Structural Basis for HIV-1 DNA Integration in the Human Genome, Role of the LEDGF/P75 Cofactor

Authors

Fabrice Michel1, Corinne Crucifix1, Florence Granger1, Sylvia Eiler1, Jean-François Mouscadet2, Sergei Korolev3, Julia Agapkina3, Rustam Ziganshin3, Marina Gottikh3, Alexis Nazabal4, Stéphane Emiliani5,6, Richard Benarous7, Dino Moras1, Patrick Schultz1, and Marc Ruff1

Organizations

1. IGBMC (Institut de Ge´ne´tique et de Biologie Moléculaire et Cellulaire), Département de Biologie et de Génomique Structurales, UDS, CNRS, INSERM, Illkirch, France
2. Laboratoire de Biotechnologie et Pharmacologie Génétique Appliquée, CNRS, ENS-Cachan, Institut d’Alembert, Cachan, France
3. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia
4. Department of Chemistry and Applied Biosciences ETH Zurich and CovalX, Technoparkstrasse, Zurich, Switzerland
5. Institut Cochin, Département des Maladies Infectieuses, Universite´ Paris Descartes, CNRS, Paris, France
6. Inserm, Paris, France
7. CellVir, Evry, France

Abstract

Integration of the human immunodeficiency virus (HIV-1) cDNA into the human genome is catalysed by integrase. Several studies have shown the importance of the interaction of cellular cofactors with integrase for viral integration and infectivity. In this study, we produced a stable and functional complex between the wild-type full-length integrase (IN) and the cellular cofactor LEDGF/p75 that shows enhanced in vitro integration activity compared with the integrase alone. Mass spectrometry analysis and the fitting of known atomic structures in cryo negatively stain electron microscopy (EM) maps revealed that the functional unit comprises two asymmetric integrase dimers and two LEDGF/p75 molecules. In the presence of DNA, EM revealed the DNA-binding sites and indicated that, in each asymmetric dimer, one integrase molecule performs the catalytic reaction, whereas the other one positions the viral DNA in the active site of the opposite dimer. The positions of the target and viral DNAs for the 3′ processing and integration reaction shed light on the integration mechanism, a process with wide implications for the understanding of viral-induced pathologies.

CovalX Technology Used

HM1
K200

Outcomes

GST-IN and His6-LEDGF were produced using E. coli BL21 (DE3) as the host strain and then transformed using pRARE that was isolated from the DE3 strain. The proteins were then incubated, expressed and purified. The purified protein complexes were subjected to cross-linking using the CovalX K200 Stabilization Kit. 10 μL of the cross-linking reagent was added at 0.6-1.2 μM of the complex and then incubated for 2 hours. The samples were mixed with a matrix (1:2 v/v) sinapic acid (10 mg/ml) containing 70% acetonitrile diluted in deionized water with 0.1% TFA). 0.5 μL of the final mixture was spotted on a MALDI target using the dried-droplet method. The samples were analyzed using a mass spectrometer that had been modified with a CovalX HM1 detection system.