Paul Lesbats

HIV-1 infection triggers multiple cellular pathways that modulate viral replication and may promote latency. Central to this process is the reverse transcription of the viral RNA into DNA and its integration into host chromatin, both processes being regulated by the cell. Emerging evidence suggests that homologous recombination (HR) proteins, especially RAD51, may influence both pre- and post-integration stages. In this study, we show that HIV-1 induces the rapid formation of RAD51 nuclear foci in a BRCA1/2-dependent manner, with RAD51 loading onto viral DNA immediately during reverse transcription. Using ChIP, imaging, and biochemical assays, we demonstrate that this process enhances viral infectivity and reverse transcription efficiency. Our findings reveal a previously unidentified cellular response that recruits repair factors to viral DNA, regulating reverse transcription and influencing viral DNA fate. This work uncovers a previously unknown role for RAD51 in modulating early HIV-1 replication, providing insights into host-virus interactions and potential therapeutic strategies.

Cellular chromatin is the first non-reversible contact point between incoming infectious agents, such as integrating viruses, and their host genomes. Retroviral integration depends on interactions between the viral intasome and host chromatin, involving interfaces between integrase, target DNA, and nucleosomal histones. While factors like LEDGF/p75 and chromatin compaction influence these interactions, the molecular mechanisms regulating access to nucleosomal interfaces remain unclear. In this study, we dissected how incoming intasomes engage nucleosome surfaces to facilitate integration. Using biochemical assays and molecular docking, we demonstrated that HIV-1 and PFV intasomes specifically bind distinct nucleosomal surfaces. Mapping these interactions onto trinucleosomes and simulating dynamic docking revealed that neighboring nucleosomes can mask functional interfaces, modulating HIV-1’s ability to access its target. Conversely, PFV intasomes are less affected by chromatin compaction due to persistent accessibility of active nucleosomal sites. These findings provide the first structural insights into how chromatin architecture influences retroviral integration. Our results highlight divergent strategies by HIV-1 and PFV to engage chromatin, revealing how nucleosome properties govern the formation of active strand transfer complexes and emphasizing the evolution of virus-specific adaptation to chromatin landscapes.

L’intégrase (IN) du VIH-1 est une enzyme clé catalysant l’insertion de l’ADN proviral dans le génome cellulaire au sein d’un complexe nucléoprotéique d’intégration.Les nombreux efforts apportés à l’étude du mécanisme d’intégration ont permis d’accumuler de multiples données sur ce processus complexe. Cependant plusieurs questions essentielles demeurent sans réponses en particulier concernant l’architecture fonctionnelle des complexes d’intégration de VIH-1. En effet même si les complexes actifs pour l’intégration sont relativement bien définis leur chronologie précoce de formation demeure inconnue. De même les phases tardives de leur association au substrat naturel d’intégration que constitue la chromatine restent floues. Enfin le rôle des facteurs du complexe de préintégration (CPI) dans la régulation des ces mécanismes doit être déterminé.Mon travail de thèse s’est reposé sur trois axes s’articulant autour de ces questions:-Comment est régulée la dynamique des phases précoces d’attachement des oligomères d’intégrase sur les extrémités virales ?-Quel est l’impact de la structure de l’ADN cible et de la chromatine sur l’association active des complexes d’intégration ?-Quel(s) rôle(s) joue(nt) les facteurs du CPI dans ces phases ?

Selection of a suitable chromatin environment during retroviral integration is a tightly regulated process. Most retroviruses, including spumaretroviruses, require mitosis for nuclear entry. However, whether intrinsic chromatin dynamics during mitosis modulates retroviral genome invasion is unknown. Previous work uncovered critical interactions of prototype foamy virus (PFV) Gag with nucleosomes via a highly conserved arginine anchor residue. Yet, the regulation of Gag–chromatin interaction and its functional consequences for spumaretrovirus biology remain obscure. Here, we investigated the kinetics of chromatin binding by Gag during mitosis and proviral integration in synchronized cells. We showed that alteration of Gag affinity for nucleosome binding induced untimely chromatin tethering during mitosis, decreased infectivity, and redistributed viral integration sites to markers associated with late replication timing of chromosomes. Mutant Gag proteins were, moreover, defective in their ability to displace the histone H4 tail from the nucleosome acidic patch of highly condensed chromatin. These data indicate that the chromatin landscape during Gag–nucleosome interactions is important for PFV integration site selection and that spumaretroviruses evolved high-affinity chromatin binding to overcome early mitosis chromatin condensation.

