title: Structural and functional role of INI1 and LEDGF in the HIV-1 preintegration complex.
abstract: Integration of the HIV-1 cDNA into the human genome is catalyzed by the viral integrase (IN) protein. Several studies have shown the importance of cellular cofactors that interact with integrase and affect viral integration and infectivity. In this study, we produced a stable complex between HIV-1 integrase, viral U5 DNA, the cellular cofactor LEDGF/p75 and the integrase binding domain of INI1 (INI1-IBD), a subunit of the SWI/SNF chromatin remodeling factor. The stoichiometry of the IN/LEDGF/INI1-IBD/DNA complex components was found to be 4/2/2/2 by mass spectrometry and Fluorescence Correlation Spectroscopy. Functional assays showed that INI1-IBD inhibits the 3' processing reaction but does not interfere with specific viral DNA binding. Integration assays demonstrate that INI1-IBD decreases the amount of integration events but inhibits by-product formation such as donor/donor or linear full site integration molecules. Cryo-electron microscopy locates INI1-IBD within the cellular DNA binding site of the IN/LEDGF complex, constraining the highly flexible integrase in a stable conformation. Taken together, our results suggest that INI1 could stabilize the PIC in the host cell, by maintaining integrase in a stable constrained conformation which prevents non-specific interactions and auto integration on the route to its integration site within nucleosomes, while LEDGF organizes and stabilizes an active integrase tetramer suitable for specific vDNA integration. Moreover, our results provide the basis for a novel type of integrase inhibitor (conformational inhibitor) representing a potential new strategy for use in human therapy.
authors.affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire, Département de Biologie Structurale intégrative, Université de Strasbourg, U596 INSERM, UMR7104 CNRS, Illkirch, France.
authors.forename: Benoit
authors.initials: B
authors.lastname: Maillot
authors.forename: Nicolas
authors.initials: N
authors.lastname: Lévy
authors.forename: Sylvia
authors.initials: S
authors.lastname: Eiler
authors.forename: Corinne
authors.initials: C
authors.lastname: Crucifix
authors.forename: Florence
authors.initials: F
authors.lastname: Granger
authors.forename: Ludovic
authors.initials: L
authors.lastname: Richert
authors.forename: Pascal
authors.initials: P
authors.lastname: Didier
authors.forename: Julien
authors.initials: J
authors.lastname: Godet
authors.forename: Karine
authors.initials: K
authors.lastname: Pradeau-Aubreton
authors.forename: Stéphane
authors.initials: S
authors.lastname: Emiliani
authors.forename: Alexis
authors.initials: A
authors.lastname: Nazabal
authors.forename: Paul
authors.initials: P
authors.lastname: Lesbats
authors.forename: Vincent
authors.initials: V
authors.lastname: Parissi
authors.forename: Yves
authors.initials: Y
authors.lastname: Mely
authors.forename: Dino
authors.initials: D
authors.lastname: Moras
authors.forename: Patrick
authors.initials: P
authors.lastname: Schultz
authors.forename: Marc
authors.initials: M
authors.lastname: Ruff
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D020285:Cryoelectron Microscopy; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005454:Fluorescence Polarization; D019427:HIV Integrase / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology / Q000502:physiology*; D006801:Humans; D013058:Mass Spectrometry; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000378:metabolism*; D011487:Protein Conformation; D000071796:SMARCB1 Protein; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors / Q000378:metabolism*; D016662:Virus Integration / Q000502:physiology*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; C504649:PSIP1 protein, human; D000071796:SMARCB1 Protein; C513266:SMARCB1 protein, human; D014157:Transcription Factors; D019427:HIV Integrase