L’infection par le virus de l’hépatite B (HBV) est problème majeur de santé publique mondial car, en dépit d’un vaccin efficace, les traitements curatifs actuels ne permettent pas l’élimination complète du virus. Comprendre les mécanismes de réplication du virus et son rôle dans la survenue du cancer hépatocellulaire (CHC) reste un enjeu majeur.Le rôle de la protéine HBx dans l’infection par HBV et l’oncogenèse viro-induite, reste mal connu, malgré un grand nombre de publications, car les fonctions décrites jusqu'alors sont limitées à des contextes d’études particuliers, souvent loin des conditions physiologiques.Mes travaux de thèse reposent sur l’utilisation de modèles d’études proches de la physiologie naturelle d’une infection par HBV, notamment des cellules primaires infectables in vitro. J’ai pu démontrer lors de mon étude que HBx est indispensable à la réplication de HBV, et qu’il agit essentiellement via son interaction avec DDB1 pour contrer la restriction du virus due au complexe SMC5/6, en induisant sa dégradation. Ce facteur de restriction permet de bloquer la transcription de l’ADN viral au niveau épigénétique. Ce nouveau rôle inattendu de SMC5/6 ouvre de nombreux axes de recherche, notamment sur les mécanismes de restriction des virus à ADN épisomal. SMC5/6 est connu pour son implication dans les voies de réparation de l’ADN : la dernière partie de ce manuscrit montre que sa dégradation dans les cellules infectées, altère ces mécanismes et sensibilise les cellules aux dommages à l’ADN, induits notamment par la radiothérapie. La présence de HBx dans les CHC pourrait ainsi s’avérer un atout pour le traitement du CHC / Hepatitis B virus (HBV) infection is a major health problem worldwide as (1) despite an effective preventive vaccine over 240 million individuals are chronically infected and (2) the actual viral suppressive treatments available do not eliminate viral DNA from cells. Thus, infected individuals are at a high risk of developing hepatocellular carcinoma (HCC) and understanding viral replication mechanisms and how it impacts on hepatocarcinogenesis is a major challenge.The role of the HBx protein, notably in viral replication and oncogenic processes, is the subject of many publications. However, many studies have often used non-physiological infection conditions. My thesis project has addressed these limitations by using cellular models, including primary human hepatocytes which can be infected by HBV, to investigate HBx’s role in these processes. I have shown that HBx is indispensable for HBV replication and that HBx associates with the infected cell’s DDB1/ E3 ubiquitin complex to target its Smc5/6 complex for degradation via the proteasome. These studies have identified that the Smc5/6 complex is a novel viral restriction factor that acts at an epigenetic level to block viral replication. This unexpected role of SMC5/6 has led to new research into the evolutionary conservation of restriction factors for episomal DNA viruses. As SMC5/6 is implicated in DNA Damage Repair (DDR), the last section of my thesis reports how SMC5/6 degradation in infected cells can sensitise cells to the cell killing effects of DNA damaging agents such as ionizing radiation and hydroxyurea. These results open-up possibilities for HCC treatment where HBx expression may be of therapeutic benefit

La drosophile est un excellent modèle pour l’étude des mécanismes moléculaires de l’immunité innée, y compris les virus. Elle a permis la caractérisation de mécanismes de défense immunitaire conservés au cours de l’évolution, tel que les voies Toll et IMD qui régulent l’expression des peptides antimicrobiens induits en réponse aux infections fongiques et bactériennes. Deux types de réponse sont impliqués dans le contrôle des infections virales chez la drosophile : une réponse inductible et l’ARN interférence. Nous avons montré que l’ARN interférence est un mécanisme global de défense antivirale puisqu’il contrôle l’infection par un virus à ADN, en plus des virus à ARN tel que le virus C de la drosophile (DCV). Le virus DCV, apparenté aux Picornaviridae, est un pathogène naturel de la drosophile. Nous avons également observé que la résistance de mouches contrôles à l’infection par le virus DCV est dépendante du fond génétique. Elle est d’ailleurs corrélée à des polymorphismes présents dans un gène porté par le chromosome III : le gène pastrel. Nos expériences de perte et gain de fonction indiquent que ce gène code pour un facteur de restriction viral, bloquant l’infection par le virus DCV mais aussi par le virus de la paralysie du cricket (CrPV). Cette restriction