TARGETING FOREIGN PROTEINS TO HUMAN-IMMUNODEFICIENCY-VIRUS PARTICLES VIA FUSION WITH VPR AND VPX

The human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vpr and Vpx proteins are packaged into virions through virus type-specific interac tions with the Gag polyprotein precursor, To examine whether HIV-1 Vpr (Vpr1) and HIV-2 Vpx (Vpx2) could be used to target foreign proteins to the HIV particle, their open reading frames were fused in frame wit h genes encoding the bacterial staphylococcal nuclease (SN), an enzyma tically inactive mutant of SN (SN), and chloramphenicol acetyltransfe rase (CAT), Transient expression in a T7-based vaccinia virus system d emonstrated the synthesis of appropriately sized Vpr1-SN/SN and Vpx2- SN/SN fusion proteins which, when coexpressed with their cognate p55( Gag) protein, were efficiently incorporated into virus-like particles, Packaging of the fusion proteins was dependent on virus type-specific determinants, as previously seen with wild-type Vpr and Vpx proteins. Particle-associated Vpr1-SN and Vpx-SN fusion proteins were enzymatic ally active, as determined by in vitro digestion of lambda phage DNA, To determine whether functional Vpr1 and Vpx2 fusion proteins could be targeted to HIV particles, the gene fusions were cloned into an HIV-2 long terminal repeat/Rev response element-regulated expression vector and cotransfected with wild-type HIV-1 and HIV-2 proviruses. Western blot (immunoblot) analysis of sucrose gradient-purified virions reveal ed that both Vpr1 and Vpx2 fusion proteins were efficiently packaged r egardless of whether SN, SN, or CAT was used as the C-terminal fusion partner, Moreover, the fusion proteins remained enzymatically active and were packaged in the presence of wild-type Vpr and Vpx proteins. I nterestingly, virions also contained smaller proteins that reacted wit h antibodies specific for the accessory proteins as well as SN and CAT fusion partners. Since similar proteins were absent from Gag-derived virus-like particles and from virions propagated in the presence of an HN protease inhibitor, they must represent cleavage products produced by the viral protease, Taken together, these results demonstrate that Vpr and Vpx can be used to target functional proteins, including pote ntially deleterious enzymes, to the human or simian immunodeficiency v irus particle. These properties may be exploitable for studies of HIV particle assembly and maturation and for the development of novel anti viral strategies.