The host restriction factor TRIM5α mediates a post-entry, pre-integration block to retroviral infection that depends upon recognition of the viral capsid by the TRIM5α PRYSPRY domain. The two predominant alleles of rhesus macaque TRIM5α (rhTRIM5αQ and rhTRIM5αTFP) restrict HIV 1, but cannot restrict the macaque-adapted virus SIVmac239. To investigate how TRIM5α recognizes retroviral capsids, we exploited the differential sensitivities of these two viruses to identify gain-of-sensitivity mutations in SIVmac239, and we solved the structure of the SIVmac239 capsid N-terminal domain. When mapped onto this structure, single amino acid substitutions affecting both alleles were in the β-hairpin. In contrast, mutations specifically affecting rhTRIM5αTFP surround a highly conserved patch of amino acids that is unique to capsids of primate lentiviruses. This "patch" sits at the junction between the binding sites of multiple cellular cofactors (cyclophilin A, Nup-358 cyclophilin A-like domain, Nup-153 and CPSF6). Differential restriction of these alleles is due to a Q/TFP polymorphism in the first variable loop (V1) within the PRYSPRY domain. Q reflects the ancestral state (present in the last common ancestor of Old World primates) and has remained unmodified in all but one lineage of African monkeys, the Cercopithecinae. While Q-alleles can be found among some Cercopithecinae primates, in others Q has been replaced by a G or overwritten by a two amino acid insertion (giving rise to TFP in macaques). In one lineage, the Q to G substitution was later followed by an adjacent 20 amino acid duplication. We found that these modifications in TRIM5α specifically impart the ability to restrict Cercopithecinae SIVs without altering β-hairpin recognition. At least twice Cercopithecinae TRIM5αs independently evolved to target the same conserved patch of amino acids in capsid. Based on these findings, we propose that the β-hairpin is a retrovirus associated molecular pattern widely exploited by TRIM5α proteins, while recognition of the cofactor binding region was driven by the emergence of the ancestors of modern Cercopithecinae SIVs. Distribution on the Cercopithecinae phylogenetic tree indicates that selection for these changes in TRIM5α V1 began 11-16 million years ago, suggesting that primate lentiviruses are at least as ancient.

The APOBEC3 (Apo3) family of proteins are single stranded (ss) DNA cytosine deaminases (C → U). They are grouped into two different structural groups, the single catalytic domain Apo3 enzymes (Apo3A, Apo3C, and Apo3H) and the double catalytic domain Apo3 enzymes (Apo3B, Apo3D, Apo3F, and Apo3G). Apo3G has been implicated in protection from HIV proliferation by becoming encapsidated into budding HIV virions and subsequently mutationally inactivating the synthesized provirus. This largely occurs in the absence of HIV viral infectivity factor (Vif) which mediates the ubiquitination and degradation of Apo3G. Apo3G is a processive enzyme, able to catalyze numerous deaminations in a 5'CCC motif in a single interaction with a substrate. There is a paucity of biochemical data on other Apo3 family members. We performed basic biochemical assays that determined the relative specific activities, processivity, cytosine motif preferences, and binding affinities for DNA, of Apo3A and Apo3H using synthetic DNA substrates in deamination assays. We found Apo3A to be an enzyme with low processivity and Apo3H to be a highly processive enzyme; both of which deaminate a 5'TC motif. Using a reconstituted HIV replication assay we assessed if processivity is needed for efficient restriction of HIV. We were able to demonstrate that each, Apo3G, Apo3A, and Apo3H were able to catalyze deaminations during in vitro reverse transcription. The mutation profile of both Apo3A and Apo3H showed that the 5'TC motif preference was less effective compared to Apo3G in triggering missense and nonsense mutations in the HIV protease active site coding sequence. Nuclear DNA can become deaminated by the related Apo3 family member activation-induced deaminase (AID), when it is present in the nucleus of activated B cells. Apo3A and Apo3H are located in the nucleus but the extent of the damage they cause has only recently been investigated. Here we used an in vitro transcription assay to determine the efficiency of Apo3A and Apo3H deamination during transcription and found that, like AID, they are highly capable of causing deaminations during transcription. Taken together, the results presented here demonstrate that processivity is not necessary for an Apo3 enzyme to catalyze deaminations during HIV reverse transcription and that Apo3A and Apo3H can catalyze deaminations during DNA transcription that could damage host genomic DNA. These results imply a potential cost for maintaining nuclear deaminases.

