L’intégration de la copie ADN du VIH-1 dans le génome d’une cellule infectée est une étape essentielle du cycle réplicatif de ce rétrovirus. Elle est réalisée par une enzyme virale, l’intégrase, qui constitue une cible privilégiée des stratégies antivirales. Différentes études suggèrent une régulation de la sélectivité d’intégration par la chromatine et la transcription. La protéine cellulaire LEDGF/p75, activateur transcriptionnel, interagissant à la fois avec l’intégrase et la chromatine constitue une parfaite illustration de cette régulation.Mon projet de thèse a été d’étudier les liens entre LEDGF/p75, la chromatine et la transcription au cours de l’intégration du VIH-1.Tout d’abord, mes travaux ont permis de valider in vitro l’interaction entre LEDGF/p75 et son partenaire TOX4, récemment identifié par notre équipe. J’ai également montré que le domaine de TOX4, fixant LEDGF/p75, inhibe in vitro l’intégration sur matrice chromatine en présence de LEDGF/p75, suggérant un rôle inhibiteur de TOX4 à l’étape d’intégration du VIH-1.Ensuite, j’ai mis au point des protocoles in vitro de couplage entre l’intégration et la transcription. L’utilisation d’extraits nucléaires pour transcrire par l’ARN polymérase II n’étant pas compatible avec le processus d’intégration, j’ai utilisé l’ARN polymérase T7 purifiée comme machinerie de transcription et étudié les conséquences sur l’intégration. Bien que l’ARN synthétisé inhibe l’intégration, j’ai pu montrer que le passage d’une ARN polymérase sur la matrice d’intégration n’affecte pas l’efficacité globale d’intégration.Enfin, la transcription modifiant la topologie de l’ADN, mon dernier objectif a été d’étudier in vitro l’effet de ce paramètre sur l’intégration. En utilisant des plasmides de différentes formes topologiques comme substrats accepteurs d’intégration, j’ai montré que l’intégration est favorisée sur les formes surenroulées négativement. J’ai également prouvé que cette sélectivité est indépendante de la présence de LEDGF/p75.Mes travaux constituent une étape dans la connaissance des bases moléculaires et mécanistiques de la sélectivité d’intégration du VIH-1 pouvant déboucher sur l’établissement de nouvelles stratégies antirétrovirales. / The integration of the DNA copy of HIV-1 in an infected cell is an essential step of the replication cycle of the retrovirus. It’s performed by a viral enzyme, called integrase, which constitutes a major target of antiviral strategies. Several studies suggest a regulation of integration selectivity by chromatin and transcription. The cellular protein LEDGF/p75, a transcriptional activator, interacting with both integrase and chromatin perfectly illustrates this regulation. My thesis project was to study the links between LEDGF/p75, chromatin and transcription during HIV-1 integration.First, my work validated in vitro the interaction between LEDGF/p75 and its partner TOX4, recently identified by our team. I have also shown that the TOX4 domain, interacting with LEDGF/p75, inhibits integration in vitro on chromatin templates in the presence of LEDGF/p75, suggesting an inhibitory role of TOX4 during the HIV-1 integration step.Then, I developed several in vitro protocols coupling HIV-1 integration and cellular transcription. As RNA polymerase II transcription machinery from Hela nuclear extracts prevents the integration process, I used purified T7 RNA polymerase to perform transcription and studied its consequences on integration. I could show that the synthetized RNA inhibits integration but that the transcription process per se does not affect global integration efficiency on the transcribed template. Since transcription modifies DNA topology, my last goal was to study the effect of this parameter on integration in vitro. Using plasmids of different topological forms as integration acceptor substrates, I showed that integration is enriched in negative supercoiled plasmids. I also proved that this selectivity is independent of the presence of LEDGF/p75. My work constitutes an initial step in understanding the molecular and mechanistic basis of HIV-1 integration selectivity that can lead to the establishment of new antiretroviral strategies.

