IN-VITRO SELECTION AND CHARACTERIZATION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 (HIV-1) ISOLATES WITH REDUCED SENSITIVITY TO HYDROXYETHYLAMINO SULFONAMIDE INHIBITORS OF HIV-1 ASPARTYL PROTEASE

Human immunodeficiency virus type 1 (HIV-1) variants with reduced sens itivity to the hydroxyethylamino sulfonamide protease inhibitors VB-11 ,328 and VX-478 have been selected in vitro by two independent serial passage protocols with HIV-1 in CEM-SS and MT-4 cell lines. Virus popu lations with greater than 100-fold-increased resistance to both inhibi tors compared with the parental virus have been obtained, DNA sequence analyses of the protease genes from VB-11,328- and VX-478-resistant v ariants reveal a Sequential accumulation of point mutations, with simi lar resistance patterns occurring for the two inhibitors. The deduced amino acid substitutions in the resistant protease are Leu-10-->Phe, M et-46-->Ile, Ile-47-->Val, and Ile-50-->Val. This is the first observa tion in HIV protease resistance studies of an Ile-50-->Val mutation, a mutation that appears to arise uniquely against the sulfonamide inhib itor class. When the substitutions observed were introduced as single mutations into an HIV-1 infectious done (HXB2), only the Ile-50-->Val mutant showed reduced sensitivity (two- to threefold) to VB-11,328 and VX-478, A triple protease mutant infectious clone carrying the mutati ons Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val, however, showed much greater reduction in sensitivity (14- to 20-fold) to VB-11,328 and VX -478. The same mutations were studied in recombinant HIV protease. The mutant protease Ile-50-->Val displays a much lower affinity for the i nhibitors than the parent enzyme (less than or equal to 80-fold), The protease triply mutated at Met-46-->Ile, Ile-47-->Val, and Ile-50-->Va l shows an even greater decrease in inhibitor binding (less than or eq ual to 270-fold). The sulfonamide-resistant HIV protease variants rema in sensitive to inhibitors from other chemical classes (Ro 31-8959 and L-735,524), suggesting possibilities for clinical use of HN protease inhibitors in combination or serially.