Influenza A virus (IAV) is a major threat to human health with seasonal epidemics, occasional pandemics and emergence of new highly pathogenic strains from the animal reservoir. Our laboratory has shown that the human Annexin A6 (AnxA6) interacts with the IAV M2 proton channel and limits production of progeny IAV from infected cells. We have found that overexpression of AnxA6 impairs morphogenesis and release of progeny viruses. These findings are supported by another study showing that AnxA6 has a critical role in the late endosomal cholesterol balance and affects IAV replication and propagation in AnxA6-overexpressing cells. However, the molecular mechanism responsible for restriction of IAV morphogenesis by AnxA6 is still unclear. AnxA6 is a calcium-dependent phospholipid-binding protein which plays a major role in cellular events such as regulation of cholesterol homeostasis and membrane organisation or repair. AnxA6 is also implicated in the regulation of intracellular signalling pathways required for IAV infection. In this study, we used a combination of virology, cellular biology and biochemistry approaches to decipher the restriction mechanism of IAV by human AnxA6. We found that AnxA6 down-regulates M2 viral protein expression and impairs viral morphogenesis and budding. We also found that AnxA6 regulates chemokines and cytokines expression during viral infection, suggesting that AnxA6 triggers an innate immune response to IAV by modulating signalling pathways required for viral replication. Finally, we observed that IAV down-regulates AnxA6 expression at mRNA level during early stages of infection and at protein level during late infection, suggesting that IAV has developed a strategy to respond to AnxA6 restriction mechanism during viral infection. We conclude that it is essential to better understand the interaction between human AnxA6 and IAV to elucidate the potential of AnxA6 as an antiviral candidate.

Les protéines IFITM (« InterFeron Inducible TransMembrane proteins »), et en particulier les membres 1, 2 et 3, sont des facteurs de restriction antiviraux dont l’expression est induite par le système immunitaire inné en réponse à une infection virale. Elles inhibent la réplication de nombreux virus pathogènes pour l’homme parmi lesquels figurent le virus de la grippe A, le VIH (Virus de l’Immunodéficience Humaine) de type 1 ou encore le virus de l’hépatite C. Ces virus entrent dans la cellule hôte, soit par fusion directe avec la membrane plasmique, soit par la voie de l’endocytose. Il est à présent communément admis que les protéines IFITM, localisées au sein des membranes plasmiques et endolysosomales, agissent en inhibant la fusion des membranes virales et cellulaires, empêchant par conséquent l’entrée du virus dans la cellule et donc sa réplication. D’autre part, dans le cas du VIH, leur incorporation dans les particules virales produites par la cellule hôte diminuerait la capacité de ces particules à infecter de nouvelles cellules cibles. Cependant, les mécanismes moléculaires par lesquels les protéines IFITM interfèrent avec le cycle viral ne sont pas encore clairement définis.Parmi les membres de la famille IFITM, IFITM3 est celui qui présente l’effet antiviral le plus systématique selon les différentes études. Il constitue donc un modèle de référence pour étudier la famille IFITM.Déterminer la structure ainsi que la topologie membranaire d’IFITM3 sous sa forme active rendrait alors possible la réalisation d’études fonctionnelles, dont les résultats contribueraient sans nul doute à élucider le(s) mécanisme(s) par le(s)quel(s) IFITM3 exerce son activité antivirale.C’est pourquoi, nous nous sommes tout d’abord attelés à reconstituer IFITM3 au sein de membranes artificielles (liposomes, nanodisques), car contrairement aux micelles de détergent, ces membranes artificielles peuvent mimer l’environnement natif des protéines membranaires et par conséquent, offrir de plus grandes chances de les y étudier sous leur forme active. Nous avons ensuite procédé à la caractérisation biochimique et biophysique d’IFITM3 et avons mis en évidence la formation de dimère de la protéine ainsi que de plus grandes espèces oligomériques. L’analyse structurale d’IFITM3 reconstituée en nanodisques par RMN nous a quant à elle permis d’identifier une courte région hélicoïdale dans la région N-terminale extramembranaire d’IFITM3 encore jamais décrite auparavant et pouvant correspondre à un motif d’internalisation. Nous avons en outre observé, par microscopie électronique à coloration négative, de potentiels effets d’IFITM3 sur la courbure de la membrane de liposomes qui pourraient être à l’origine de son action inhibitrice sur la fusion virale. Et enfin, nous avons montré au travers d’expériences TEVC que lorsqu’IFITM3 est présente dans l’environnement extracellulaire d’ovocytes de xénope, celle-ci est capable d’engendrer des fuites ioniques au travers de la membrane des ovocytes qui pourraient résulter soit, d’une déstabilisation de la membrane par IFITM3 soit, d’une formation de pores membranaires par la protéine. / The host cell first line of defence against viral infections induces the production of interferons. These interferons are then released in the surrounding medium where they bind to target cells and induce the expression of hundreds of genes so called interferon-stimulated genes (ISGs). The interferon inducible transmembrane proteins IFITM are part of the products of these ISGs. IFITM1, 2 and 3 are antiviral factors able to restrict the replication of a broad variety of enveloped viruses, such as influenza virus, HIV-1 (Human Immunodeficiency Virus) and Hepatitis C virus. These viruses enter in the host cell either by direct fusion with the cell membrane or by endocytosis. IFITM proteins contain two membrane regions for insertion or interaction with plasma and endolysosomal membranes where they block the fusion of virus particles with cellular membranes by a mechanism which is still undefined. In addition, their incorporation into new HIV virions, in virus producing cells, has been correlated with decreased infectivity.Among the IFITM protein family members, IFITM3 is the one showing the most recurrent antiviral effect in the different studies. Therefore it represents a good model to study the whole IFITM family.The determination of its structure and membrane topology is crucial to be able to clarify, through structure-based functional studies, the mechanism(s) by which IFITM3 interfere with the viral cycle.Here we characterized and we studied IFITM3 structure and membrane topology in a lipidic environment close to its native environment such as liposomes and nanodiscs. We demonstrated that IFITM3 can self-associate to form at least a dimer. Some higher order associations of IFITM3 have been observed after its reconstitution into liposomes and big size nanodiscs. We discovered by NMR in solution that the N-terminal region of IFITM3 contains a small helical region, never described until now, which could correspond to an internalization motif. We also observed by negative staining electron microscopy some liposomal membrane curvature changes that could be assigned to the presence of IFITM3 in these liposomes. And we discovered through TEVC experiments that IFITM3 addition in the extracellular environment of xenopus oocytes produces ion leaks through the oocyte membrane which could result either from membrane destabilization or from a pore formation.

