Abstract
BackgroundInsertion of retroviral genome DNA occurs in the chromatin of the host cell. This step is modulated by chromatin structure as nucleosomes compaction was shown to prevent HIV-1 integration and chromatin remodeling has been reported to affect integration efficiency. LEDGF/p75-mediated targeting of the integration complex toward RNA polymerase II (polII) transcribed regions ensures optimal access to dynamic regions that are suitable for integration. Consequently, we have investigated the involvement of polII-associated factors in the regulation of HIV-1 integration.ResultsUsing a pull down approach coupled with mass spectrometry, we have selected the FACT (FAcilitates Chromatin Transcription) complex as a new potential cofactor of HIV-1 integration. FACT is a histone chaperone complex associated with the polII transcription machinery and recently shown to bind LEDGF/p75. We report here that a tripartite complex can be formed between HIV-1 integrase, LEDGF/p75 and FACT in vitro and in cells. Biochemical analyzes show that FACT-dependent nucleosome disassembly promotes HIV-1 integration into chromatinized templates, and generates highly favored nucleosomal structures in vitro. This effect was found to be amplified by LEDGF/p75. Promotion of this FACT-mediated chromatin remodeling in cells both increases chromatin accessibility and stimulates HIV-1 infectivity and integration.ConclusionsAltogether, our data indicate that FACT regulates HIV-1 integration by inducing local nucleosomes dissociation that modulates the functional association between the incoming intasome and the targeted nucleosome.
Highlights
Insertion of retroviral genome DNA occurs in the chromatin of the host cell
INviral DNA complexes were checked by in vitro concerted integration activity. These fractions were incubated with cellular protein extracts from HIV-1 permissive HeLa P4 cells previously counterselected against beads coupled to DNA alone to limit the unspecific selection of potential DNA and avidin binders
Given the requirement of open chromatin structure for efficient HIV-1 integration, we propose that the activated integration observed in cells inhibited or knockdown for FACT, results from an increased access of nucleosomes for the HIV-1 intasome as supported by our in vitro analysis
Summary
Insertion of retroviral genome DNA occurs in the chromatin of the host cell. This step is modulated by chromatin structure as nucleosomes compaction was shown to prevent HIV-1 integration and chromatin remod‐ eling has been reported to affect integration efficiency. The functional association between retroviral intasomes and chromatin has been shown to be modulated both by chromatin and intasome structures, thereby governing their preference for specific target DNA flexibility and nucleosome density both in vitro and in vivo depending on the retroviral genus [8]. This leads to a specific and distinct requirement for chromatin structures depending on the viral integration machinery. Previous works showed that, while Avian Sarkoma Leukosis Virus (ASLV) and HIV-1 integration was preferred in regions of the chromatin with low nucleosomes density both in vitro and in vivo, PFV and Murine Leukemia Virus (MLV) integration accommodate more different chromatin structures with a significant preference for regions of high nucleosomes density [8]
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