title: Proteolytic activity of human immunodeficiency virus Vpr- and Vpx-protease fusion proteins.
abstract: In addition to Gag, Pol, and Env, primate lentiviruses encode other virion-associated proteins, including Vpr, Vpx, and Vif. Vpr- and Vpx-staphylococcal nuclease and chloramphenicol acetyltransferase fusion proteins incorporate into human immunodeficiency virus (HIV) virions and retain enzyme activity when expressed in trans with HIV proviruses (Wu et al., J. Virol. 69, 3389, 1995). To explore whether the viral protease (PR) could be expressed as a proteolytically active fusion protein, the HIV PR coding region was fused in-frame with the HIV-2 vpx and HIV-1 vpr genes. Using a vaccinia virus-T7 expression system, the Vpx-PR fusion protein was expressed and formed homodimers. Coexpression with Pr55Gag demonstrated that Vpx-PR possessed Gag-specific proteolytic activity and inhibited the production of Gag virus-like particles. Trans-expression of a PR-Vpr fusion protein with HIV-1 provirus caused a profound reduction in viral protein expression and virion production. Importantly, the PR-Vpr fusion protein caused a similar level of inhibition and intracellular cleavage of Pr55Gag precursor protein when coexpressed with protease defective HIV-1 provirus. The inhibitory effect of PR-Vpr expression on virion production was markedly greater than that of PR alone. These results indicate that Vpr arguments the intracellular proteolytic activity of PR when expressed as a fusion protein and thus may be relevant for the expression of PR in intracellular immunization strategies against HIV infection. Moreover, the ability to express and package enzymatically active PR-Vpr fusion protein, independent of Gag/Pol, may provide a novel means to study enzyme function.
authors.affiliation: Department of Medicine, University of Alabama at Birmingham 35294, USA.
authors.forename: X
authors.initials: X
authors.lastname: Wu
authors.forename: H
authors.initials: H
authors.lastname: Liu
authors.forename: H
authors.initials: H
authors.lastname: Xiao
authors.forename: J C
authors.initials: JC
authors.lastname: Kappes
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D004279:DNA, Viral; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism; D016336:Gene Products, vpr / Q000235:genetics / Q000378:metabolism*; D016333:HIV Protease / Q000235:genetics / Q000378:metabolism*; D017320:HIV Protease Inhibitors / Q000494:pharmacology; D015497:HIV-1 / Q000201:enzymology* / Q000254:growth & development; D015498:HIV-2 / Q000737:chemistry / Q000201:enzymology*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009025:Morpholines / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics / Q000378:metabolism*; D054325:vpr Gene Products, Human Immunodeficiency Virus
chemical_list: D004279:DNA, Viral; D015683:Gene Products, gag; D016336:Gene Products, vpr; D017320:HIV Protease Inhibitors; D009025:Morpholines; D010455:Peptides; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; C074286:VPX protein, Human immunodeficiency virus 2; D054334:Viral Regulatory and Accessory Proteins; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D054325:vpr Gene Products, Human Immunodeficiency Virus; C072111:L 689502; D016333:HIV Protease