The zinc fingers of HIV nucleocapsid protein NCp7 direct interactions with the viral regulatory protein Vpr.
The 96-amino acid protein Vpr functions as a regulator of cellular processes involved in human immunodeficiency virus, type 1 (HIV-1) life cycle, in particular by interrupting cells division in the G2 phase. Incorporation of Vpr in the virion was reported to be mediated by the C-terminal domain of the Pr55(Gag) polyprotein precursor, which includes NCp7, a protein involved in the genomic RNA encapsidation and p6, a protein required for particle budding. To precisely define the Gag and Vpr sequences involved in this protein-protein interaction, NCp7, p6, and Vpr as well as a series of derived peptides were synthesized using Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. Binding assays were carried out by Far Western experiments and by competition studies using (52-96)Vpr immobilized onto agarose beads. The results show that interaction between NCp7 and Vpr occurs in vitro by a recognition mechanism requiring the zinc fingers of NCp7 and the last 16 amino acids of Vpr. Moreover, NCp10, the equivalent of NCp7 in Moloney murine leukemia virus but not polysine inhibits Vpr-NCp7 complexation. Interestingly enough, Vpr was found to interact with Gag, NCp15, and NCp7 but not with mature p6 in vitro. In vivo mutations in NCp7 zinc fingers in an HIV-1 molecular clone led to viruses with important defects in Vpr encapsidation. Together, these results suggest that NCp7 cooperates with p6 to induce Vpr encapsidation in HIV-1 mature particles. The NCp7-Vpr complex could also be important for interaction of Vpr with cellular proteins involved in cell division.
de Rocquigny
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Département de Pharmacochimie Moléculaire et Structurale, INSERM U266, CNRS URA D 1500, UFR des Sciences Pharmaceutiques et Biologiques, 75270 Paris Cedex 06, France.
Petitjean
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P
Tanchou
V
V
Decimo
D
D
Drouot
L
L
Delaunay
T
T
Darlix
J L
JL
Roques
B P
BP
D000595:Amino Acid Sequence; D001665:Binding Sites; D002213:Capsid / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D016336:Gene Products, vpr / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D014764:Viral Proteins; D014771:Virion / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; D016336:Gene Products, vpr; C071884:NCP7 protein, Human immunodeficiency virus 1; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C083002:p6 gag protein, Human immunodeficiency virus 1