Ser283 to release this inhibition. 2, 3, 4) PLK1 full activation is depedent of activating kinases such as Aurora A. 5) Binding of phosphopeptide to the hinge between the two PBs results in the activation of PLK1. 6) Activated PLK1 could further phosphorylate phosphopeptides. 7) APC/c complexe recognizes the D-box within PLK1 IDL, mediating its degradation through ubiquitylation (8).All the steps described here occur possibly at different localizations and cell-cycle stages, explaining the multimodal way of activating PLK1. Adapted from S.Zitouni et al., Nature Reviews, 2014.

Figure 33 :

 33 Figure33: Model of ubiquitylationdependent release of PLK1. During anaphase, PLK1 is located to the kinetochores. Ubiquitylation of its Lys492 by the CUL3-KLHL22 complex leads to PLK1 release from kinetochores and relocalization to microtubules, resulting in normal sister chromatids separation. Adapted from S.Zitouni et al., Nature Reviews, 2014.

Figure 34 :

 34 Figure 34: Schematic representation of PLK1 funcations and localizations during the cell-cycle. As mentionned in the text, PLK1 is involved in nearly every step of the mitosis while other PLKs members are restricted to specific stages and localizations. Strating from G2, PLK1 is found near the centrosome. In prophase and metaphase, PLK1 localizes at kinetochores and at the spindle pole while in anaphase and telophase it relocalizes to central spindles. Extracted from S. Zitouni et al., Nature Reviews, 2014.

Figure 35 :

 35 Figure 35: PLK1 and the DNA damage response. CDC14B activates the APC/c complex component CDH1, which promotes the degradation of PLK1 and cycle arrest. Consequently, WEE1 is activated and CDC25 is inactivated through a signalling cascade that involves the Ser protein kinase ATM and the Ser/Thr protein kinase ATR, and the dual function checkpoint kinases Ser/Thr protein kinase Chk1 and Chk2. ATM and ATR, which are closely related kinases that are activated by DNA damage, initiate the DNA damage checkpoint by phosphorylating several key proteins. Once activated, the checkpoint leads to cell-cycle arrest and DNA repair or apoptosis. ATM is activated by double-strand breaks and phosphorylates Chk2, whereas ATR is activated by single-strand breaks and phosphorylates Chk1. PLK1 indirectly inactivates p53 function to control the DNA damage response. During DNA repair, CDK1 is reactivated to enable cell-cycle progression. PLK1 initiates repair by phosphorylating DNA repair protein RAD51 homologue 1 (RAD51), which triggers the degradation of WEE1 and claspin, leads to the inactivation of Chk1 and Chk2 and promotes the phosphorylation of tumour suppressor p53 binding protein 1 (p53BP1), which triggers its dissociation from DNA. Dashed arrows indicate non-functional pathways. P, phosphate; Ub, ubiquitin. Extracted from S. Zitouni et al., Nature Reviews, 2014.

N

  -Myc overexpression is associated with poor prognosis and resistance to therapy in human cancers. Moreover, pharmacological strategies targeting directly N-Myc are still elusive. In 2016, Xiao et al., identified a reciprocal activation between PLK1 and N-Myc. PLK1 promotes ubiquitindependent degradation of Fbw7 by directly phosphorylating it, thus counteracting Fbw7-dependent degradation of N-Myc. In turn, through its transcription factor activity, N-Myc enhances PLK1

3. 3 . 8 . 4 .

 384 Interplays between PLK1 and PIV5 Paramyxoviruses are negative-sense, single-stranded RNA viruses. The replication is dependent on the viral phosphoprotein (P) and the large (L) protein, with phosphorylation of P being known to regulate viral gene expression. The PIV5 is a prototypical paramyxovirus and by contrast to others examples presented before, PLK1 was shown to downregulate viral replication by phosphorylating the P protein. However, increased viral gene expression correlates with higher cell

Figure 36 :

 36 Figure 36: PLK1 interplays with several viruses, mostly increasing their replication.

1 :

 1 In 2016, Arbutus Biopharma published final results from a phase 1/2 clinical trial regarding the anti-tumor activity of intravenous siRNA targeting PLK1, TKM-080301 in subjects with advanced hepatocellular carcinoma. More specifically, TKM-080301 is a type of LNP formulation, referred to as SNALP (Stable Nucleic Acid Lipid Particles) containing siRNA targeting PLK1. In this so called chemorefractory cancer, administration of siPLK1 resulted in increased partial response and inhibition of tumor growth. However, due to adverse effects, TKM-080301 was considered to have suboptimal efficacy (NCT02191878). In another phase 1 clinical trial, TKM-080301 safety and effectiveness were evaluated for primary or secondary liver cancer and was administrated by hepatic arterial infusion (NCT01437007). Although some issues are still matters of concern (delivery, toxicity, cost, and biological barriers), siRNA definitively opens news perspectives for drug development.Table VI: Several PLK1 inhibitors being clinically trialed or in early development for anti-cancer therapies. Hypothesis and ObjectivesIn highly HBV endemic regions, 70-80% of hepatocellular carcinoma cases are HBVattributable. Despite the HBV vaccine, the World Health Organization reports 240 million people are chronically infected with HBV. Current treatments to control chronic HBV infection for reducing the incidence of liver cancer are ineffective, thus new and effective therapies are needed.Polo-like-kinase 1 (PLK1), which plays pivotal roles in mitosis and is over-expressed in many human cancers, represents a promising druggable target in oncology. Focusing on HBV, it was shown that the X protein (HBx) could activate PLK1 in murine cell transformation models. Our and collaborators recent studies have identified a positive link between PLK1 activation and HBV replication. The interplay between PLK1 and HBV replication was described using the HepAD38 cellular model of HBV replication. In this context, the HBV DNA is stably integrated into the host genome, under control of a Tet-off expression system. Transcription of HBV pregenomic RNA (pgRNA), the template of viral replication, is initiated by tetracycline removal. It has been shown that in HBV-replicating HepAD38 cells, increased PLK1 expression correlates with down-regulation of two proteins that are components of chromatin modifying complexes; SUZ12 protein of the PRC2 complex, and ZNF198 of the LSD1-CoREST-HDAC1 complex. PLK1 inhibition was described to inhibit HBV replication by reducing viral transcription. How PLK1 regulates HBV transcription remains unknown. Yet it remained to be determined whether PLK1 could also play a role for HBV replication in non-dividing hepatocytes. The goals of my thesis project were to investigate if: 1) PLK1 was activated upon HBV infection in non-dividing hepatocytes 2) PLK1 plays proviral role towards HBV 3) PLK1 inhibition could serve as new antiviral strategy Ancillary studies: 1) Decipher the mechanisms of PLK1-HBV interplays 2) Study the potential link between PLK1/HBV and PLK1/HBV-related HCC To this extent, I used relevant models that resemble physiologic HBV infection, such as of Primary Human Hepatocytes (PHH), dHepaRG and liver-humanized FRG mice, where HBV replicates in non-transformed and non-dividing cells, thus enabling the study of the interphasic role of PLK1, irrespective of its well-established cell division implication.

FIG. 1 .

 1 FIG. 1. HBV infection activates PLK1. dHepaRG cells (A-C) or PHHs (D) were infected with a low dose (100 vge/cell) or high dose (1000 vge/cell) of HBV. (A) Cells were harvested at the indicated time points, and RNA was extracted and subjected to RT-qPCR. Fold induction of the mRNA expression level of PLK1 and HBV was normalized to housekeeping genes, compared with mock infection. (B) Immunoblotting of PLK1 and phosphorylated PLK1 (pPLK1-S137 and pPLK1-T210) using whole cell extract of mock-or HBV-infected dHepaRG cells isolated at the indicated time points postinfection. Quantification by chemiluminescence was performed with a ChemiDoc XRS1 system (Biorad). (C) Immunofluorescence microscopy of the indicated proteins with and without HBV infection in dHepaRG cells at different times postinfection. Cells were fixed with 2% paraformaldehyde and stained with the indicated antibodies. (D) Immuno-blots of PLK1 and phosphorylated PLK1 using whole cell extract from mock-or HBV-infected PHHs.

2 .

 2 FIG. 2. Effect of BI-2536 on HBV replication in nondividing and nontransformed dHepaRG cells. dHepaRG cells were infected with 100 vge/cell of HBV for 7 days followed by treatment with increasing concentration of PLK1 inhibitor BI-2536, as indicated, for 3 days. (A) Secreted HBsAg and HBeAg quantified by ELISA from supernatants of HBV-infected dHepaRG cells, day 10 post-infection. Total RNA and DNA extracted from infected cells, day 10 postinfection, were analyzed by way of HBV-specific RT-qPCR or qPCR, normalized to housekeeping genes, and compared with mock-treated cells. The results are presented as a fold change in expression or secretion compared with untreated controls and are expressed as the mean 6 standard error of the mean of at least three independent experiments. (B) Cell viability of HepaRG cells measured using Cell Titer Glo One Solution Assay, with increasing concentration of BI-2536, as indicated. DMSO and puroMycin were used as controls. (C) Immunofluorescence microscopy of the indicated proteins with and without BI-2536 in infected dHepaRG cells. (D) Mock and infected dHepaRG cells were treated with biochemicals or transfected by siRNA (25 nM) targeting either HBV (siHBV) or PLK1 (siPLK1) for 3 days. Cell lysates were ana-lyzed by way of native agarose gel electrophoresis, transferred onto enhanced chemiluminescence membrane, and immunoblotted with HBc antibody. (D.i) Immunoblotting with quantification below. No Txt 5 no treatment; TFV 5 tenofovir at 10 lM; Bay41 5 Bay41-4109 at 10 lM; AT130 5 AT130 at 10 lM; BI-2536 at the indicated concentration. (D.ii) Quantification of two indepen-dent experiments by chemiluminescence with a ChemiDoc XRS1 system (Biorad).

4 .

