Contributors: Rollin, Bertrand; Virologie et pathogenèse virale (VPV); Université Claude Bernard Lyon 1 (UCBL); Université de Lyon-Université de Lyon-Centre National de la Recherche Scientifique (CNRS); Rétrovirus et Transfert Génétique; Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM); Pathogénie des infections à lentivirus; Institut National de la Santé et de la Recherche Médicale (INSERM)-IFR48; Institut des sciences biologiques - CNRS Biologie (INSB-CNRS)-Institut des sciences biologiques - CNRS Biologie (INSB-CNRS); Laboratoire de Biotechnologie et Pharmacogénétique Appliquée (LBPA); École normale supérieure - Cachan (ENS Cachan)-Centre National de la Recherche Scientifique (CNRS); le SIDA (ANRS; AC14-2 'HIV-1 Integrase and Preintegration Complex'). S.V. received an ANRS fellowship, and S.P. received a fellowship from the French ECS Association (Ensemble contre le SIDA). We are deeply grateful to Simone Peyrol and Isabelle Leparc-Goffart (Centre d'Imagerie de la Faculte´ de Me´decine Laennec) and to Paul Paulet (Centre Re´gional d'Imagerie Cellulaire de Montpellier) for significant contributions to the EM specimen processing, image digitalization, and photography. We are also grateful to Jean-Claude Cortay for valuable advice on pT7-7 cloning, fast-performance liquid chromatography, and protein purification. We thank Roger Monier, Catherine Dargemont, Robert Vigne, Etienne Decroly, Gilles Que´rat, and Corinne Ronfort for fruitful discussions during this study. We are indebted to Franc¸ois Grateau, Hospices Civils de Lyon, for the financing of the MegaView II TEM camera and automatic MT-X ultramicrotome (RMC EM Products Group, Ventana Medical Systems, Inc., Tucson, Ariz.).; Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM); INSB-INSB
نبذة مختصرة : 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.
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