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 p r55(gag) and Pr160(gag-pol) polyproteins. The processing is mediated s pontaneously by the viral protease unit (PR) contained within the Pr16 0(gag-pol) precursor. However, little is known about the mechanism of this process. The expression in Escherichia coil and the isolation of a 14-kDa HIV-1 PR ''miniprecursor'' with Ala(28) mutated to serine has permitted study of the mechanism for cleavage at the N-terminus of th e protease. The miniprecursor is active against a synthetic peptide su bstrate, and its specific activity is near that of the mutant mature p rotease. The rate of conversion of radiolabeled precursor to mature pr otease is quantitated by measuring the amounts of the two radiolabeled proteins separated by SDS-PAGE. The apparent first-order conversion r ate constant, k(app,) is dependent on miniprecursor concentration indi cating a second-order reaction and suggesting an interdimeric processi ng mechanism. A significant first-order rate constant is observed when the plot of k(app) versus initial precursor concentration is extrapol ated to zero. This observation suggests the presence of an alternative processing mechanism involving a single active precursor dimer. The p resence of both mechanisms is an advantage for the virus to ensure pro cessing under various conditions.