 4 FIG. 4. Effect of PLK1 knockdown on HBV replication in dHepaRG cells and PHHs. PHHs or dHepaRG cells infected with 100 vge/ cell of HBV for 7 days were transfected with siRNA (5 or 25 nM) targeting PLK1 (siPLK1) or HBV (siHBV) or HCV (siHCV), as indi-cated. (A, C) Total RNA and DNA were extracted and subjected to RT-qPCR or qPCR with PLK1 or HBV primers. Quantification of PLK1 mRNA and HBV DNA is relative to absence of siRNA transfection (no siRNA). Results represent the mean 6 standard error of the mean of at least three independent experiments. (B) Immunofluorescence microscopy of the indicated proteins, with and without siPLK1 transfection in HBV-infected dHepaRG cells. Cells were fixed with 2% paraformaldehyde at day 10 postinfection, and stained with the indi-cated antibodies. (D) Representative Southern blotting. DNA was extracted from infected PHHs transfected by the indicated siRNA.

6 .

 6 FIG. 6. Effect of BI-2536 on cap-sid formation in liver-humanized HBV-infected FRG mice. Immunohistochemistry staining (A) and native capsid migration assays (B) employing liver samples from the same mice as in Figure 5. (A) Rep-resentative immunohistochemistry staining with anti-HBc antibody (Dako) and FAH antibody for non-treated and BI-2536 treated livers. (B) Capsid migration assay employ-ing extracts from mock-treated FRG mice (mouse numbers M#1-M#4) and BI-2536 treated animals (mouse numbers M#5-M#38). (C) Cumulative quantification of capsid migration signals from panel B.

  FIG. 8. HBc is a phosphorylation substrate of PLK1 in vitro. In vitro PLK1 kinase assays of wild-type (WT) HBc and site-directed mutants. (A) Recombinant HBc or immunoaffinity purified HBc isolated from HepaRG-TR-HBc cells and PLK1 were used in in vitro PLK1 kinase assays. Reactions were performed with (1) or without (2) PLK1 inhibitor BI-2536 (500 nM), as indicated, in the presence of c 32 P-ATP, and analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. (B) Amino acid sequence of Cterminal domain (CTD) of HBc. Eight S/T residues are present in HBc CTD. CDK2 SP sites are indicated in red color, and putative PLK1 phosphorylation sites in blue. (B-E) In vitro kinase assays of WT HBc and site-directed mutants with (1) or without (2) addition of BI-2536, as indicated. HBc 3A contains serine to alanine substitutions of residues 155, 162, and 170. HBc 3D contains serine to aspartate substitutions of the same residues. Commassie Blue staining of the same gel used for autoradiog-raphy is shown. Relative intensity quantified by ImageJ software is the ratio of signal from in vitro kinase reaction signal versus the corresponding signal of Commassie Blue-stained band, expressed relative to WT (n = 3).

3 : 4 :

 34 Specificity of anti-HBc antibodies. Three antibodies (Ab8637 (mouse monoclonal), Ab140243 (rabbit polyclonal), and Dako-B0586 (rabbit polyclonal)) were tested in western blotting against assembled HBV nucleocapsid (obtained from a cell lysate of HepG2215 cells, which contain an HBV transgene and feature an intracellular HBV life cycle; NI means lysate from non infected cells (i.e; HepG2 cells)) run into agarose gel or denaturated HBcAg run in SDS-PAGE. Ab8637 is the only antibody tested capable to recognize only assembled nucleocapsids. The 2 polyclonal antibodies recognized both from of HBcAg. Effect of BI 2536 on establishment of HBV infection in dHepaRG. A) Diagram of infection and treatment protocol. B) Differentiated HepaRG cell were treated with increasing concentration of BI 2536 for 3 days, then infected with 100 vge/cell of HBV for 7 days.Secreted antigens HBsAg and HBeAg were quantified by ELISA from supernatants of HBV infected dHepaRG cells at day-10 p.i. Total RNA and DNA extracted from infected cells on day-10 p.i. were analyzed by HBV-specific RT-qPCR or qPCR, normalized to housekeeping genes, and compared to mock-treated cells. Fold change in expression or secretion was quantified relative to untreated controls, and are mean ± SEM of at least 3 independent experiments. C) Immunofluorescence microscopy of indicated proteins, +/-transfection of siPLK1 in HepaRG cells. siRNA was transfected for 3 days prior to infection and cells were fixed on days-3 or 7 p.i.

5 :Supplementary Figure 7 :

 57 Limited over-expression of PLK1 leads to an increase of HBV replication in dHepaRG. A) Differentiated HepaRG-TR-PLK1 CA cells were induced by the addition of tetracycline (0, 16, 80 400, 1000 or 2000 ng/mL) for 72 hours and PLK1 levels analysed by immunoblotting. B) dHepaRG-TR-PLK1 CA cells were infected with 100 vge/cell of HBV for 7 days followed by induction of PLK1 by the addition of indicated amount of tetracyline for 3 days. HBeAg secretion was analysed by ELISA. Total DNA was extracted and HBV DNA was measured by qPCR. Results are shown as fold change in HBV DNA as compared to control cell line. Results are represented as the mean ± SEM of 2 independent experiments. end of treatment (or mock-treatment) (i.e. weeks 7 and 8). C) ALAT was dosed in sera of mice at weeks 7 and 8. Effect of IFN-α and entecavir on HBV replication in liver humanized HBV-infected FRG mice. Eleven mice, engrafted with human PHH for two months, were infected with HBV (5.10e8 vge/mouse). At week-4 post infection treatment was started in 8 mice and 3 other mice were mock treated. IFN-(25 g/kg) was injected twice a week by IP injections, for 4 weeks.

  '. LNP-siRNAs were administrated to cells by direct dilution into the culture medium. Cells were exposed to LNPs for 24 hours, then washed with culture medium to remove excess of LNPs. Treatment were repeated as indicated in figure legends.

(

  Autobio Diagnostics) according to the manufacturer's instructions. Total intracellular DNAs and RNAs as well as secreted viral DNAs/RNAs were purified from infected cells using NucleoSpin® 96 Tissue kit, NucleoSpin® 96 RNA kit, NucleoSpin® 96 virus kit (Macherey-Nagel) respectively, according to the manufacturer's instructions. Reverse-transcription of Thesis Adrien FOCA 141 RNA into cDNA was performed with the SuperScript® III First-Strand Synthesis kit (Invitrogen)

- Figure 1 .

 1 Evaluation of the efficacy and toxicity of LNP siPLK1 in primary human hepatocytes. PHHs were infected with HBV genotype D at a multiplicity of infection of 200vge/cell. From 4 days post-infection (p.i.), cells were treated every 3-4 days with increasing LNP siPLK1 concentrations or the indicated control molecules (LNP siHBV and LNP siCtrl were used at 200ng/mL and 10μg/mL of puromycin served as positive death control, Tenofovir (TFV) was used at a concentration of 10μM). Cells and supernatants were harvested at day-13 p.i. for subsequent analyses. (A) RNAs were extracted and levels of PLK1 mRNAs (relative to PRNP mRNAs) analyzed by RT-qPCR. Results were normalized to the Mock-Treated condition. (B) Proteins were extracted from cytosol and nuclear compartments with NE-PER Nuclear and Cytoplasmic kit. Thirty μg of nuclear fraction for each condition were analyzed by western blot. Lamin B1 served as loading control for nuclear proteins while β-Tubulin served as control for cytoplasm contamination (a representative blot is shown). (C) ATP production, reflecting cell viability, was measured using Cell TiterGlo One Solution Assay. (D) Phase microscopy pictures of PHH at the end of the experiment acquired on an Olympus IX70 microscope, objective X40. Scale bar (white) corresponds to 500μM. Results presented in graphs are the mean +/-SEM (standard error of the mean) of 3 independent experiments (3 donors of PHH) each performed in biological triplicate. P ≤ 0.05*, P ≤ 0.01**, and P ≤ 0.001*** were considered statistically significant.

- Supporting Figure 1 .

 1 Efficacy of LNP siPLK1 and toxicity profile in HepG2 cells. (A) HepG2 cells were treated with increasing doses of LNP siPLK1, ranging from 1.3ng/mL to 133ng/mL. RNA were extracted, reverse transcribed and PLK1 mRNA quantified using specific qPCR. Normalized to housekeeping gene and relative to Mock-Treated condition. (B) Cell viability was assessed with CellTiter Blue reagent and plotted among with knockdown of PLK1 RNA under various LNP siPLK1 concentrations.

  observed a dose dependent decrease of intracellular HBV DNAs and RNAs in HBV-infected PHH or dHepaRG cells treated with LNP siPLK1 (Fig. 2D, Fig S2D). The highest dose of LNP siPLK1 resulted in a decrease of 55% and 60% of HBV DNA and RNA respectively (Fig. 2D, Fig S2D). LNP siHBV treatment resulted in a stronger effect, with 80% and 90% loss, whereas only viral DNA amount decreased with TFV (around 60%) as expected. The lower inhibition of intracellular DNA amount for all molecules tested, as compared to what observed with secreted DNA is likely due to the detection of remaining input HBV DNA from HBV inoculum as discussed below. Interestingly and contrary to what we described with SM-PLK1i [6], we observed a decrease in the levels of HBV RNAs upon LNP siPLK1 treatment of HBV-infected hepatocytes without observing any significant changes in cccDNA amount (analyzed by qPCR

- Figure 2 .

 2 Antiviral activity of LNP siPLK1 in non-transformed and non-dividing HBV-infected PHH. PHH were infected with HBV genotype D at a multiplicity of infection of 200vge/cell and treated with the indicated molecules as shown (A) on the schemactic representation of the experiment. LNP siHBV and LNP siCtrl were used at 200ng/mL and TFV at 10μM. At the end of the experiment, (B) RNAs and DNAs from the cell supernatants were extracted, digested or not with DNaseI or RNaseH and analyzed by HBV-specific RT-qPCR or qPCR. (C) Levels of secreted ApoB, HBeAg and HBsAg were assessed by ELISA. (D) Intracellular total RNAs and DNAs were extracted and analyzed by HBV-specific RT-qPCR or qPCR. Results presented in graphs are the mean +/-SEM of 3 independent experiments (3 donors of PHH) each performed in biological triplicate and are presented as ratio compared to the Mock-Treated condition. P ≤ 0.05*, P ≤ 0.01**, and P ≤ 0.001*** were considered statistically significant.

- Figure 3 . 2 .

