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(Received for publication, November 7, 1995, and in revised form, April 30, 1996)
From Vertex Pharmaceuticals Incorporated,
Cambridge, Massachusetts 02139
Passage of human immunodeficiency virus type-1
(HIV-1) in T-lymphocyte cell lines in the presence of increasing
concentrations of the hydroxylethylamino sulfonamide inhibitor VX-478
or VB-11328 results in sequential accumulation of mutations in HIV-1
protease. We have characterized recombinant HIV-1 proteases that
contain these mutations either individually (L10F, M46I, I47V, I50V) or
in combination (the double mutant L10F/I50V and the triple mutant
M46I/I47V/I50V). The catalytic properties and affinities for
sulfonamide inhibitors and other classes of inhibitors were determined.
For the I50V mutant, the efficiency
(kcat/Km) of processing
peptides designed to mimic cleavage junctions in the HIV-1 gag-pol
polypeptide was decreased up to 25-fold. The triple mutant had a 2-fold
higher processing efficiency than the I50V single mutant for peptide
substrates with Phe/Pro and Tyr/Pro cleavage sites, suggesting that the
M46I and I47V mutations are compensatory. The effects of mutation on
processing efficiency were used in conjunction with the inhibition
constant (Ki) to evaluate the advantage of the
mutation for viral replication in the presence of drug. These analyses
support the virological observation that the addition of M46I and I47V
mutations on the I50V mutant background enables increased survival of
the HIV-1 virus as it replicates in the presence of VX-478. Crystal
structures and molecular models of the active site of the HIV-1
protease mutants suggest that changes in the active site can
selectively affect the binding energy of inhibitors with little
corresponding change in substrate binding.
Volume 271, Number 30,
Issue of July 26, 1996
pp. 17979-17985
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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