Structural and functional role of INI1 and LEDGF in the HIV-1 preintegration complex

PLoS One. 2013 Apr 11;8(4):e60734. doi: 10.1371/journal.pone.0060734. Print 2013.

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.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / metabolism*
  • Chromosomal Proteins, Non-Histone / metabolism*
  • Cryoelectron Microscopy
  • DNA-Binding Proteins / metabolism*
  • Fluorescence Polarization
  • HIV Integrase / metabolism*
  • HIV-1 / enzymology
  • HIV-1 / physiology*
  • Humans
  • Mass Spectrometry
  • Models, Molecular*
  • Multiprotein Complexes / metabolism*
  • Protein Conformation
  • SMARCB1 Protein
  • Spectrometry, Fluorescence
  • Transcription Factors / metabolism*
  • Virus Integration / physiology*

Substances

  • Adaptor Proteins, Signal Transducing
  • Chromosomal Proteins, Non-Histone
  • DNA-Binding Proteins
  • Multiprotein Complexes
  • PSIP1 protein, human
  • SMARCB1 Protein
  • SMARCB1 protein, human
  • Transcription Factors
  • HIV Integrase

Grant support

This work was supported by grants from the CNRS, the INSERM, SIDACTION, the French National Agency for Research against AIDS (ANRS), the SPINE 2 European Project (FP6 Contract N° QLG2- CT-2002-00988 and 031220), the French Infrastructure for Integrated Structural Biology (FRISBI, ANR-10-INSB-05-01) and Instruct, part of the European Strategy Forum on Research Infrastructure (ESFRI) supported by national members subscription. B.M. was supported by a doctoral fellowship from ANRS. N.L. was supported by a postdoctoral fellowship from ANRS. K.P.A. was supported by a postdoctoral fellowship from SIDACTION. V.P. was supported by ANR “RETROCONTROL” grant AO2012–2016 and SIDACTION AO2011–2013. P.S. acknowledges support from the Association pour la Recherche sur le Cancer (ARC) and the Fondation pour la Recherche Médicale (FRM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.