 32 LNP siPLK1 does not affect cccDNA amount. (A) Intracellular cccDNA specific Taqman qPCR [19] from PHH samples from Fig. 2, relative to Mock-Treated. (B) Representative Southern blot of pooled n=3 total DNA from Fig. Two micrograms of DNA was loaded by condition and processed as described [22]. EcoRI digestion serves as control as it allows cccDNA linearization of HBV genotype D (shift from coiled 2.2kb band to full lengh 3.2kb linear DNA). (C) Quantification of Southern blot presented in panel B using image Lab software. Mitochondrial DNA was used as loading control and served for normalization. P ≤ 0.05*, P ≤ 0.01**, and P ≤ 0.001*** were considered statistically significant.

- Figure 4 .

 4 Pre-infection treatment with LNP siPLK1 in primary human hepatocytes. Experiments conducted on three independent PHH batches. LNP siHBV and LNP siCtrl were used at 200ng/mL and TFV at 10μM. PreS1 peptide at 100nM served as guenine entry inhibitor. Results presented in graphs are the mean of 3 independent experiments with SEM. (A) Experimetal design of the study. (B) RNA were extracted, reverse-transcribed and subjected to specific PLK1 qPCR. Normalization was done on PrP and quantification is relative to PLK1 expression in Mock-Treated condition. (C) Quantification of secreted ApoB, HBeAg and HBsAg by ELISA using supernatants of HBV infected PHH, relative to Mock-Treated. (D) RNA and DNA from the supernatant were extracted, followed by a digestion step, were analyzed by HBV-specific RT-qPCR or qPCR, normalized to housekeeping genes, and compared to Mock-Treated cells. (E) Intracellular total RNA and DNA extracted from HBV infected cells were analyzed by HBV-specific RT-qPCR or qPCR, normalized to housekeeping genes, and compared to Mock-Treated. (D) Intracellular cccDNA specific

- Figure 5 .

 5 Localization of HBV RNAs under LNP siPLK1 treatment. (A) Total RNA were extracted, reverse transcribed and submitted to HBV specific qPCR. Normalized on housekeeping gene and relative to Mock-Treated condition. (B) Cytosolic and nuclear compartments were recovered using a homemade protocol adapted from Weil et al.

Figure 37 :

 37 Figure 37: Hypotetic model of PLK1 activation based on innate immune sensing of HBV. Subviral particles and/or naked capsids are known to be recognized by TLR2/TLR4, thereby activating the NFkB pathway. HBs has been shown to interact directly with the β 2 glycoprotein I (β2GPI) via the TLR4/Myd88 route and through the activation of NFkB by phosphorylation of Ser32/36 by IKKa[START_REF] Jing | HBsAg/β2GPI activates the NF κB pathway via the TLR4/MyD88/IκBα axis in hepatocellular carcinoma[END_REF]. NFkB is a well described transcription factor of PLK1, leading to increase its transcription. PLK1 was identified as a gammaBD kinase, which negatively regulates IKK activation[START_REF] Higashimoto | Regulation of I(kappa)B kinase complex by phosphorylation of (gamma)-binding domain of I(kappa)B kinase (beta) by Polo-like kinase 1[END_REF] and could explain how HBV shut down innate immune response quickly after infection.

  found to co-immunoprecipitates with HBc in HepaD38 cells. All these observations and bibliographic data led us to hypothesis that PLK1 may play a role in HBV life cycle by regulating the encapsidation/reverse transcription of the pgRNA via a phosphorylation process of the HBV core protein. We confirmed our hypothesis and have mapped PLK1 phosphorylation sites on HBc C-ter domain to be S168, S176, and S178 (reported in the first study) but requiring prior phosphorylation at positions S155, S162 and S170, sites shown to be phosphorylated by CDK2(Ludgate et al., 2012)(Figure38).

Figure 38 :

 38 Figure38: HBc CTD primary sequence, highlithing important sites of phosphorylation. In red are the three SP sites described by Ludgate and colleagues to be phosphorylated by CDK2. They represent a first wave of phosphorylation that is required for PLK1 to interact and further phosphorylate the CTD of HBc (in blue). Extracted from the review I have written on the "The diverse functions of the hepatitis B core/capsid protein (HBc) in the viral life cycle: Implications for the development of HBc-targeting antivirals". Please confer to Appendix review(Diab et al., 2018) for precisions.

Figure 39 :

 39 Figure 39: Schematic representation of the lipid nanoparticles technology. The large spherical structure can be coated with PEG and different molecules (A), depending on the cell type to target, and contains thousands of nanoparticles. Inside each nanoparticle, a nucleic acid can be incorporated, such as siRNA (E). This technology allows efficient delivery of the coumpond with limited side effects. Extracted from Arbutus Biopharma portofolio.

Figure 40 :

 40 Figure 40: Hypotetic model of PLK1-inhbition release under HBV infection. In non-infected hepatocytes (left), HNF4α acts as a repressor of PLK1 transcription, thereby promoting the quiescence of the hepatocytes and the differentiation[START_REF] Walesky | Hepatocyte Nuclear Factor 4 alpha Deletion Promotes Diethylnitrosamine-induced Hepatocellular Carcinoma in Mice[END_REF]. Under HBV infection (right), HNF4α is found to bind the cccDNA where it promotes the viral transcription[START_REF] Palumbo | IL6 Inhibits HBV Transcription by Targeting the Epigenetic Control of the Nuclear cccDNA Minichromosome[END_REF]. The dashed black line represents the relocalization of HNF4α from genomic DNA to the cccDNA, thus, releasing the inhibition of PLK1. In turn, PLK1 act as a proviral factor either by increasing the cccDNA activity or by promoting stability/trafficking/maturation of the viral RNAs.

Figure 41 :

 41 Figure 41: Two models presenting how PLK1 could be involved in HBV-related carcinogenesis. Top:Upon HBV infection, PLK1 is overexpress and stay at an elevated level in a long-lasting way. This alters the hepatocytes and microenvironment landscape by promoting proliferation, DNA damages, chromosomic instabilities … and eventually along with the occurrence of satellite events, lead to cellular transformation. In this model PLK1 acts as a key driver of carcinogenesis. Bottom: Initially the situation remain unchanged, PLK1 is overexpressed due to HBV infection. However, once the infection is stably establish, the PLK1 level returns to a basal state untill a driver carcinogenic event occurs such as p53 mutation, wich lead in an indirect manner to PLK1 overexpression. In this situation PLK1 overexpression plays a role of satellite event towards transformation process and is just the results of high-proliferative rate of uncontrolled cells. In both cases it is of importance to note that with our actual technology, we can pretend in best cases to detect tumour approximately 10 years after the emergence of the first transformed cell.

Fig. 1 .

 1 Fig. 1. Stages of the HBV life cycle regulated by HBc. 1) HBc regulates transport and nuclear release of the viral genome(Blondot et al., 2016). 2) HBc associates with cccDNA(Guo et al., 2011). 3) HBc modulates host gene expression[START_REF] Xie | Multi-omics analyses reveal metabolic alterations regulated by hepatitis B virus core protein in hepatocellular carcinoma cells[END_REF]. 4) HBc is required for pgRNA encapsidation(Nassal, 1992). 5) HBc is required for reverse transcription(Lewellyn and

Fig. 2 .

 2 Fig. 2. Arginine-rich motifs of di erent viral proteins exhibit an abundance of arginine residues within short 10-20 residues. The arginine-rich domain of HBc has distinct nuclear localization (NLS) and cytoplasmic retention signals (CRS)(Li et al., 2010).

Fig. 3 .

 3 Fig. 3. HBc C-terminal domain (CTD) is a phosphorylation sub-strate for CDK2 (SP sites at positions 155, 162 and 170) and PLK1 (phosphorylation sites at positions 168, 176 and 178), as shown by Ludgate et al. (2012) and Diab et al. (2017), respectively.

Fig. 4 .

 4 Fig. 4. Top view of an HBc hexamer in complex with CAM sulfamoylbenzamide SBA_R01, PDB: 5T2P (Left) and CAM heteroaryldihydropyrimidine HAP_R01 PDB: 5WRE (Zhou et al., 2017) (Right).

  Martin Berke, b Karen Vergauwen, b Adrien Foca, a Koen Vandyck, b Frederik Pauwels, b Fabien Zoulim, a,c David Durantel a a INSERM, U1052, Cancer Research Center of Lyon, Université de Lyon (UCBL1), CNRS UMR_5286, Centre Léon Bérard, Lyon, France b Janssen Research and Development, Beerse, Belgium c Liver Unit, Hospices Civils de Lyon, Lyon, France

FIG 1

 1 FIG 1 Effect of JNJ-827 and JNJ-890 on capsid assembly in tubo and in cellulo. (A) Structures of JNJ-827 and JNJ-890. (B) EM images of in tubo capsid formation obtained using the indicated amounts of molecules as described in Materials and Methods. (C) Graphs of SEC analyses performed with the same amounts of compounds as in panel B. Compound 1 (Cp 1) and Cp 2 correspond to inactive controls of JNJ-827 and JNJ-890. (D) Differentiated HepaRG were infected with HBV (500 virus genome equivalent [vge]/ml) and then treated from day 7 postinfection for 7 days every 2 or 3 days (three treatments) with 10 μM concentrations of the indicated compounds. Noninfected (NI) and infected not treated (NT) controls were also added. Protein lysates were loaded and run in native conditions on an agarose gel, the proteins were transferred onto an enhanced chemiluminescence membrane, and HBc was detected by chemiluminescent immunoblotting with an anti-HBc antibody. The blot is representative of three experiments. (E) Quantification (means plus the standard errors of the mean [SEM]; n = 3) of the capsid signal by chemiluminescence determined using a ChemiDoc XRS+ System (Bio-Rad).

FIG 2

 2 FIG 2 Antiviral properties of JNJ-827 and JNJ-890 in PHHs. PHHs were mock infected (NI condition) or infected with HBV (200 vge/ml), cultivated during 5 days postinfection, and then not treated (NT condition) or treated three times for 7 days (treatment every 2 or 3 days) with 10 μM concentrations of the indicated compound. (A) Intracellular HBV total DNA, cccDNA, total RNA, and PreC/pgRNA levels were quantified by qPCR, qPCR FRET (fluorescence resonance energy transfer), and quantitative reverse transcription-PCR (RT-qPCR), respectively. (B) The HBeAg, HBsAg, and ApoB levels in cell culture supernatant were monitored by ELISA. Extracellular HBV DNA and RNA levels were quantified by qPCR or RT-qPCR after nucleic acid extraction from supernatant. The results are means plus the SEM (n = 3) performed with three different PHH donors. Statistics were determined using a two-tailed Mann-Whitney test, with the following calculated probabilities: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; and nd, not determined.

