THE ZINC FINGERS OF HIV NUCLEOCAPSID PROTEIN NCP7 DIRECT INTERACTIONSWITH THE VIRAL REGULATORY PROTEIN VPR

The 96-amino acid protein Vpr functions as a regulator of cellular pro cesses involved in human immunodeficiency virus, type 1 (HIV-1) life c ycle, in particular by interrupting cells division in the G(2) phase, Incorporation of Vpr in the virion was reported to be mediated by the C-terminal domain of the pr55(Gag) polyprotein precursor, which includ es NCp7, a protein involved in the genomic RNA encapsidation and p6, a protein required for particle budding, To precisely define the Gag an d Vpr sequences involved in this protein-protein interaction, NCp7, p6 , and Vpr as well as a series of derived peptides were synthesized usi ng Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry, Binding assays wer e carried out by Far Western experiments and by competition studies us ing (52-96)Vpr immobilized onto agarose beads, The results show that i nteraction between NCp7 and Vpr occurs in vitro by a recognition mecha nism requiring the zinc fingers of NCp7 and the last 16 amino acids of Vpr, Moreover, NCp10, the equivalent of NCp7 in Moloney murine leukem ia virus but not polysine inhibits Vpr-NCp7 complexation, Interestingl y enough, Vpr was found to interact with Gag, NCp15, and NCp7 but not with mature p6 in vitro. In vive mutations in NCp7 zinc fingers in an HIV-1 molecular clone led to viruses with important defects in Vpr enc apsidation. 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.