INCORPORATION OF VPR INTO HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 - ROLE OF CONSERVED REGIONS WITHIN THE P6 DOMAIN OF PR55(GAG)

The 96-amino acid Vpr protein is the only virion-associated regulatory protein encoded by the human immunodeficiency virus type 1 (HIV-1). V pr incorporation into the viral particle is most likely due to an inte raction with a viral structural protein. Recent data have shown that D NA encoding for the p55 Gag precursor protein (pr55(gag)) is minimal v iral genetic information necessary for Vpr incorporation. Other studie s have suggested that the p6 portion of pr55(gag), which is unique to lentiviruses, is involved in Vpr incorporation. To investigate the mec hanism of incorporation of Vpr into HIV-1 virions, COS-7 cells were co transfected with ptrENV, an expression vector that encodes all of the HIV-1 regulatory proteins including Rev and Vpr, and different constru cts of pIIIgagCAR, a rev-dependent Gag expression plasmid that encodes Pr55(gag) and the viral protease. Virions produced from gag construct s containing a premature p6 termination codon at positions Leu-1, Ser- 17, Tyr-36, or Leu-44 lacked detectable Vpr. In contrast, gag construc ts with double Pro-10-Pro-11 substitutions for Leu-10-Leu-11 or a prem ature termination codon at position Pro-49 of p6 were still able to in corporate Vpr, however, with lower efficiency than wild type. The muta tions described in this study affected directly two short regions with in the p6 domain, which are highly conserved among primate immunodefic iency viruses. Our results suggest that the conserved (P-T/S-A-P-P) an d (L-X-S-L-F-G) motifs located near the N-terminus and C-terminus, res pectively, of the p6 domain of Gag are critical for Vpr incorporation into HIV-1 virions.