FIG 3

 3 FIG 3 Effect of JNJ-827 and JNJ-890 on HBV infection establishment in dHepaRG cells and PHHs. PHH (A and B) or dHepaRG (C and D) cells were pretreated for 24 h with 10 μM concentrations of the indicated CAMs or 100 nM preS1 peptide entry inhibitor and then inoculated with HBV (200 vge/ml) in the presence of drug for 24 h. One day after infection, the medium was changed, and the cells were cultured for 1 week. For the TDF control, cells were treated twice after infection. A noninfected (NI) control was included. (A and C) Intracellular HBV total DNA, cccDNA, and total RNA levels were quantified by qPCR, qPCR FRET, and RT-qPCR, respectively. (B and D) HBeAg, HBsAg, and ApoB levels in cell culture supernatant were monitored by ELISA. The results are means plus the SEM (n = 3) performed with three different PHH donors or three different differentiations of HepaRG. Statistics were determined using a two-tailed Mann-Whitney test, with the following calculated probabilities: *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001.

FIG 4

 4 FIG 4 Antiviral properties of JNJ-827 and JNJ-890 in dHepaRG cells: short-term versus long-term treatment. dHepaRG were infected with HBV (200 vge/ml), cultured for 1 week, and then treated for 7 days (treatment each 2 or 3 days; 3 treatments in total) (A and B) or 1 month (treatment each 2 or 3 days; 15 treatments in total) (C and D) with a 10 μM concentration of the indicated compound. The antiviral effects of the compounds were monitored at endpoint. (A and C) Intracellular HBV total DNA, total RNA, and PreC/pgRNA levels were quantified by qPCR and RT-qPCR. (B and D) HBeAg, HBsAg, and ApoB levels in cell culture supernatant were monitored by ELISA. Extracellular HBV DNA and RNA levels were quantified by qPCR or RT-qPCR after nucleic acid extraction from the supernatant. The results are means plus the SEM (n = 3) performed with three different differentiations of HepaRG. Statistics were determined using a two-tailed Mann-Whitney test, with the following calculated probabilities: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; and nd, not determined.

FIG 5

 5 FIG 5 Effect of JNJ-827 and JNJ-890 on HBeAg biogenesis and/or secretion. HepaRG-TR-HBe cells were treated with tetracycline (1 μg/ml) for 48 h to allow expression of HBeAg from tetracycline-regulated promoter and then treated for 5 days (two treatments) with 10 μM concentrations of the indicated compounds. (A) The HBeAg secretion level in cell culture supernatant was monitored by ELISA. (B) Intracellular HBeAg amount was also monitored by ELISA. For panels A and B, the results are means plus the SEM (n = 3), and the statistics were determined using a two-tailed Mann-Whitney test (**, P < 0.01). (Ci) Evaluation of intracellular and extracellular HBeAg levels by immunoblotting (results from a representative experiment [n = 2]). (Cii and iii) Quantification of extracellular and intracellular HBeAg signal by chemiluminescence done with a ChemiDoc XRS+ System (Bio-Rad). The results are means plus the SEM (n = 2).

4 .

 4 Antiviral properties of JNJ-827 and JNJ-890 in primary human 26 hepatocytes. PHH were mock (NI condition) or infected with HBV (200 vge/mL), cultivated 27 during 5 days post infection, then non-treated (NT condition) or treated three times for 7 28 days (treatment every 2/3 days) with 10 μM of indicated compound. (A, D, G) CccDNA, was 29 quantified by qPCR. (B, E, H) HBeAg, HBsAg and ApoB levels in cell culture supernatant were 30 monitored by ELISA. (C, F, I) Extracellular HBV DNA and RNA levels were quantified by qPCR 31 or RT-qPCR after nucleic acid extraction from supernatant. Results obtained with 3 different 32 PHH donors/batches are represented independently. Results are the mean + SD of technical 33 replicates (n=2 or 3).

34 Supplementary figure 5 . 7 .Supplementary figure 8 .

 34578 Determination of JNJ-827 EC50 on cccDNA establishment. PHH were pre-treated for 24h with increasing amount of JNJ-827 ranging from 5 nM to 10 μM then inoculated with HBV (200 vge/mL) in presence of drug for 24h. One day after infection, medium was changed and cells were cultured for one week. Intracellular cccDNA and HBV total DNA levels were quantified by qPCR FRET and qPCR respectively. HBeAg and HBsAg Effect of JNJ-827 and JNJ-890 on HBV infection establishment in 57 dHepaRG. Differentiated HepaRG cells were pre-treated for 24h with 10 μM of indicated 58 CAMs or 100 nM of preS1 peptide entry inhibitor, then inoculated with HBV (200 vge/mL) in 59 presence of drug for 24h. One day after infection, medium was changed and cells were week. For the TDF control, cells were treated twice after infection. A noninfected (NI) control was included. (A, C, E) CccDNA, was quantified by qPCR. (B, D, F) HBeAg, HBsAg and ApoB levels in cell culture supernatant were monitored by ELISA. Results obtained with 3 different HepaRG differentiations are represented independently. Results are the mean + SD of technical replicates (n=2 or 3). Antiviral properties of JNJ-827 and JNJ-890 in dHepaRG: short versus long-term treatment; Part 1: short-term results. dHepaRG were infected with HBV (200 vge/mL), cultured during one week then treated for 7 days (treatment each 2/3 days; 3 treatments in total) with 10 μM of indicated compound. The antiviral effects of the compounds were monitored at end point. (A, C, E) HBeAg, HBsAg and ApoB levels in cell culture supernatant were monitored by ELISA. (B, D, F) Extracellular HBV DNA and RNA levels were quantified by qPCR or RT-qPCR after nucleic acid extraction from supernatant. Results obtained with 3 different HepaRG differentiations are represented independently.Results are the mean + SD of technical replicates (n=2 or 3).

9 .

 9 Antiviral properties of JNJ-827 and JNJ-890 in dHepaRG: short 84 versus long-term treatment; Part 2: long-term results. dHepaRG were infected with HBV 85 (200 vge/mL), cultured during one week then treated for one month (treatment each 2/3 86 days; 15 treatments in total) with 10 μM of indicated compound. The antiviral effects of the 87 compounds were monitored at end point. (A, C, E) HBeAg, HBsAg and ApoB levels in cell 88 culture supernatant were monitored by ELISA. (B, D, F) Extracellular HBV DNA and RNA 89 levels were quantified by qPCR or RT-qPCR after nucleic acid extraction from supernatant. 90 Results obtained with 3 different HepaRG differentiations are represented independently. 91 Results are the mean + SD of technical replicates (n=2 or 3individuals are chronically infected with hepatotropic viruses (WHO),

Figure 1 .

 1 Figure 1. HBc protein is the viral determinant involved in the early inhibition of dsRNA-mediated IFN responses in hepatocytes. (A) PHH or (B) differentiated HepaRG were exposed/infected with 1000 vge/cell of wild type or UV-inactivated HBV for the indicated time, then stimulated with poly(I:C)-LMW (10 μg/mL) for 4h. After total RNA purification, the expression of innate immunity genes (IFN-/3, IL-29, OAS-1 and IL-6) was analysed by RT-qPCR and compared to the mock control normalized to 100%. Proliferative HepaRG cells were either mock-or lipo-transfected for 4 hours with (C) low or (D) high amounts of complete HBV inoculum, nucleocapsids or recombinant HBcAg (100 or 1000 vge/cell and HBcAg at 50 or 500 pg /cell) prior to poly(I:C) stimulation (4h). Expression of indicated genes was analysed by RT-qPCR and compared to the mock control normalized to 100%. (E) dHepaRG expressing in a tetracycline dependent manner (Tet at 10 μg/mL; 48h of expression of the corresponding protein) either HBc , HBs, HBx, or HBe were stimulated with poly(I:C) for 4h. Then IFN-/3 gene expression as well as IL-6 or IP-10 secretion were monitored by RT-qPCR or ELISA. (F) Differentiated HepaRG-TR-HBc and HepaRG-TR-T7 cells were treated or not for 24h with tetracycline (Tet, 10 μg/mL) prior to stimulation with poly (I:C) for 4h and gene expression analysed by RT-qPCR and compared to the non-tettreated cells normalized to 100%. (G) HepaRG-TR-HBc cells were transfected with the pIFN-l3p-Luc plasmid, induced or not with tetracycline for 48h, then stimulated with poly(I:C) for 4h. Luciferase activity was analysed on cell lysate with a Luminoskan Ascent. All results are represented as the mean ± SEM of at least 3 independent experiments.

Figure 3 :

 3 Figure 3: HBc protein interacts rapidly with innate gene promoters to inhibit dsRNA-mediated IFN response in hepatocytes. (A) Differentiated HepaRG-TR-HBc and HepaRG-TR-T7 cells were treated for 4h, 24h and 48h with tetracycline (10 μg/mL), or (B) dHepaRG-TR-HBc cells were treated or not for 72h with Bayer41-4109 (1 μM) or AT130 (3 μM) prior to be stimulated with tetracycline (10 μg/mL) for 24h, and ChIP-qPCR was performed with homemade anti-HBc (or isotopic antibodies) and specific primers for IFN-6, IL-29, OAS-1 and IL-6 promoters. The percentage of input enrichment was normalized on the nontetracycline stimulated HepaRG-TR-HBc cells and on HKG promoter. (C) PHH were infected with a complete HBV inoculum for 2h, 4h 8h, or 12 days and ChIP-qPCR performed as in (A and B). (D) PHH were infected with a complete HBV inoculum for 8h in the presence or absence or nocodazole, and ChIP-qPCR was performed as in (A and B). All results are represented as the mean ± SEM of 3 independent experiments.