Selection of a suitable chromatin environment during retroviral integration is a tightly regulated and multilayered process that involves interplay between viral and host factors. However, whether intrinsic chromatin dynamics during mitosis modulate retroviral genome invasion is currently poorly described. Direct interaction between the spumaretrovirus prototype foamy virus (PFV) Gag protein and cellular chromatin has been described as a major determinant for integration site selection. A previous Gag chromatin-binding site (CBS)–nucleosome co-crystal structure revealed an interaction with the histone H2A-H2B acidic patch via a highly conserved arginine anchor residue. Yet, the molecular mechanisms regulating Gag-chromatin capture during PFV infection remain obscure. Here, we investigated the kinetics of Gag-chromatin interactions during mitosis and proviral integration of PFV-infected synchronized cells. Using Gag CBS variant viruses, we showed that alteration of Gag affinity for nucleosome binding induced untimely chromatin tethering during mitosis, decreased infectivity and redistributed viral integration sites to markers associated with late replication timing of host chromosomes. Mutant Gag proteins were moreover defective in their ability to displace the histone H4 tail from the nucleosome acidic patch of highly condensed mitotic chromatin. These data indicate that the mitotic chromatin landscape during Gag–nucleosome interactions hosts PFV integration site selection determinants and that spumaretroviruses evolved high-affinity chromatin binding to overcome early mitosis chromatin condensation for optimal viral DNA tethering, integration and infection.

The past decade illuminated the H2A-H2B acidic patch as a cornerstone for both nucleosome recognition and chromatin structure regulation. Higher-order folding of chromatin arrays is mediated by interactions of histone H4 tail with an adjacent nucleosome acidic patch.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent responsible for the recent coronavirus disease 2019 (COVID-19) pandemic. Productive SARS-CoV-2 infection relies on viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). Indeed, viral entry into cells is mostly mediated by the early interaction between the viral spike protein S and its ACE2 receptor. The S/ACE2 complex is, thus, the first contact point between the incoming virus and its cellular target; consequently, it has been considered an attractive therapeutic target. To further characterize this interaction and the cellular processes engaged in the entry step of the virus, we set up various in silico, in vitro and in cellulo approaches that allowed us to specifically monitor the S/ACE2 association. We report here a computational model of the SARS-CoV-2 S/ACE2 complex, as well as its biochemical and biophysical monitoring using pulldown, AlphaLISA and biolayer interferometry (BLI) binding assays. This led us to determine the kinetic parameters of the S/ACE2 association and dissociation steps. In parallel to these in vitro approaches, we developed in cellulo transduction assays using SARS-CoV-2 pseudotyped lentiviral vectors and HEK293T-ACE2 cell lines generated in-house. This allowed us to recapitulate the early replication stage of the infection mediated by the S/ACE2 interaction and to detect cell fusion induced by the interaction. Finally, a cell imaging system was set up to directly monitor the S/ACE2 interaction in a cellular context and a flow cytometry assay was developed to quantify this association at the cell surface. Together, these different approaches are available for both basic and clinical research, aiming to characterize the entry step of the original SARS-CoV-2 strain and its variants as well as to investigate the possible chemical modulation of this interaction. All these models will help in identifying new antiviral agents and new chemical tools for dissecting the virus entry step.

Foamy viruses (FV) are retroviruses belonging to the Spumaretrovirinae subfamily. They are non-pathogenic viruses endemic in several mammalian hosts like non-human primates, felines, bovines, and equines. Retroviral DNA integration is a mandatory step and constitutes a prime target for antiretroviral therapy. This activity, conserved among retroviruses and long terminal repeat (LTR) retrotransposons, involves a viral nucleoprotein complex called intasome. In the last decade, a plethora of structural insights on retroviral DNA integration arose from the study of FV. Here, we review the biochemistry and the structural features of the FV integration apparatus and will also discuss the mechanism of action of strand transfer inhibitors.

Retroviral integration site selection: a running Gag? –