Conserved cysteines of the human immunodeficiency virus type 1 protease are involved in regulation of polyprotein processing and viral maturation of immature virions.
We investigated the role of the two highly conserved cysteine residues, cysteines 67 and 95, of the human immunodeficiency virus type 1 (HIV-1) protease in regulating the activity of that protease during viral maturation. To this end, we generated four HIV-1 molecular clones: the wild type, containing both cysteine residues; a protease mutant in which the cysteine at position 67 was replaced by an alanine (C67A); a C95A protease mutant; and a double mutant (C67A C95A). When immature virions were produced in the presence of an HIV-1 protease inhibitor, KNI-272, and the inhibitor was later removed, limited polyprotein processing was observed for wild-type virion preparations over a 20-h period. Treatment of immature wild-type virions with the reducing agent dithiothreitol considerably improved the rate and extent of Gag processing, suggesting that the protease is, in part, reversibly inactivated by oxidation of the cysteine residues. In support of this, C67A C95A virions processed Gag up to fivefold faster than wild-type virions in the absence of a reducing agent. Furthermore, oxidizing agents, such as H2O2 and diamide, inhibited Gag processing of wild-type virions, and this effect was dependent on the presence of cysteine 95. Electron microscopy revealed that a greater percentage of double-mutant virions than wild-type virions developed a mature-like morphology on removal of the inhibitor. These studies provide evidence that under normal culture conditions the cysteines of the HIV-1 protease are susceptible to oxidation during viral maturation, thus preventing immature virions from undergoing complete processing following their release. This is consistent with the cysteines being involved in the regulation of viral maturation in cells under oxidative stress.
Davis
D A
DA
HIV and AIDS Malignancy Branch, Medicine Branch, National Cancer Institute, Bethesda, Maryland 20892, USA. dadavis@helix.nih.gov
Yusa
K
K
Gillim
L A
LA
Newcomb
F M
FM
Mitsuya
H
H
Yarchoan
R
R
D000409:Alanine; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D019556:COS Cells; D017124:Conserved Sequence; D003545:Cysteine / Q000378:metabolism*; D003959:Diamines; D004229:Dithiothreitol; D015683:Gene Products, gag / Q000378:metabolism; D016333:HIV Protease / Q000378:metabolism*; D017320:HIV Protease Inhibitors / Q000494:pharmacology; D015497:HIV-1 / Q000378:metabolism* / Q000502:physiology; D006801:Humans; D008867:Microtomy; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D011498:Protein Precursors / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D014771:Virion / Q000502:physiology; D019065:Virus Assembly
D003959:Diamines; D015683:Gene Products, gag; D017320:HIV Protease Inhibitors; D009842:Oligopeptides; D011498:Protein Precursors; D011506:Proteins; C059767:p55 gag precursor protein, Human immunodeficiency virus 1; D016333:HIV Protease; D003545:Cysteine; C077822:kynostatin 272; D000409:Alanine; D004229:Dithiothreitol