Figure 4 :

 4 Figure 4: HBc affects host gene epigenetic status to inhibit IFN response in hepatocytes. (A) Differentiated HepaRG-TR-HBc cells were treated for 4h, 24h, or 48h with tetracycline (10 μg/mL), or (B) PHH were infected by 100 vge/cell of HBV for 24h, then histone modifications, recruitment of the histone methyl-transferase EZH2, and binding of HBc to IFN-6, IL-29, OAS-1, and IL-6 promoters were assessed by ChIP-qPCR, using either none, control, anti-H3K27Ac, anti-H3K27me3, anti-EZH2, or anti-HBc antibodies. The percentage of input enrichment was normalized either to the non-tetracycline stimulated HepaRG cell lines (A) or to mock infected PHH (B) and on HKG promoter. All results are represented as the mean ± SEM of 3 independent experiments.

Suppl. Fig. 6 .

 6 Innate immune gene expression and ChIP analysis with mouse liver samples. Expression of indicated innate immune genes and level of HBV RNA in high (A) and low (B) viremia mice as measured by RT-qPCR. Results of ChIP-qPCR experiments done with anti-HBc and primers specific to indicated promoters in high (C) and low (D) viremia mice.

Figure 6 :

 6 Figure 6: Long-term inhibition of hepatocyte innate response by HBV core protein.Persistently HBVinfected liver-humanized FRG mice were sacrificed 8 weeks post-infection and livers store at -80°C after snap freezing in liquid nitrogen. ChIP-qPCR was performed using none, control, antiH3K4me3, anti-H3K27Ac, anti-H3K27me3, anti-EZH2, or anti-HBc antibodies and specific primers for IFN-6, IL-29, OAS-1, IL-6, GAPDH and DHFR promoters. The percentage of input enrichment was normalized to the non-infected mice. All results are represented as the mean ± SEM of 3 independent experiments performed with 3 independent extraction of the same liver samples.

Figure 7 :

 7 Figure 7: HBc inhibitor treatment potentiates the anti-HBV effect of TLR3-L.Differentiated HepaRG cells were infected with HBV (100 vge/cell) and left untreated for 7 days. Infected cells were treated twice (at days 7 and 10 p.i.) with various concentration of an HBc inhibitor (BAY41-4109) as indicated, then co-treated (day 13) for 72 h with various concentrations of BAY41-4109 and poly (I:C). Secretion of HBeAg was monitored in the supernatant of cells at the end of treatment (day 16) (A), and intracellular HBV DNA accumulation by qPCR (B). All results are represented as the mean ± SEM of 3 independent experiments. On panels (Ci and Cii),

  Antibodies and others reagents. Most of chemicals and control antibodies were purchased from Sigma Aldrich. Poly (I:C) low-molecular weight (LMW), poly (I:C) (LMW)/Lyovec, and other ligands were from Invivogen. Antibodies for ChIP experiments were purchased from Diagenode (anti-H3K27me3 pAb-069-050, anti-H3K4me3 pAb-003-050, anti-H3K27Ac pAb-

  Cells were lysed in TTB buffer (10 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.5% Triton X-100, pH 7.5) for total proteins extraction or in NE-PER Nuclear and

2 .

 2 Loss of PRC2function in a subpopulation of HBV replicating cells de-represses expression of a hepatic cancer stem cell (hCSC)-like gene signature (EpCAM, BAMBI, CD44, CD133, Oct4, Nanog, and Sox2)3 . Furthermore, DDX5 which facilitates HOTAIR/PRC2-mediated transcriptional repression of these genes, is downregulated during HBV infection. Significantly, re-expression of these hCSC genes and downregulation of DDX5 are linked to poor prognosis HBV-associated liver cancer.Our ongoing studies have generated data which show: 1) DDX5 is a roadblock to hCSC formation. Specifically, downregulation of DDX5 allows hepatosphere formation, expression of Wnt-induced pluripotency genes, decreased sensitivity to cisplatin and Sorafenib, and abrogation of anti-tumor immunity. 2) Downregulation of DDX5 during HBV infection occurs via induction of oncomirs miR17~92 and miR106b~25. Interestingly, downregulation of DDX5 enhances virus biosynthesis via an unknown mechanism. 3) Inhibitors of miR17~92 and miR106b~25 reverse loss of DDX5 and other known tumor suppressor targets (PTEN, SMAD7, LKB1), and preliminary studies show these miR inhibitors (anti-miRs) reduce HBV replication. These observations suggest development of these anti-miRs as anti-viral and antitumor molecular tools.

Table I :

 I List of HBV viral mRNAs transcripted from cccDNA.…………………………………………………………....35

Table II :

 II List of the eight HBV proteins.………………………………………………………………………………………......35

Table III :

 III Incidence of drug resistance during antiviral therapy.……………………………………………………....52

Table IV :

 IV List of targeted therapies evaluated in phase 3 trials in HCC.…………………………………………....73

Table V :

 V Comparison of PLK1 gene expression between cancers and normal tissue.………………………..90

Table VI :

 VI Several PLK1 inhibitors being clinically trialed or in early development for anti-cancer therapies.………………………………………………………………………………………………………………………………...........99

Chapter 1: Hepatitis B Virus 1.1. HBV discovery: from Hippocrate to Blumberg. A story where jaundice and leukemia act as protagonists

Table III : Incidence of drug resistance during antiviral therapy.

 III Data are presented in %. No evidence of resistance has been shown after 8 years of TDF treatment (long term treatment with TDF for chronic hepatitis B infection is safe and well tolerated and associated with durable virologic response with no detectable resistance: 8 year results from two phase 3 trials).

	Drug	Year 1	Year 2	Year 3	Year 4	Year 5
	Lamivudine	24	38	49	67	70
	Adefovir	0	3	11	18	29
	Entecavir	0.2	0.5	1.2	1.2	1.2
	Entecavir (LAM resistant)	6	15	36	46	51
	Telbivudine	4	17	-	-	-

Table IV :

 IV

List of targeted therapies evaluated in phase 3 trials in HCC. Cabozantinib is not presented in this table as the first result came from a clinical trial published in July 2018. Extracted from

Forner A et al., The Lancet, 2018.

Table V : Comparison of PLK1 gene expression between cancers and normal tissue. Mean

 V

	Cholangiocarcinoma	24.3
	Uterine corpus endometrial carcinoma	21.3
	Lung squamous cell carcinoma	20.8
	Glioblastoma	12.4
	Hepatocellular carcinoma	11.7
	Breast invasive carcinoma	11.3
	Esophageal carcinoma	10.2

PLK1 expression in cancers/mean PLK1 expression in normal tissue. Adapted from

Liu et al., Translational Oncology, 2016. 3.3.7.1.

PLK1 interconnects with several cancer-associated pathways 3.3.7.1.1. PLK1 inhibits tumour suppressors

  INSERM U1052, Cancer Research Center of Lyon, Lyon, France; 2 University of Lyon, Universit' e Claude-Bernard, UMR_S1052, UCBL, Lyon, France; 3 Department of Basic Medical Sciences and Purdue Center for Cancer Research, Purdue University, West Lafayette, IN; 4 CIRI-International Center for Infectiology Research, Team EVIR, INSERM, U1111, Universit' e Claude Bernard Lyon 1, CNRS, UMR5308, Ecole Normale Sup' erieure de Lyon, Univirsity of Lyon, Lyon, France; 5 Hepato-Gastroenterogy Unit, Croix-Rousse Hospital, Hospices Civils de Lyon, Lyon, France; 6 Labex DEVweCAN, Lyon, France.

0×10 05 1.0×10 06 1.0×10 07 1.0×10 08 Viremia in mice

			W4
	Mock-treated	IFN- (25 g/kg/tw)	ETV (2,5 g/day)

W8

Viral genome copy/mL M HEPATOLOGY, VOL. 66,

NO. 6, 2017

Table 1

 1 Kinases that phosphorylate HBc.

	Thesis Adrien FOCA

TABLE 1

 1  The anti-HBV activity of AT130, JNJ-827, BAY41, and JNJ-890 in stable HBV-replicating cells was tested in a dose-response assay. Intracellular (HepG2.117 cells) and extracellular (HepG2.2.15 cells) HBV DNA was extracted, and DNA levels were assessed using qPCR. In each experiment, the EC50s were determined based on the mean inhibition from two wells per compound concentration. The cytotoxicity for HepG2 cells was assessed using a resazurin readout. EC50, 50% effective concentration; CC50, 50% cytotoxic concentration; Intra, intracellular; Extra, extracellular; n, number of experiments; ND, not done. The half-maximal effective concentration (EC 50 ) was determined in HepG2.117 cells (intracellular EC 50 [iEC 50 ]) and in HepG2.2.15 cells (extracellular EC 50 [eEC 50 ]) after quantification of total intracellular or extracellular HBV DNA by quantitative PCR (qPCR). The mean iEC 50 values for JNJ-827, JNJ-890, BAY41, and AT130 were 4.7, 66, 67, and 1,020 nM, respectively, whereas the eEC 50 values were 5.7, <391, 101, and 127 nM, respectively (Table

	AT130	Intra	1,020 (120-4,620), n = 24	ND	>50,000, n = 11	ND	>49
		Extra	127 (<98-207), n = 4			ND	>393
	JNJ-827	Intra Extra	4.7 (2.1-11), n = 55 5.7 (<3.9-9.8), n = 11	>25,000 (>5,000->25,000), n = 3	>25,000, n = 11	>5,319 >4,386	>5,319 >4,386
	BAY41	Intra	67 (21-216), n = 467	33,900 (21,400->50,000),	35,400 (10,600->120,000),	506	528
		Extra	101 (25-216), n = 99	n = 3	n = 56	336	350
	JNJ-890	Intra Extra	66 (63-70), n = 2 <39, n = 2	ND	>100,000, n = 4	ND ND	>1,515 >256
		Anti-HBV activity and cytotoxicity of JNJ-827 and JNJ-890.

a

  and an engineered HepaRG-TR-HBe cell line expressing only HBeAg in a tetracyclinedependent manner (obtained by double transduction with lentiviral vectors [from Invitrogen; T-Rex System] containing tetracycline repressor and HBe expression transgenes [a detailed method is available on request]) were cultured as previously described[START_REF] Devwecan | [END_REF]. Primary human hepatocytes (PHHs) were isolated from surgical liver resections, after informed consent of patients (kindly provided by M. Rivoire[CLB, Lyon]; agreements DC-2008-99 and DC-2008-101) as previously described (62) and cultured in complete William's medium supplemented with 1.8% of DMSO. Differentiated HepaRG (dHepaRG) cells and PHHs were infected as previously described (63), with HBV genotype D inoculum prepared either from HepG2.2.15 or HepAD38 cells. The multiplicities of infection (expressed as virus genome equivalent/ cell) are indicated in the figure legends.