No description available.

SERINC5 is a restriction factor for retroviruses, antagonized by Nef of primate lentiviruses, by glycoGag of Moloney Murine Leukaemia Virus (MoMLV) and by S2 of Equine Infectious Anaemia virus (EIAV). In addition, SERINC5 sensitizes HIV-1 to neutralizing antibodies (nAbs) targeting the MPER in gp41. However, since the identification of SERINC5 as an inhibitor of retrovirus infectivity, many features of the host factor await clarification, notably the molecular mechanisms of restriction and viral counteraction. Furthermore, SERINC5 cellular role beyond restriction is still obscure. This thesis explores multiple aspects of the mutual antagonism governing the SERINC5 interplay with retroviruses. We first describe a contribution towards the determination of the structure of SERINC5 and the identification of the determinants crucial for antiviral activity, virus sensitization to neutralization and counteraction by retroviruses. By performing a structure-based mutagenesis screening, we identified SERINC5 ECL3, ECL5 and the interface between subdomains as regions essential for inhibition of HIV-1 infectivity and virus sensitization to 4E10 and 2F5 nAbs. The simultaneous impairment of both SERINC5 antiviral effects indicates that they are mechanistically related and support the hypothesis of a SERINC5-mediated impairment of the envelope glycoproteins. We included a comparative analysis of the antiviral activity of human SERINC paralogs and their sensitivity to retroviral counteraction. It has been previously established that SERINC3 inhibits HIV-1 infectivity less potently than SERINC5, while SERINC2 has no antiviral effects. We report here that similarly to SERINC3, SERINC1 is endowed with a modest antiviral activity; in contrast, SERINC4 severely inhibits HIV-1 infectivity, despite being poorly expressed. Irrespectively of their antiretroviral potency, all SERINC proteins are incorporated into virus particles. Interestingly, we observed that virion-associated SERINC2 is specifically cleaved by the viral protease, but proteolysis does not explain the lack of antiretroviral effects. Furthermore, SERINC5 and SERINC2 have different glycomic profiles, but diverse post-translational modification is irrelevant for their opposite activity against HIV-1. In addition, we reported that human SERINCs are differently targeted by retroviral counteracting factors, with SERINC5 being the paralog most efficiently downregulated, while SERINC1 being completely resistant. A cysteines cluster within ICL4 emerged as the major determinant of SERINC5 responsiveness to different nef alleles, while it proved irrelevant for internalization by MoMLV glycoGag and EIAV S2, indicating that diverse retroviral counteractors likely target the host factor differently. Though SERINC5 ICL4 harbours multiple motifs governing SERINC5 sensitivity to antagonization, insertion of this loop within SERINC2 was not enough to transfer susceptibility to Nef activity, suggesting that the overall conformation of the protein is essential for downregulation by Nef. Importantly, the cysteine stretch within ICL4 is palmitoylated, suggesting that this modification may be important for counteraction by the lentiviral factor. SERINC5 and CD4 downregulation by Nef are functionally related, as they both require AP-2 mediated endocytosis. However, regions in Nef selectively governing SERINC5 internalization are unknown. We reported here that Phe90 within Nef αA-helix genetically uncouples the activities on SERINC5 and CD4, being selectively involved in SERINC5 downregulation. In parallel, we explored SERINC5 antagonization by different glycoGag alleles and observed that the ability to target the host factor is not conserved across divergent γ-retroviruses. Finally, we observed that HIV-1 may evade SERINC5 restriction by direct cell-to-cell infection, suggesting that the host factor may have a broader role in retroviral spreading, requiring the evolution and the conservation of active viral counteraction. To this end, we preliminary investigated a positive contribution of SERINC5 to intracellular signalling.

