Interaction of human immunodeficiency virus type 1 Vif with Gag and Gag-Pol precursors: co-encapsidation and interference with viral protease-mediated Gag processing.
Interactions of human immunodeficiency virus type 1 (HIV-1) Vif protein with various forms of Gag and Gag-Pol precursors expressed in insect cells were investigated in vivo and in vitro by co-encapsidation, co-precipitation and viral protease (PR)-mediated Gag processing assays. Addressing of Gag to the plasma membrane, its budding as extracellular virus-like particles (VLP) and the presence of the p6 domain were apparently not required for Vif encapsidation, as non-N-myristoylated Deltap6-Gag and Vif proteins were co-encapsidated into intracellular VLP. Encapsidation of Vif occurred at significantly higher copy numbers in extracellular VLP formed from N-myristoylated, budding-competent Gag-Pol precursors harbouring an inactive PR domain or in chimaeric VLP composed of Gag and Gag-Pol precursors compared with the Vif content of Pr55Gag VLP. Vif encapsidation efficiency did not seem to correlate directly with VLP morphology, since these chimaeric VLP were comparable in size and shape to Pr55Gag VLP. Vif apparently inhibited PR-mediated Pr55Gag processing in vitro, with preferential protection of cleavage sites at the MA-CA and CA-NC junctions. Vif was resistant to PR action in vitro under conditions that allowed full Gag processing, and no direct interaction between Vif and PR was detected in vivo or in vitro. This suggested that inhibition by Vif of PR-mediated Gag processing resulted from interaction of Vif with the Gag substrate and not with the enzyme. Likewise, the higher efficiency of Vif encapsidation by Gag-Pol precursor compared with Pr55Gag was probably not mediated by direct binding of Vif to the Gag-Pol-embedded PR domain, but more likely resulted from a particular conformation of the Gag structural domains of the Gag-Pol precursor.
Bardy
Martine
M
Laboratoire de Virologie et Pathogénèse Virale, CNRS UMR-5537, Faculté de Médecine RTH Laennec de Lyon, 7 rue Guillaume Paradin, 69372 Lyon Cedex 08, France1.
Gay
Bernard
B
Laboratoire de Virologie et Pathogénèse Virale, CNRS UMR-5537, Faculté de Médecine RTH Laennec de Lyon, 7 rue Guillaume Paradin, 69372 Lyon Cedex 08, France1.
Pébernard
Stéphanie
S
Laboratoire de Virologie et Pathogénèse Virale, CNRS UMR-5537, Faculté de Médecine RTH Laennec de Lyon, 7 rue Guillaume Paradin, 69372 Lyon Cedex 08, France1.
Chazal
Nathalie
N
Laboratoire de Virologie et Pathogénèse Virale, CNRS UMR-5537, Faculté de Médecine RTH Laennec de Lyon, 7 rue Guillaume Paradin, 69372 Lyon Cedex 08, France1.
Courcoul
Marianne
M
Unité de Pathogénie des Infections à Lentivirus, INSERM U-372, Campus de Luminy, Marseille, France2.
Vigne
Robert
R
Unité de Pathogénie des Infections à Lentivirus, INSERM U-372, Campus de Luminy, Marseille, France2.
Decroly
Etienne
E
Unité de Pathogénie des Infections à Lentivirus, INSERM U-372, Campus de Luminy, Marseille, France2.
Boulanger
Pierre
P
Laboratoire de Virologie et Pathogénèse Virale, CNRS UMR-5537, Faculté de Médecine RTH Laennec de Lyon, 7 rue Guillaume Paradin, 69372 Lyon Cedex 08, France1.
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D015685:Fusion Proteins, gag-pol / Q000235:genetics / Q000378:metabolism*; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D016346:Gene Products, vif / Q000235:genetics / Q000378:metabolism*; D016333:HIV Protease / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D014771:Virion / Q000378:metabolism; D019065:Virus Assembly; D054320:vif Gene Products, Human Immunodeficiency Virus
D015685:Fusion Proteins, gag-pol; D015683:Gene Products, gag; D016346:Gene Products, vif; D011498:Protein Precursors; D011994:Recombinant Proteins; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D054320:vif Gene Products, Human Immunodeficiency Virus; D016333:HIV Protease