Antiviral treatment of PHHs and dHepaRG cells. JNJ-827, JNJ-890, and TDF (used at 10 μM), as well as PreS1

  The amounts of HBeAg and HBsAg in the supernatant and sera were quantified by commercially available ELISA (Autobio, Ltd., China) according to the manufacturer's instructions. An intracellular HBeAg ELISA was performed after cell lysis in a buffer containing 400 mM potassium acetate, 25 mM KHEPES (pH 7.2), 15 mM magnesium acetate, 1% (vol/vol) NP-40, and 0.5% (wt/vol) deoxycholate supplemented with 1 mM phenylmethylsulfonyl fluoride and 1X protease inhibitor cocktail. anti-rabbit antibody (Invitrogen) for PML and 20S subunit detections, both diluted at 1:1,000 in 1X PBS. Counterstaining for the detection of nucleic acid was performed using Hoechst 33258 (Life Technologies) diluted at 1:5,000 in the Alexa Fluor mix. Cells were washed four times with wash buffer, and coverslips were mounted using mounting medium (Dako). Images were obtained with a confocal microscope Leica TSC-SP5X. Statistical analysis. Statistical analyses were conducted using nonparametric Mann-Whitney tests using GraphPad Prism software 6.0. Significance is denoted in the figures as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001.

	Native agarose gel migration assay and immunoblotting. The intracellular formation/accumula-tion of HBV
	nucleocapsid in infected dHepaRG cells was performed from cells lysed in a buffer containing 50 mM Tris-HCl
	(pH 8), 1 mM EDTA, and 1% (wt/vol) NP-40. Cell lysates were centrifuged for 1 min at 10,000 rpm and 4°C.
	The supernatants were recovered and incubated at least2h at 37°C with 10 μg/ml of a DNase I/RNase A mix
	in buffer containing 20 mM Mg(CH3COO)2. Lysates were loaded into a native 1.2% agarose gel, and
	electrophoresis was performed in 1X Tris-HCl acetate EDTA (TAE) buffer. Capillary transfer of proteins to ECL
	membrane was performed in 1X Tris-HCl/NaCl/EDTA (TNE) buffer. Immuno-blotting was conducted
	according to a standard protocol with an antibody against HBcAg (Dako, catalog no. B0586) at 1:1,000.
	For the detection of HBeAg in cell supernatants, supernatants were 10X concentrated by trichloro-acetic
	acid (Sigma) precipitation and subjected to immunoblotting with Dako B0586 antibody at 1:1,000. An antibody

directed against 13-actin (Sigma-Aldrich, catalog no. A1978) diluted at 1:5,000 was used for the normalization of results. Immunofluorescence staining. dHepaRG cells were fixed with 2% formaldehyde in 1X phosphate-buffered saline (PBS) for 20 min at room temperature and permeabilized/blocked with 0.1% (vol/vol) Triton X-100 (Sigma), 1% (wt/vol) bovine serum albumin (BSA; Euromedex), and 10% (vol/vol) fetal bovine serum (Thermo Scientific) in 1X PBS at room temperature for 30 min. Primary antibodies against HBcAg (Ab8637; used at 1:500), which only recognizes assembled capsid, promyelocytic leukemia protein (PML; Abcam, catalog no. ab31101; used at 1:200), and the proteasomal 20S subunit (Pierce, catalog no. PA1-977; used at 1:200) were diluted in 1X PBS and incubated 1 h at room temperature. Cells were washed four times with wash buffer (1X PBS with 0.1% Triton X-100) and subsequently incubated at room temperature for 1 h with either Alexa Fluor 488-conjugated goat anti-mouse antibody (Invitrogen) for HBcAg detection or Alexa Fluor 594-conjugated goat

MicroRNA inhibitors rescuing RNA helicase DDX5 as HBV antiviral and anti-HCC tools

  Saravana KK Mani*, Jiazeng Sun*, Zhibin Cui*, Adrien Foca#, David Durantel#, Yen-Hsing Li**, Philip S Low** and Ourania Andrisani*. *Basic Medical Sciences and **Chemistry Departments, Purdue Center for Cancer Research Purdue University, West Lafayette, IN 47907 and #Cancer Research Center of Lyon (CRCL) UMR Inserm 1052 -CNRS 5286, Lyon Cedex 03 Chronic hepatitis B virus (HBV) infection is linked to liver cancer pathogenesis. Despite the HBV vaccine, 250 million people globally are chronically infected with HBV. Current treatments are ineffective. New approaches are needed targeting mechanisms that regulate both virus biosynthesis and liver cancer pathogenesis.

Gadot (Plateforme Anatomopathologie Recherche, Centre L' eon B' erard, Lyon, France) for the IHC analyses.

Michael M.C. Lai, M.D., Ph.D.[1][2][3] Wen-Chi Su, Ph.D.1,2

Thesis Adrien FOCA

Acknowledgment:

We thank Lydie Lefranc¸ois, Judith Fresquet, and Maud Michelet for isolation of PHHs, as well as the staff from Michel Rivoire's surgery room for providing us with liver resection. We also thank Jean-Franc¸ois Henry, Nadine Aguilera, and Jean-Louis Thoumas from the animal facility (Plateau de Biologie Experimental de la Souris, ENS de Lyon), as well as Gise `le Froment and B' en' edicte Eckel for their technical help in handling mice. We thank the Vectorology platform (INSERM U1089, Nantes) for the production of the adeno-uPA vector and Nicolas

ACKNOWLEDGEMENTS

The authors thank Maud Michelet, Océane Floriot, Jenifer Molle, Laura Dimier, Anaëlle Dubois and Judith Fresquet for PHHs isolation as well as the staff from Michel Rivoire's surgery room for providing us with fresh liver resections.

Acknowledgments

Supported by NIH grant DK044533-19 to OA, the Chateaubriand Fellowship from the French Embassy in Washington DC to AD, and grants from ANRS (French national agency for research on AIDS and viral hepatitis; several grants from CSS4) and INSERM core grants to FZ and DD. We also acknowledge funding by the DEVweCAN LABEX (ANR-10-LABX-0061) of the "Université de Lyon", within the program "Investissements d'Avenir" (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR) to FZ and DD. The authors thank Dr. RL Hullinger for his critical review and editing of this manuscript.

ACKNOWLEDGMENTS

We thank Maud Michelet and Jennifer Molle for help with the isolation of primary human hepatocytes and the staff from Michel Rivoire's surgery room for providing assistance with liver resection. This study was supported by research grants from Janssen, ANRS (French National Agency for Research on AIDS and Viral Hepatitis; grants from CSS4), FINOVI (Foundation for Innovation in Infectiology), and INSERM. This study was also indirectly supported by the DEVweCAN LABEX (ANR-10-LABX-0061) of the Université de Lyon, within the program Investissements d'Avenir (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). This study was mainly sponsored by Janssen. INSERM U1052 also received institutional funding from INSERM, as well as external grants from ANRS, and DEVweCAN LABEX (ANR-10-LABX-0061) of the Université de Lyon, within the program Investissements d'Avenir (ANR-11-IDEX-0007) operated by the ANR. T.L., A.F., F.Z., and D.D. have nothing to declare. J.M.B., K.V., K.V., and F.P. are employees of Janssen.

Acknowledgments

The authors would like to thank Lydie Lefrançois and Judith Fresquet for the isolation of primary human hepatocytes, as well as the staff from Pr Michel Rivoire's surgery room for providing us with liver resection. This work was supported by grants from ANRS (French national agency for research on AIDS and viral hepatitis; several grants from CSS4), FINOVI (Foundation for innovation in infectiology; project call n°#4), FRM (Foundation for medical research; DEQ20110421327), and Hoffmann-La-Roche (U.S.A. division) and by INSERM core grants. This work was also supported by the DEVweCAN LABEX (ANR-10-LABX-0061) of the "Université de Lyon", within the program "lnvestissements d'Avenir" (ANR-11-IDEX-0007) operated by the French National Research Agency (ANR). REMERCIEMENTS Je tenais tout d'abord à remercier les membres de mon jury d'avoir accepté de lire mon manuscrit et d'avoir apporté leur critique constructive à mes travaux de thèse. Merci aux Dr. Kremsdorf, Dr. Neuveut et Dr. Lupberger. Merci au Pr. Baumert. Merci à vous, Pr. Bernet, qui m'avez donné le goût de la recherche et avez accepté la présidence de mon jury. Merci au Pr. Zoulim de m'avoir acceuilli au sein de son laboratoire.

Merci à toi David, qui a toujours su être là malgré un emploi du temps plus que chargé ! Merci pour les discussions scientifiques, les digressions, les parenthèses de parenthèses (de parenthèses …) et les parties de Bad' endiablées ! Merci à tous les membres du laboratoire avec qui j'ai partagé ces dernières années.

List of Abbreviations aa amino acid

Author names in bold designate shared co-first authorship.

36) Pickar A, Zengel J, Xu P, Li Z, He B. Mumps virus nucleopro-tein enhances phosphorylation of the phosphoprotein by polo-like kinase 1. J Virol 2016;90:1588-1598. 37) Sun D, Luthra P, Li Z, He B. PLK1 down-regulates parainflu-enza virus 5 gene expression. PLoS Pathog 2009;5:e1000525. 38) Liu X. Targeting polo-like kinases: a promising therapeutic approach for cancer treatment. Transl Oncol 2015;8:185-195.

Supporting Information

Polo-like-kinase 1 is a proviral host-factor for hepatitis B virus replication ---------------------------------------------------------------------------------------------------------------------------Supporting Figure 2. Antiviral activity of LNP siPLK1 in non-transformed and non-dividing dHepaRG cells. The data were generated the same way as in Figure 2.

Supplementary figures and figure fegends

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Supplementary figures and figure legends

Novel potent capsid assembly modulators regulate multiple steps of the Hepatitis B virus life-cycle

Summary

Hepatitis B virus (HBV), as many other viruses, has evolved different strategies to evade immune responses to establish and maintain viral persistence. In particular, HBV does not seem to induce any measurable IFN and pro-inflammatory responses during acute infection. The absence of innate immune response could be due to an impaired detection of HBV by the host innate sensors or could result from an active evasion via the prompt establishment of inhibitory mechanisms.

