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Accueil du site > Animations Scientifiques > Séminaires 2008 > Regulation of HIV-1 integration by chromatin structure and LEDGF/p75 protein, in vitro studies

Regulation of HIV-1 integration by chromatin structure and LEDGF/p75 protein, in vitro studies

par Webmaster - 23 janvier 2008

Orateur :

Marc Lavigne, Unité de Virologie Structurale, Institut Pasteur, CNRS, Paris

Salle :


Sujet :

Integration of retroviral cDNA into the genome of the infected cell is an essential step of HIV-1 replication cycle. Understanding the molecular mechanisms that regulate the efficiency and selectivity of integration will have a major impact on the development of new antiviral strategies. Integration is performed by a virus-encoded enzyme, called integrase and can be reproduced in vitro, using a recombinant enzyme and naked DNA fragments as viral substrate and cellular host template. However, the actual target of integration is chromatin and the structure and components of this large nucleoprotein complex are suspected to control in vivo the efficiency and selectivity of integration. Furthermore, several cellular proteins interacting with both integrase and chromatin also regulate integration. One of these cofactors is the lens epithelium-derived growth factor LEDGF/p75, that is essential for HIV-1 integration and infectivity.

Our goal is to determine the molecular mechanisms involved in cellular regulation of HIV-1 integration, with a particular focus on chromatin and LEDGF/p75. We have established an in vitro integration assay on various reconstituted chromatin templates. Using this assay, we have observed that chromatin remodeling by the SWI/SNF complex doesn’t change the efficiency of integration but affects its selectivity. In collaboration with Alan Engelman (Harvard University, Boston USA), we have shown that LEDGF/p75 stimulates HIV-1 integrase activity on both naked DNA and reconstituted polynucleosome templates. On both templates, activation of integration depends on the LEDGF/p75-integrase interaction. However, a differential requirement for the DNA and chromatin binding elements of LEDGF/p75 is observed when using naked DNA versus polynucleosomes. With naked DNA, the complete removal of these elements is required to ablate cofactor function. With polynucleosomes, activation mainly depends on the PWWP domain, and to a lesser extent on nearby AT-hook DNA binding motifs. GST pull down assays furthermore reveal a role for the PWWP domain in binding to nucleosomes. These results are completely consistent with recent ex vivo studies that characterized the PWWP and integrase-binding domains of LEDGF/p75 as crucial for restoring HIV-1 infection to LEDGF-depleted cells. Our studies therefore establish novel in vitro conditions, highlighting chromatinized DNA as target acceptor templates, for physiologically relevant studies of LEDGF/p75 in lentiviral cDNA integration.

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