APOBEC3A (Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like editing complex 3 A) appartient à la famille des cytidines désaminases, qui clivent les cytidines en uraciles. APOBEC3G (le modèle de la famille) est un facteur de restriction du VIH : son incorporation dans la particule virale lui permet de désaminer le génome viral néoformé, entrainant hypermutations et dégradation de l’ADN viral. A3A, au contraire, n’est pas incorporée dans la particule virale : la protéine, exprimée spécifiquement dans les cellules myéloïdes de façon inoffensive pour la cellule, agit de la même façon qu’A3G en s’attaquant au virus dès son entrée dans la cellule.Physiologiquement, dans les cellules non-myéloïdes, les APOBEC3 désaminent l’ADN cellulaire simple brin, dont les uraciles sont retirés par UNG2. Les sites abasiques sont clivés par la machinerie de réparation de l’ADN, conduisant parfois à des cassures double-brin et à la mort de la cellule.L’objectif de la thèse était de comprendre cette différence de comportement selon le type cellulaire. Pour cela, des lignées cellulaires inductibles pour A3A ont été créées en cellules HeLa et U937 (monocytaire). Les données obtenues indiquent qu’A3A, partiellement nucléaire, édite l’ADN des cellules en division, conduisant à des dommages à l’ADN, à la production d’espèces réactives de l’oxygène (ROS) et à la mort des cellules. Les cellules différenciées ne présentent pas ce type de dommages, et cela s’explique par une localisation différente de la protéine. Ces résultats permettent de faire pour la première fois le lien entre dommages à l’ADN induits par un membre de la famille des A3 et production de ROS, et donc à l’induction d’une activation immunitaire. Cette activation pourrait avoir des implications dans l’infection ainsi que dans les processus tumorigéniques. / APOBEC3A (Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like editing complex 3 A) belongs to a family of cytidine deaminases that can edit cytidines to uraciles. APOBEC3G (model protein for the family) is a restriction factor for HIV: since it’s incorporated in the viral particle, it can deaminate the newly formed viral genome leading to hypermutation and viral DNA degradation.A3A is not incorporated in the viral particle: this protein is specifically expressed in myeloid cells where it is harmless for the cell and edits the DNA of the incoming viral particle in the same way than A3G.Physiologically, in non-myeloid cells, APOBEC3s deaminate single strand cellular DNA and the resulting uraciles are cut out by UNG2. These abasic sites are cleaved by the DNA repair machinery and can generate double strand breaks that will result in cell death.The objective of the thesis was to understand this difference of behaviour between different cell types. For that purpose, A3A-inducible cell lines were created in HeLa and U937 (monocytic) cells. The results obtained indicate that partially nuclear A3A edits the genomic DNA of cycling cells, leading to DNA damage, to the production of reactive oxygen species (ROS) and to cell death. Differentiated cells do not present this type of damage and that phenotype can be explained by a different localization of the protein.These results link for the first time DNA damage induced by a member of the A3 proteins family to ROS production and to induction of an immune activation. This activation could have implications in infection as well as in tumorigenic processes.

