CHARACTERIZATION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 MUTANTS WITH DECREASED SENSITIVITY TO PROTEINASE-INHIBITOR RO-31-8959

A human immunodeficiency virus type 1 (HIV-1) variant with highly redu ced susceptibility to Ro 31-8959, an inhibitor of the viral proteinase , has been selected by repeated passage of wild-type virus in CEM cell s in the presence of increasing concentrations of the inhibitor. Pepti de sequences of the proteinase of selected virus were obtained from pr oviral DNA. Sequence comparison to wild-type (wt) proteinase demonstra ted two amino acid substitutions in the resistant virus, a Gly to Vat exchange at position 48 and a Leu to Met exchange at position 90. Furt hermore, sequences of intermediate passage virus suggest contributions from positions 12, 36, 57, and 63 in early steps of resistance develo pment. The selected virus showed a ca. 40-fold increase in 50% inhibit ory concentration of Po 31-8959. Growth kinetics of resistant virus we re comparable to wild-type virus and the resistant genotype proved to be stable in the absence of inhibitor. Directed mutagenesis of the HIV -I HXB2 proteinase al positions 48 and 90 suggested that each mutation alone led to a moderate decrease in sensitivity of the recombinant vi rus to proteinase inhibitor. However, a recombinant virus carrying bot h mutations in the proteinase gene showed a significant reduction in i ts sensitivity to Ro 31-8959 thus proving the importance of these exch anges for the resistance phenotype. (C) 1995 Academic Press, Inc.