KINETIC CHARACTERIZATION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 PROTEASE-RESISTANT VARIANTS

Passage of human immunodeficiency virus type-1 (HTV-1) in T-lymphocyte cell lines in the presence of increasing concentrations of the hydrox ylethylamino sulfonamide inhibitor VX-478 or VB-11328 results in seque ntial accumulation of mutations in HIV 1 protease, We have characteriz ed recombinant HTV-1 proteases that contain these mutations either ind ividually (L10F, M46I, I47V, I50V) or in combination (the double mutan t L10F/I50V and the triple mutant M46I/I47V/I50V). The catalytic prope rties and affinities for sulfonamide inhibitors and other classes of i nhibitors were determined, For the I50V mutant, the efficiency (k(cat) /K-m) of processing peptides designed to mimic cleavage junctions in t he HIV-1 gag-pol polypeptide was decreased up to 25-fold. The triple m utant had a a-fold higher processing efficiency than the I50V single m utant for peptide substrates with Phe/Pro and Tyr/Pro cleavage sites, suggesting that the M46I and I47V mutations are compensatory, The effe cts of mutation on processing efficiency were used in conjunction with the inhibition constant (K-i) to evaluate the advantage of the mutati on for viral replication in the presence of drug. These analyses suppo rt the virological observation that the addition of M46I and I47V muta tions on the I50V mutant background enables increased survival of the HIV-1 virus as it replicates in the presence of VX-478, Crystal struct ures and molecular models of the active site of the HTV-1 protease mut ants suggest that changes in the active site can selectively affect th e binding energy of inhibitors with little corresponding change in sub strate binding.