apparait dans les premières heures après infection. La région C-terminale de la protéine Pastrel est nécessaire à son activité antivirale ainsi qu’à sa localisation dans les cellules. La protéine Pastrel co-localise avec le Rouge de Nil, un marqueur des gouttelettes lipidiques. Ainsi, nos résultats suggèrent un lien entre le métabolisme lipidique et le blocage d’une infection virale chez la drosophile. / Since the discovery of the evolutionarily conserved TOLL and IMD pathways, involved in antifungal and antibacterial immune responses, the fruit fly Drosophila melanogaster is used as a model to study the molecular mechanisms of innate immunity. To defend against viral pathogens, Drosophila relies on two main facets: the RNA interference (RNAi) pathway and virus specific inducible responses. We show that the RNAi pathway plays a role in the control of a DNA virus, in addition to RNA viruses. We also observe that the fly genetic background can modulate the resistance to infection by Drosophila C virus (DCV), a natural pathogen of Drosophila. This resistance to DCV infection is correlated with polymorphisms in a gene named pastrel,localized on the left arm of the third chromosome. Our loss- and gain-of-function experiments indicate that pastrel encodes a molecule opposing infection by picorna-like viruses DCV and also Cricket Paralysis virus (CrPV), raising the question of the mechanism involved. This restriction appears early after infection. The Cterminal region of Pastrel protein is important for its antiviral activity and its localization in vesicular structures co-localizing with Nile Red staining, a marker for lipid droplets. Altogether, our data suggest a connection between lipid droplets and restriction of viral infection in Drosophila, as already described in mammals between the restriction factor Viperin, present on lipid droplets, and the replication of the human pathogen Hepatitis C Virus.

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APOBEC3 (A3) proteins are a family of host-encoded cytidine deaminases that protect against retroviruses and other viral intruders. Retroviruses, unlike other viruses, are able to integrate their genomic proviral DNA within hours of entering host cells. A3 proteins hinder retroviral infectivity by editing retroviral replication intermediates, as well as by inhibiting retroviral replication and integration through deamination-independent methods. These proteins thus constitute the first line of immune defense against endogenous and exogenous retroviral pathogens. The overall goal of my Master's project was to better understand the critical role A3 proteins play in restricting inter- and intra-host transmission of retroviruses. There are two specific aspects that I focused on: first, investigating the role of mouse APOBEC3 (mA3) in limiting the zoonotic transmission of murine leukemia retroviruses (MLVs) in a rural environment; second, to identify the molecular features in MLVs that confer susceptibility or resistance to deamination by mA3. For the first part of my project, we collected blood samples from dairy and production cattle from four different geographical locations across Canada. We then designed a novel PCR screening strategy targeting conserved genetic regions in MLVs and Mouse Mammary Tumor Virus (MMTV) and MMTV-like betaretroviruses. Our results indicate that 4% of animals were positive for MLV and 2% were positive for MMTV. Despite crossing the species barrier by gaining entry into bovine cells, our study also demonstrates that the bovine A3 protein is able to potently inhibit the spread of these murine retroviruses in vitro. The next question we asked was whether mA3 could also mutate and restrict murine endogenous retroviruses and thereby partake in limiting zoonotic transmission. Moloney MLV and AKV MLV are two highly homologous murine gammaretroviruses with opposite sensitivities to restriction by mA3: MoMLV is resistant to restriction and deamination while AKV is sensitive to both. Design of MoMLV/AKV hybrid viruses enabled us to map the region of mA3 resistance to the region encoding the glyco-Gag accessory protein. Site-directed mutagenesis then allowed us to correlate the number of N-linked glycosylation sites with the level of resistance to deamination by mA3. Our results suggest that Gag glycosylation is a possible viral defence mechanism that arose to counteract the evolutionary pressure imposed by mA3. Overall, my projects show the important role A3 proteins play in intrinsic immunity, whether defending the host from foreign retroviral invaders or endogenous retroviral foes.