In a previous work, we showed that HBV virion could trigger a very early inhibition of the hepatocyte-mediated IFN response immediately after infection. Here, we demonstrate that HBV core protein (HBc) is the main viral component involved in this inhibitory process.

After HBV entry and migration of nucleocapsids to the nuclear pore, HBc enters the nucleoplasm, where, after binding to target promoters, it impairs the activation of specific innate immunity gene expression upon dsRNA sensors engagement. The recruitment of EZH2 histone methyltransferase is responsible for the establishment of repressive epigenetic histone marks (H3K27) at specific ISG-related HBc-bound-promoters, affecting IFN gene response immediately after the onset of infection. This inhibitory mechanism is maintained during persistent infection thanks to the recycling of nucleocapsids to the nucleus.

Interestingly, we also found that this inhibition is at work in non-parenchymal cells (Kupffer and liver endothelial sinusoidal cells) into which HBV does penetrate and deliver HBc to the nucleus compartment.

HBV has evolved a strategy to impair IFN response immediately after the onset of infection by using the incoming virion structural protein HBc to regulate gene expression by epigenetic mean.

tune cellular innate response and to finally enable the orchestration of subsequent adaptive response. In turn, viruses, in particular those that progress to chronicity, have evolved many strategies to evade this IFN response to their own benefit [START_REF] Bowie | Viral evasion and subversion of pattern-recognition receptor signalling[END_REF][START_REF] Heim | Innate immunity and HCV[END_REF][START_REF] Manel | Hiding in plain sight: how HIV evades innate immune responses[END_REF][START_REF] Randall | Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures[END_REF].

In this respect, HBV is a peculiar virus, as, in contrast to HIV or HCV, it does not seem to induce a measurable systemic production of IFN neither in the early [START_REF] Dunn | Temporal analysis of early immune responses in patients with acute hepatitis B virus infection[END_REF][START_REF] Stacey | Induction of a striking systemic cytokine cascade prior to peak viremia in acute human immunodeficiency virus type 1 infection, in contrast to more modest and delayed responses in acute hepatitis B and C virus infections[END_REF] nor in the late phase of human infection [START_REF] Rehermann | Immunological aspects of antiviral therapy of chronic hepatitis B virus and hepatitis C virus infections[END_REF]. If the results obtained with human samples are subjected to caution, because of the difficulty to get robust and homogeneous cohort of patients during the acute phase of infection, the experiments performed in self-resolving HBV-infected chimpanzees have suggested that innate immunity genes were not up-regulated in their liver when HBV viremia was exponentially rising (i.e. few weeks post infection) (Wieland et al., 2004). Since then, HBV was considered as a "stealth" virus, which does not induce IFN response, likely due to a lack of detection or activation, by innate sensors (pattern recognition receptors; PRRs) [START_REF] Wieland | Interferon prevents formation of replication-competent hepatitis B virus RNA-containing nucleocapsids[END_REF]. Corroborating this, a close look at the viral life cycle indeed suggests that viral PAMPs (i.e. pathogen associated molecular patterns), in particular viral nucleic acids, are not highly exposed to PRRs (Seeger et al., 2015). However, a stealth virus can also be a virus that is able to actively inhibit nascent responses.

Substantiating this hypothesis, our recent work, performed in vitro in infected primary human hepatocytes (PHH) or closely related in HepaRG cells {Luangsay, 2015 #96}, has shown that HBV could be weakly detected by PRRs in hepatocytes; however, HBV was capable to actively and rapidly counteract nascent innate response. The inhibitory mechanism involved a viral component present in HBV virions, but was neither due to HBsAg nor HBeAg {Luangsay, 2015 #95}. The core protein (HBc) and HBV polymerase, with a stoichiometry of 240 unit to 1 in favour of HBc (Seeger et al., 2015), were potentially the two viral proteins composing the infectious virions that could mediate this inhibitory effect.

In this respect, it was shown that over-expression of the viral polymerase could both inhibit RIGI/MDA5 and STING-mediated IFN-α induction by either interfering with DDX3, a regulating component of the IRF3 kinase complex [START_REF] Wang | Hepatitis B virus polymerase blocks pattern recognition receptor signaling via interaction with DDX3: implications for immune evasion[END_REF] or the K63-linked ubiquitination of STING {Liu, 2015 #98}. Other studies have also suggested, using a reporter gene and a non-physiologic expression system, that HBc could inhibit the expression of IFNα and MxA by directly interfering with To test this hypothesis, we exposed dHepaRG cells to standardized amounts (100 or 1000 vge/cell) of HBV complete inoculum (i.e. containing HBV virions and subviral particles (SVPs; HBsAg and HBeAg)), non-enveloped nucleocapsids only (NCs, gradient-purified), or recombinant HBc (recHBc). To allow their cell penetration, NCs and recHBc, were delivered by lipo-transfection as previously reported [START_REF] Rabe | Lipid-mediated introduction of hepatitis B virus capsids into nonsusceptible cells allows highly efficient replication and facilitates the study of early infection events[END_REF]. Twenty four hours later, cells were stimulated by poly (I:C) for an additional 4h after washing the excess of viral inoculum, nucleocapsids or core proteins. Transfection of dHepaRG cells with complete HBV inoculum, as for that obtained with a conventional infection, could also lead to a significant inhibition of poly (I:C)-mediated induction of the expression of IFN-α, IL-29, OAS-1, and IL-6 genes (Figures 1C and1D). Importantly, similar results were obtained with both transfected NCs and recHBc, with the exception that induction of IL-6 gene expression was not affected by these two forms of HBV core protein. This suggests that HBc per se is responsible for the specific inhibition of the IFN response, whereas a different type of inhibition of NF-KB-mediated pro-inflammatory response by other HBV proteins could be at play (Luangsay et al., 2015b). To further demonstrate the prominent role of HBc in the early inhibition of hepatocyte IFN response, we engineered an inducible HepaRG cell line expressing HBV core protein under the control of tetracycline (i.e.). Similar cell lines were also generated for HBs, HBe and HBx expression as well as a control cell line expressing the T7 polymerase protein (Figure S1). In HepaRG-TR-HBc cells, HBc was detected as early as 2 hours post-induction, was not secreted, reached a plateau of expression by 48-72 h post-induction, and was mainly localized within the nucleus of cells (Figure S2). Following HBc, HBs, HBe, or HBx mock-or tet-induction, cells were stimulated for 4h with TLR3-L to measure IFN-α gene expression, as well as IP10 and IL-6 secretion. Among the four inducible cell lines, only that expressing HBc showed a reduced induction of IFN-α gene expression and IP10 secretion following stimulation, thus confirming that HBc is the main contributor to this inhibition (Figures 1E).

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Similar results were also obtained with other prototypic genes of the IFN response, and with a RIGI/MDA5 ligand (poly (I:C)-LMW-Lyovec) (Figure 1F), or using an IFN-α luciferase reporter construct transfected in HBc-expressing cells (Figure 1G). These results corroborated with those obtained in dHepaRG cells exposed to purified HBV nucleocapsids or recHBc (Figures 1C and1D). Similarly, in this cell line, IL-6 expression or secretion was not affected by HBc and interestingly none of the other viral protein tested (HBs, HBx, HBe) could modulate its secretion as well (Figures 1E and1F). The fact that, in our experimental conditions, HBc did not seem to regulate HBV-mediated NF-KB-dependent proinflammatory pathways was further confirmed by using other TLR agonists (TLR1/2, TLR3, RIGI/MDA5, TLR4, TLR5, TLR6, and TLR8 agonists) involved in NF-KB dependent IL-6 induction (RNA and protein levels) (Figures S3A andS3B). Importantly, the inhibition of the poly(I:C)-mediated induction of IFN and ISG genes, observed in HBc expressing cells, was not due to a modification of expression of dsRNA sensors in the time rame of the experiment (Figures S3C andS3D). Altogether, these results suggest that HBc is an early inhibitor of the IFN response in hepatocytes exposed to HBV.

248

Nuclear localization of HBc is required to efficiently impair dsRNA-mediated IFN response

It has been previously proposed that HBc could be mainly localized in the nucleus of hepatocytes in patients with high viremia (i.e. IT/low inflammatory phase or HBeAg+ patients in CAH phase), whereas a cytosolic or mixed subcellular localization was observed in patients well-advanced in CAH phase (HBeAg negative patients). This suggested that the nuclear localization of HBc could be associated with a high viremia and a poor/lack of immune response (Akiba et al., 1987;[START_REF] Chevallier-Queyron | Immunohistochemistry as a tool for chronic hepatitis diagnosis[END_REF]Chu and Liaw, 1987). By performing ISH staining on liver biopsies from a cohort of 45 patients in CAH ( 18HBeAg-positive and 27 HBeAg-negative patients), correlations between the viral load and percentage of hepatocytes positive for HBcAg in the nucleus (Figure S4A), and between HBeAg positivity and percentage of hepatocytes positive for HBcAg in the nucleus (Figure S4B) were found. Our results confirmed that high viremia seems to correlate with a higher localization of HBcAg within the nucleus of infected hepatocytes. This result is consistent with the fact that the binding of HBc to specific innate gene promoters could result in a direct or indirect recruitment of EZH2, which in turn contributed to the establishment of epigenetic repressive marks by tri-methylation of H3 at position K27. Therefore, these results strongly suggest that the inhibition of IFN response is due to a very early epigenetic reprogramming of innate immunity genes caused by the active transport into the nucleus and binding of incoming HBc to their promoters. In order to determine whether HBc could be directly responsible for the recruitment of EZH2 on target promoters, we attempted to co-immunoprecipitate HBc and EZH2 using specific antibodies, but failed to detect interaction between these proteins in HepaRG-TR-HBc cell lines, as well as in HBV-infected PHH. We also analyzed the effect of HBV infection on EZH2 expression. In both HBV-infected PHH (Figure 5A-5C) and dHepaRG (Figures 5D and5E), we found an increased expression of EZH2, as compared to kinetically-paired and non-infected hepatocyte. It is worth noting that the weak/lack of HBc detection in HepaRG cells is due to very low level of HBV replication in this model as compared to PHH. shown in infected hepatocytes or in the liver of infected mice, HBc was found on IFN-α and OAS-1 promoters, and not on IL-6 one in both cell types (Figure 8D).

