Project 3- Study of the cellular responses to pathogenic genomes mobility

Integration of the viral genome into the cell involves the formation of a complex between integrase and viral DNA called intasome (for a review see (1)) which will transit inside the nucleus where it will catalyze integration. This process requires cellular factors, such as CPSF6 and LEDGF / p75, which help to transport the complex to areas of transcribed chromatin suitable for integration. After nuclear import of viral DNA into the integration complex and its integration into the host DNA, the integration locus must be repaired in order to establish a stable infection. All the results of the literature suggest that cellular DNA repair machinery actively participates in the stability of the infection process by acting at the level of the integration step while playing a restrictive role by limiting viral replication. In this context, interactions have been reported between IN and factors of the RAD family involved in the repair of DNA double strand breaks. The recent development of PCR-based methodologies making it possible to specifically quantify the repaired and unrepaired forms of the genome have also made it possible to confirm the involvement of the non-homologous repair pathway (NHEJ) and of DNA-PK in the post- integrative stages.

The work carried out in the laboratory has been able to demonstrate a role of the hRAD51 recombinase in the regulation of the early phases of virus replication and its integration. The selection of specific partners of the functional complexes formed between the IN and the viral DNA  have, moreover, led to the identification of new potential cofactors of the intasome, in addition to the cofactors already proven, like LEDGF / p75. The selection of cellular partners of the LEDGF / p75 • IN • DNAviral complex led us in particular to identify several proteins that interact specifically (directly or indirectly) with this complex. Among them, the BRCA1 and BRCA2 proteins caught our attention because they are part of the same homologous repair pathway involving hRAD51 previously identified. Interestingly, these proteins make it possible to load RAD51 on repair sites, in particular in chromatin regions transcribed by RNA polymerase II (extensive data from the literature) thanks to the ϒH2Ax modification made possible by the action of the histone chaperone FACT also identified as integration partner. This FACT complex participates in PolII transcription but also in the modulation of the chromatin structure for DNA repair as well as for the integration of HIV-1. Strikingly, LEDGF / p75 forms a tripartite complex with IN and FACT and can promote DNA repair by homologous recombination involving hRAD51, BRCA1 / 2 and which is also regulated by the accessibility of chromatin. Thus, all of these factors enriched in the PolII regions could constitute a protein network regulating and coordinating the different phases of integration: targeting, catalysis, dissociation of the complex and then site repair. We propose in this project to investigate this control network summarized in Figure 1.

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