Like all viruses, the replication of HIV-1 relies heavily on host proteins due to its limited genome products. HIV-1 integrase (IN) catalyzes the integration of viral DNA into host genome and also impacts other steps of viral replication cycle, all of which are assisted by various cellular proteins. Among them, LEDGF/p75 acts as the IN-to-chromatin tethering factor. However, whether other cellular cofactors also participate in this process still remains elusive. To gain insight into the mechanism of action of HIV-1 IN during viral integration, we used a previously described IN/yeast lethality system and our results revealed that the HIV-1 IN-induced yeast lethality absolutely required its chromatin binding ability. Since there is no yeast homolog of LEDGF/p75, it raises the possibility that IN may recruit other cellular cofactors for its chromatin targeting. Consistently, further analysis in mammalian cells indicated that HIV-1 IN was able to mediate chromatin binding independent of IN-LEDGF/p75 interaction and that HIV-1 fitness relied more on chromatin binding than LEDGF/p75 binding of IN. These data greatly enrich our current knowledge on the dynamic interplay within the ternary complex IN/LEDGF/chromatin. HIV-1 exploits multiple cellular cofactors not only to facilitate viral replication, but also to evade the host defense system in favor of the virus. IN is known to be an unstable protein, degraded by the host ubiquitin-proteasome pathway. To investigate how IN avoids the host degradation machinery in the context of viral infection, we showed that IN interacted with host protein Ku70 and protected itself from the Lys48-linked polyubiquitination proteasomal pathway. More importantly, Ku70 was shown to be incorporated into the progeny virus in an IN-dependent manner, and both cell- and virus- associated Ku70 were essential for HIV-1 replication. Finally, the data demonstrated that the interactions between HIV-1 IN and host cofactors can be regulated through its SUMO-interacting motifs (SIMs). Three putative SIMs (72VILV75; 200IVDI203 and 257IKII260) in IN were examined and shown to be essential for IN-LEDGF/p75 but not IN-Ku70 interaction. In summary, this study advances our knowledge of the interaction network between IN and its cofactors, which would have important implications for the design of anti-HIV drugs.

Like all viruses, the replication of HIV-1 relies heavily on host proteins due to its limited genome products. HIV-1 integrase (IN) catalyzes the integration of viral DNA into host genome and also impacts other steps of viral replication cycle, all of which are assisted by various cellular proteins. Among them, LEDGF/p75 acts as the IN-to-chromatin tethering factor. However, whether other cellular cofactors also participate in this process still remains elusive. To gain insight into the mechanism of action of HIV-1 IN during viral integration, we used a previously described IN/yeast lethality system and our results revealed that the HIV-1 IN-induced yeast lethality absolutely required its chromatin binding ability. Since there is no yeast homolog of LEDGF/p75, it raises the possibility that IN may recruit other cellular cofactors for its chromatin targeting. Consistently, further analysis in mammalian cells indicated that HIV-1 IN was able to mediate chromatin binding independent of IN-LEDGF/p75 interaction and that HIV-1 fitness relied more on chromatin binding than LEDGF/p75 binding of IN. These data greatly enrich our current knowledge on the dynamic interplay within the ternary complex IN/LEDGF/chromatin. HIV-1 exploits multiple cellular cofactors not only to facilitate viral replication, but also to evade the host defense system in favor of the virus. IN is known to be an unstable protein, degraded by the host ubiquitin-proteasome pathway. To investigate how IN avoids the host degradation machinery in the context of viral infection, we showed that IN interacted with host protein Ku70 and protected itself from the Lys48-linked polyubiquitination proteasomal pathway. More importantly, Ku70 was shown to be incorporated into the progeny virus in an IN-dependent manner, and both cell- and virus- associated Ku70 were essential for HIV-1 replication. Finally, the data demonstrated that the interactions between HIV-1 IN and host cofactors can be regulated through its SUMO-interacting motifs (SIMs). Three putative SIMs (72VILV75; 200IVDI203 and 257IKII260) in IN were examined and shown to be essential for IN-LEDGF/p75 but not IN-Ku70 interaction. In summary, this study advances our knowledge of the interaction network between IN and its cofactors, which would have important implications for the design of anti-HIV drugs.