Long-term in vivo maintenance of the HBc-mediated inhibition of IFN response

Discussion

Neither acute human [START_REF] Dunn | Temporal analysis of early immune responses in patients with acute hepatitis B virus infection[END_REF][START_REF] Stacey | Induction of a striking systemic cytokine cascade prior to peak viremia in acute human immunodeficiency virus type 1 infection, in contrast to more modest and delayed responses in acute hepatitis B and C virus infections[END_REF] or chimpanzee (Wieland et al., 2004) HBV infections, nor chronic HBV infections [START_REF] Tan | A longitudinal analysis of innate and adaptive immune profile during hepatic flares in chronic hepatitis B[END_REF] are accompanied by the production of IFN in the bloodstream of patients and the induction of innate immunity gene expression in the liver. This is in sharp contrast with chronic HCV or HIV infections, in which some interferon-stimulated genes are up-regulated and an uncontrolled IFN response could contribute to immune-pathogenesis {Heim, 2014 #90;Doyle, 2015 #89}.

As for others viruses [START_REF] Bowie | Viral evasion and subversion of pattern-recognition receptor signalling[END_REF][START_REF] Heim | Innate immunity and HCV[END_REF][START_REF] Manel | Hiding in plain sight: how HIV evades innate immune responses[END_REF][START_REF] Randall | Interferons and viruses: an interplay between induction, signalling, antiviral responses and virus countermeasures[END_REF], HBV has likely evolved mechanisms to prevent and/or escape IFN response. Using rather non-physiological cell culture models and levels of viral protein expression, several groups have pointed out the role of HBx, HBV polymerase, and HBc as potential intracellular regulator of the induction of the expression of type-I IFN [START_REF] Jiang | Mechanism of inhibiting type I interferon induction by hepatitis B virus X protein[END_REF][START_REF] Wang | Hepatitis B virus polymerase blocks pattern recognition receptor signaling via interaction with DDX3: implications for immune evasion[END_REF]Wang et al., 2010;[START_REF] Wei | The hepatitis B virus X protein disrupts innate immunity by downregulating mitochondrial antiviral signaling protein[END_REF]Wu et al., 2007b;Yu et al., 2010). Moreover secreted HBV proteins, i.e. HBsAg and HBeAg, were also shown to inhibit TLRs-mediated innate immune responses, yet by an ill-defined mechanism (Wu et al., 2007a;[START_REF] Wu | Hepatitis B virus suppresses toll-like receptor-mediated innate immune responses in murine parenchymal and nonparenchymal liver cells[END_REF]. Thesis Adrien FOCA

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In this study, using more relevant models, i.e. proper infection of primary human hepatocytes (PHH) or closely related cells (dHepaRG), we demonstrate that HBc is a key very early negative regulator of the IFN response. Indeed, we have shown that the HBc protein associated to "incoming" HBV virions can inhibit lFN response after trafficking of nucleocapsids (NCs) through the cytosol and translocation of HBc into the nucleus, where it is able to transcriptionally repress IFN-response gene expression. In a previous study, using mainly HepaRG cells, we previously showed (Luangsay et al., 2015b), and we confirmed here using PHH, that this inhibition could be seen as early as 2 hours after the onset of infection, and showed that HBc can be found on the promoter of target genes after only 4h of exposure. Others have found that, after experimental lipo-transfection of NCs to cells, the delivery of HBV genomic DNA into the nucleus takes only few minutes [START_REF] Rabe | Lipid-mediated introduction of hepatitis B virus capsids into nonsusceptible cells allows highly efficient replication and facilitates the study of early infection events[END_REF], thus confirming that HBc can be rapidly found in the nucleus of neo-infected cells, where it can play its immune-modulatory role.

HBc is capable to bind to dsDNA, including cccDNA as well as host genes (Bock et al., 2001;Guo et al., 2012), and it likely does it via the same domain required for RNA binding, which is a protamine like domain found in mammalian DNA binding protein located at the Cterminus of the protein (Seeger et al., 2015;Zheng et al., 1992). The biophysical aspect of this interaction is yet to be defined, but could involve a dimeric forms of the protein. It is worth noting that all the promoters of targeted genes contained ISRE motifs. EMSA experiment also showed that HBc could bind to ISRE-containing dsDNA (Fernandez et al., 2003).

The early action of HBc could explain why a nascent innate response, as evidenced previously (Luangsay et al., 2015b), could be stopped and why no IFN would not be produced upon infection. We have also shown that when HBV life cycle is launched via a baculoviral vector, therefore in the absence of HBc during inoculation, a strong interferon response, accompanied by eradication of infection, could be observed {Lucifora, 2010 #91}.

Altogether our observation show that HBV is indeed a "stealth" virus, not because of absence of detection, but rather because of a very early inhibition of innate immune responses. Such an early inhibition of innate immune responses via a structural protein embarked into virions and readily mobilized into the newly penetrated cells has been shown, to our knowledge, only in the case of the tegument protein (ORF52) of KHSV. This protein was indeed very recently HepaRG-TRx and HepaRG-TR-T7pol were derived by co-selection with blasticidin and zeocin.

HBV infection and lipo-transfection of recombinant HBcAg and non-enveloped

nucleocapsids. Differentiated HepaRG cells and PHH were infected with HBV or its control mock (depleted from HBsAg, HBeAg and infectious Dane particles} in complete William's medium supplemented with 4% PEG-8000 at 37°C as previously described (Hantz et al., 2009). Recombinant HBcAg and non-enveloped nucleocapsids were lipo-transfected into HepaRG cells using "Pro-Ject Protein Transfection Reagent" kit according to manufacturer's instructions (Thermo Scientific).

Humanized FRG mouse model and HBV infection.

All animal studies were reviewed and approved by a local ethical comity. The highly immunosuppressed FRG mice (Fah -/-/Rag2 - /-/Il2rg -/-), deficient for T-, B-and NK-cells were used (Azuma et al., 2007) and maintained in a specific pathogen-free facility. High quality, cryopreserved human hepatocytes were purchased (BD, Biosciences) and injected via an intra-splenic route into 2 to 3 month old mice as described previously. Liver humanized Fah -/-/Rag2 -/-/Il2rg -/-mice featuring a seric production of human albumin of at least 5mg/mL were infected with 200 μl of HBV inoculums (1.10 8 veg to 1.10 9 veg in PBS) via intra-peritoneal route (Bissig et al., 2010).

Serum was harvested every week by retro-orbital bleeding and stored at -80°C in aliquots for further antigenemia and viremia analysis. Mice were sacrificed 9 weeks post-infection and hepatic tissues were processed for RT-qPCR and ChIP analysis or fixed in formalin and embedded in paraffin for immune-staining.

PRRs stimulation. Cells were stimulated with TLR or RLR agonists and harvested for the analysis of the innate gene expression (after 4h) and cytokines production (after 6h). TRL and RLR agonist were: TLR1/2 (Pam3CSK4, 0.8 μg/ml), TLR3 (Poly (I:C)-LMH, 10 μg/ml), TLR4 (LPS, 0.4μg/ml), TLR5 (Flagellin, 0.1 μg/ml), TLR6 (FSL-1, 0.1 μg/ml), TLR7/8 (ssRNA40, 10 μg/ml), RIGI/MDA5 (Poly (I:C)-LMH-Lyovec 0.2 μg/ml).

RNA extraction and qRT-PCR.

Total RNA were purified with the Nucleospin RNA II kit according to the manufacturer's protocol (Macherey Nagel}. cDNA was obtained after reverse transcription using the Superscript® III Reverse Transcriptase (Life technologies) and qPCR was performed using the Express SYBR GreenER qPCR Supermix Universal according to the manufacturer's instructions (Life technologies} and run on the MyiQ Biorad machine. The Chromatin immunoprecipitation (ChIP) assays. Cells were fixed with formaldehyde 1% at room temperature with rocking to capture the DNA-protein interaction and cross-linking was stopped with Glycine 0.125M (Euromedex). Cells were washed 3 times with ice-cold PBS 1X then scrapped and lysed in 500μl of lysis buffer per 10 7 cells (50 mM Tris HCl pH8, 10 mM EDTA, 1% SDS and protease inhibitor cocktail (Complete EDTA-free tablets, Roche)) for 5 min in ice. Cell lysates were sonicated to reduce DNA length to 200 bp using a Bioruptor system (Diagenode). The sheared chromatin extract was then frozen in aliquots at -80 o C until use. Sheared chromatin (100 μl) was diluted 10-fold in RIPA buffer (10 mM Tris HCl pH5, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton, 0.1% SDS, O.1% sodium deoxycholate, protease inhibitor cocktail) and then pre-blocked with protein A sepharose 4B (Sigma) for 2 hours at 4°C under gentle agitation. Pre-blocked chromatin was incubated overnight with rotation at 4°C with 10μg of specific antibodies. An additional sample of lysate containing no antibody was also incubated overnight at 4°C (no antibody control).

Chromatin was then immunoprecipitated with magnetic beads (Dynabeads Protein G, Novex) for 2 h at 4°C with rotation. After several washes with RIPA buffer, cross-linking was reversed at 68°C for 2h under shaking in elution buffer (20 mM Tris HCl pH7.5, 5 mM EDTA, 50 mM NaCl, 1% SDS, 50 μg/ml proteinase K (Eurobio) and inhibitor cocktail proteases).

DNA from elution buffer was extracted with Phenol/Chloroform/Isoamyl-Alcohol and precipitated with ethanol supplemented with NaAc 3M and Glycogen 20mg/ml (Sigma) overnight at -20°C. Extracted DNA from non-immunoprecipitated fraction of no antibody control was used as internal control (total input). Extracted DNA was used as template for qPCR specific on IFN-αp, IL-29p, IL-6p, OAS-1p and GAPDHp. The percentage of input enrichment (2^tiCt (total input -antibody input)) was normalized on the Mock condition depending of the experiments (no tetracycline stimulation or no HBV infection) and also on GAPDH housekeeping gene promoter. Results are representative of 3 independents experiments. The list of primer pairs is given on Table2. receptor, providing a specific handle for delivering the anti-miRs via covalent conjugation to folate, as recently described 4 .