Proteolytic processing mechanisms of a miniprecursor of the aspartic protease of human immunodeficiency virus type 1.
The infectivity of the human immunodeficiency virus (HIV) depends upon correct proteolytic processing of viral polyprotein precursors, the Pr55gag and Pr160gag-pol polyproteins. The processing is mediated spontaneously by the viral protease unit (PR) contained within the Pr160gag-pol precursor. However, little is known about the mechanism of this process. The expression in Escherichia coli and the isolation of a 14-kDa HIV-1 PR "miniprecursor" with Ala28 mutated to serine has permitted study of the mechanism for cleavage at the N-terminus of the protease. The miniprecursor is active against a synthetic peptide substrate, and its specific activity is near that of the mutant mature protease. The rate of conversion of radiolabeled precursor to mature protease is quantitated by measuring the amounts of the two radiolabeled proteins separated by SDS-PAGE. The apparent first-order conversion rate constant, kapp, is dependent on miniprecursor concentration indicating a second-order reaction and suggesting an interdimeric processing mechanism. A significant first-order rate constant is observed when the plot of kapp versus initial precursor concentration is extrapolated to zero. This observation suggests the presence of an alternative processing mechanism involving a single active precursor dimer. The presence of both mechanisms is an advantage for the virus to ensure processing under various conditions.
Co
E
E
Protein Studies Program, Oklahoma Medical Research Foundation, Oklahoma City 73104.
Koelsch
G
G
Lin
Y
Y
Ido
E
E
Hartsuck
J A
JA
Tang
J
J
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000378:metabolism; D004792:Enzyme Precursors / Q000378:metabolism; D016333:HIV Protease / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational
D004792:Enzyme Precursors; D001224:Aspartic Acid; D016333:HIV Protease