Le Virus de l'Hépatite B (VHB) infecte 350 millions d'individus à l'échelle mondiale. Il est responsable d'hépatites aigües pouvant évoluer vers la chronicité puis le carcinome hépatocellulaire. Le génome du VHB est constitué d'un ADN partiellement bicaténaire. De par sa nature, il pourrait être sensible à l'action de certaines nucléases cellulaires qui hydrolysent l'ADN double brin. Nous avons ainsi mis en évidence la capacité de la Désoxyribonuclase I (DNase I) à être incorporée dans les virions du VHB, ce qui permet la dégradation du génome viral et la diminution de son infectivité. La DNAse I est particulièrement surexprimée en hypoxie et pourrait contribuer à l'élimination du virus chez les individus cirrhotiques. Par ailleurs, nous avons montré que la cytidine désaminase APOBEC3DE appartenant à une famille de facteurs de restriction viraux possède un rôle proviral. En effet, son association avec APOBEC3F et APOBEC3G mène à une diminution de l'activité de ces dernières et ceci favorise la réplication du VHB. La formation d'hétérodimères APOBEC3DE/APOBEC3F et APOBEC3DE/APOBEC3G semble génèrer un encombrement stérique ne permettant pas l'encaspidation d'APOBEC3F et APOBEC3G, raison pour laquelle le génome du VHB est moins muté lorsqu'APOBEC3DE est exprimée. / Hepatitis B Virus (HBV) infects 350 millions people worldwilde. It triggers accute hepatitis that can turn into cirrhosis then hepatocellular carcinoma. HBV genome is composed of a partially double-stranded DNA.Thus, it could be targeted by some cellular nucleases that hydrolyze double-stranded DNA. We have highlighted that Deoxyribunuclease I (DNase I) can be incorporated into HBV virions and degrade its genome, leading to a loss of viral infectivity. Moreover, DNase I is upregulated under hypoxia which is a caracteristic of liver cirrhosis. DNase I could be involved in HBV elimination in cirrhotic patients. In an other study, we found that APOBECDE, a cytidine deaminase of the same family than some restriction factors, has a proviral activity. Indeed, association of APOBEC3DE with APOBEC3F or APOBEC3G leads to a loss of cytidine deaminase activity and a better viral replication. When APOBEC3DE is associated with those two proteins, APOBEC3F and APOBEC3G cannot be incorporated into HBV virions. This is the reason why HBV is more infectious when APOBEC3DE is expressed.

The abundant cellular protein Cyclophilin A (CypA) was found to bind to HIV-1 capsid (CA) in 1993. Since that time, several complementary methods, including disruption of the binding interface by cyclosporine A, CA mutants, and CypA mutants, have been used to demonstrate that CypA acts within human target cells to promote HIV-1 infection. In contrast, in cells from non-human primates, CypA in target cells decreases HIV-1 infectivity, and it does so by promoting TRIM5α-mediated restriction. Using human cancer cell lines and the genetic methods available at the time, attempts to obtain evidence that CypA inhibits HIV-1 restriction by the human TRIM5α ortholog, let alone that human TRIM5α restricts HIV-1, were unsuccessful. Here we revisit the question of the mechanism by which CypA increases HIV-1 infectivity by exploiting lentiviral vectors optimized for primary human blood cells that serve as HIV-1 targets. Disruption of CA−CypA interaction is demonstrated to render HIV-1 vulnerable to endogenous human TRIM5α-mediated recognition and restriction, which occur prior to completion of reverse transcription. Identical findings were acquired with single-cycle vectors or with replication-competent viruses. Consistently, a previously identified, cyclosporine-resistant CA mutation A92E is also shown to confer resistance against restriction by human TRIM5α. Therefore, the results presented in this thesis reveal that HIV-1 exploits a host protein CypA bound to its CA to evade potent restriction by human TRIM5α. This finding not only answers a long-standing question regarding the role of CypA in HIV-1 infection, but also may reinvigorate the development of CypA inhibitors for treatment of HIV-1.

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