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Journal of Virology, December 2003, p. 12507-12522, Vol. 77, No. 23
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.23.12507-12522.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

The Human Polycomb Group EED Protein Interacts with the Integrase of Human Immunodeficiency Virus Type 1

Sébastien Violot,1 Saw See Hong,1 Dina Rakotobe,1 Caroline Petit,2,{dagger} Bernard Gay,1,{ddagger} Karen Moreau,1,§ Geneviève Billaud,1 Stéphane Priet,3 Joséphine Sire,3 Olivier Schwartz,2 Jean-François Mouscadet,4 and Pierre Boulanger1*

Laboratoire de Virologie and Pathogénèse Virale, Faculté de Médecine RTH Laennec, CNRS UMR-5537 and Université Claude Bernard Lyon 1, 69372 Lyon Cedex 08,1 Laboratoire Rétrovirus et Transfert Génétique, Institut Pasteur, 75724 Paris Cedex 15,2 Unité de Pathogénie des Infections à Lentivirus, INSERM U-372, 13276 Marseille Cedex 09,3 CNRS UMR-8532, Ecole Normale Supérieure, 94235 Cachan Cedex, France4

Received 21 April 2003/ Accepted 23 August 2003

Human EED, a member of the superfamily of WD-40 repeat proteins and of the Polycomb group proteins, has been identified as a cellular partner of the human immunodeficiency virus type 1 (HIV-1) matrix (MA) protein (R. Peytavi et al., J. Biol. Chem. 274:1635-1645, 1999). In the present study, EED was found to interact with HIV-1 integrase (IN) both in vitro and in vivo in yeast. In vitro, data from mutagenesis studies, pull-down assays, and phage biopanning suggested that EED-binding site(s) are located in the C-terminal domain of IN, between residues 212 and 264. In EED, two putative discrete IN-binding sites were mapped to its N-terminal moiety, at a distance from the MA-binding site, but EED-IN interaction also required the integrity of the EED last two WD repeats. EED showed an apparent positive effect on IN-mediated DNA integration reaction in vitro, in a dose-dependent manner. In situ analysis by immunoelectron microscopy (IEM) of cellular distribution of IN and EED in HIV-1-infected cells (HeLa CD4+ cells or MT4 lymphoid cells) showed that IN and EED colocalized in the nucleus and near nuclear pores, with maximum colocalization events occurring at 6 h postinfection (p.i.). Triple colocalizations of IN, EED, and MA were also observed in the nucleoplasm of infected cells at 6 h p.i., suggesting the ocurrence of multiprotein complexes involving these three proteins at early steps of the HIV-1 virus life cycle. Such IEM patterns were not observed with a noninfectious, envelope deletion mutant of HIV-1.

* Corresponding author. Mailing address: Laboratoire de Virologie and Pathogénèse Virale, Faculté de Médecine RTH Laennec de Lyon, 7, Rue Guillaume Paradin, 69372 Lyon Cedex 08, France. Phone: 33-4-7877-8621. Fax: 33-4-7877-8751. E-mail: Pierre.Boulanger{at}laennec.univ-lyon1.fr.

{dagger} Present address: Institut Cochin de Génétique Moléculaire, 75014 Paris, France.

{ddagger} Present address: Laboratoire Infections Rétrovirales et Signalisation Cellulaire, CNRS UMR 5121, Institut de Biologie, 34060 Montpellier, France.

§ Present address: Laboratoire Génétique et Cancer, CNRS UMR 5641 and Université Claude Bernard Lyon 1, 69373 Lyon Cedex 08, France.

Journal of Virology, December 2003, p. 12507-12522, Vol. 77, No. 23
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.23.12507-12522.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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