VIH-1, l’agent étiologique du Syndrome de l’Immunodéficience Acquise, est un rétrovirus qui infecte les cellules immunitaires et détourne leur machinerie cellulaire pour se répliquer rapidement. Lors de l’infection, le génome ARN est rétrotranscrit en ADN par la transcriptase inverse virale (RT), puis l’insertion du génome proviral dans l’ADN de la cellule hôte est une étape obligatoire du cycle viral catalysée par l’enzyme virale Intégrase (IN). L’interaction de l’IN avec son cofacteur essentiel, la protéine nucléaire LEDGF/p75, dirige l’intégration à l’intérieur de gènes dans des régions fortement exprimées de la chromatine, ce qui permet la production efficace de nouveaux virions. Les Inhibiteurs Allostériques Intégrase-LEDGF (INLAIs) sont une nouvelle classe de molécules antirétrovirales se liant à l’IN au site de liaison de LEDGF/p75. Conçus pour inhiber compétitivement l’interaction protéine-protéine IN-LEDGF/p75, ils inhibent également les activités enzymatiques de l’Intégrase et augmentent son niveau de multimérisation.Nous avons étudié plusieurs nouvelles séries d’INLAIs de la société Mutabilis, et avons pu démontrer que ces molécules inhibent l’intégration, mais ont aussi un effet antirétroviral plus puissant et indépendant de LEDGF/p75 post-intégration au cours de la maturation des virions, qui conduit à la production de virus non infectieux, ayant une morphologie excentrique caractérisée par un défaut d’encapsidation du génome viral. Lors de l’infection de cellules par ces virus, le cycle viral s’arrête à l’étape de rétrotranscription du génome viral. Nous avons montré que ces virions contiennent pourtant un génome viral stable et fonctionnel, une RT active et l’ARNtLys3 qui sert d’amorce à la rétrotranscription, et ont également conservé leur immunoréactivité pour les lymphocytes B et T. En évaluant l’impact du polymorphisme de l’IN au voisinage du site de liaison, nous avons identifié le variant polymorphe Ala125, pour lequel l’INLAI MUT-A perd concomitamment son effet sur la maturation des virions et sur la multimérisation de l’IN, tandis qu’il inhibe aussi bien l’intégration et l’interaction IN-LEDGF, prouvant que l’effet tardif des INLAIs est associé à l’induction de la multimérisation de l’IN. Nous avons pu associer la multimérisation de l’IN à une déstabilisation du dimère par les INLAIs en analysant les co-structures de MUT-A avec les intégrases polymorphes. Les INLAIs, outre leur intérêt thérapeutique sont de remarquables réactifs qui ont permis de démontrer le rôle essentiel de l’intégrase à trois étapes clés du cycle viral du VIH-1 : la rétrotranscription, l’intégration et la maturation des virions. / HIV-1, the causative agent of AIDS, is a retrovirus that infects immune cells and hijacks their cell machinery to achieve rapid replication. In the course of infection, the RNA genome is reverse transcribed into DNA by the viral Reverse Transcriptase (RT) before the obligatory insertion of the proviral genome into the host cell DNA catalyzed by the viral enzyme Integrase (IN). The interaction of IN with its essential cofactor, the nuclear protein LEDGF/p75, targets integration within gene introns in highly transcribed chromatine regions, which allows efficient production of new virions. IN-LEDGF Allosteric Inhibitors (INLAIs) are a novel class of antiretroviral molecules binding IN at the LEDGF/p75-binding site. Designed to competitively inhibit IN-LEDGF/p75 protein-protein interaction, they are also capable of inhibiting IN enzymatic activities and raising the IN multimerization level.We studied several new INLAI series from the company Mutabilis. We could demonstrate that these molecules inhibit integration, but also have a more potent, LEDGF-independent, antiretroviral effect during virion maturation, resulting in the production of non-infectious virions. Virions produced upon INLAI treatment have an eccentric morphology characterized by an encapsidation defect of the viral genome, and lead to an infection block at reverse transcription. Yet, we showed that these virions package a stable and functional viral genome, an active RT and the tRNALys3 primer for reverse transcription, and also keep their immunoreactivity towards B- and T-cell lymphocytes. When evaluating the influence of polymorphism at the edge of the binding site, we identified the IN Ala125 polymorphic variant which causes the concomitant loss of MUT-A effect on virion maturation and IN multimerization, whereas inhibition of integration and IN-LEDGF interaction are maintained. This proves that INLAIs exert their late stage effect through induction of IN multimerization. We could associate IN multimerization to INLAI-induced dimer destabilization by analyzing MUT-A co-structures with polymorphic integrases. Beside their therapeutic interest INLAIs are highly valuable reagents that allowed to demonstrate the essential role of integrase at three key steps of the HIV-1 replication cycle, reverse transcription, integration and virus maturation.