Structural and kinetic analyses of the protease from an amprenavir-resistant human immunodeficiency virus type 1 mutant rendered resistant to saquinavir and resensitized to amprenavir

Recent drug regimens have had much success in the treatment of human immuno deficiency virus (HIV)- infected individuals; however, the incidence of res istance to such drugs has become a problem that is likely to increase in im portance with long-term therapy of this chronic illness. An analysis and un derstanding of the molecular interactions between the drug(s) and the mutat ed viral target(s) is crucial for further progress in the field of AIDS the rapy. The protease inhibitor amprenavir (APV) generates a signature set of HIV type 1 (HIV-1) protease mutations associated with in vitro resistance ( M46I/L, I47V, and I50V [triple mutant]), Passage of the triple-mutant APV-r esistant HIV-1 strain in MT4 cells, in the presence of increasing concentra tions of saquinavir (SQV), gave rise to a new variant containing M46I, G48V , I50V, and I84L mutations in the protease and a resulting phenotype that w as resistant to SQV and, unexpectedly, resensitized to APV, This phenotype was consistent with a subsequent kinetic analysis of the mutant protease, t ogether with X-ray crystallographic analysis and computational modeling whi ch elucidated the structural basis of these observations. The switch in pro tease inhibitor sensitivities resulted from (i) the I50V mutation, which re duced the area of contact with APV and SQV; (ii) the compensating I84L muta tion, which improved hydrophobic packing with APV; and (iii) the G-to-V mut ation at residue 48, which introduced steric repulsion with the P3 group of SQV, This analysis establishes the fine detail necessary for understanding the loss of protease binding for SQV in the quadruple mutant and gain in b inding for APV, demonstrating the powerful combination of virology, molecul ar biology, enzymology, and protein structural and modeling studies in the elucidation and understanding of viral drug resistance.