title: Chromatinized templates reveal the requirement for the LEDGF/p75 PWWP domain during HIV-1 integration in vitro.
abstract: Integration is an essential step in the retroviral lifecycle, and the lentiviral integrase binding protein lens epithelium-derived growth factor (LEDGF)/p75 plays a crucial role during human immunodeficiency virus type 1 (HIV-1) cDNA integration. In vitro, LEDGF/p75 stimulates HIV-1 integrase activity into naked target DNAs. Here, we demonstrate that this chromatin-associated protein also stimulates HIV-1 integration into reconstituted polynucleosome templates. Activation of integration depended on the LEDGF/p75-integrase interaction with either type of template. A differential requirement for the dominant DNA and chromatin-binding elements of LEDGF/p75 was however observed when using naked DNA versus polynucleosomes. With naked DNA, the complete removal of these N-terminal elements was required to abate cofactor function. With polynucleosomes, activation mainly depended on the PWWP domain, and to a lesser extent on nearby AT-hook DNA-binding motifs. GST pull-down assays furthermore revealed 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.
authors.affiliation: Department of Virology, Unit of Structural Virology, Pasteur Institute, 25 rue du Dr Roux, 75724 Paris cedex 15, France.
authors.forename: Yaïr
authors.initials: Y
authors.lastname: Botbol
authors.forename: Nidhanapati K
authors.initials: NK
authors.lastname: Raghavendra
authors.forename: Shaila
authors.initials: S
authors.lastname: Rahman
authors.forename: Alan
authors.initials: A
authors.lastname: Engelman
authors.forename: Marc
authors.initials: M
authors.lastname: Lavigne
mesh_terms: D004247:DNA / Q000378:metabolism; D019427:HIV Integrase / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology / Q000235:genetics*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000378:metabolism; D009707:Nucleosomes / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013698:Templates, Genetic; D016662:Virus Integration
chemical_list: D036341:Intercellular Signaling Peptides and Proteins; D009707:Nucleosomes; C119380:lens epithelium-derived growth factor; D004247:DNA; D019427:HIV Integrase