RESISTANCE TO 2',3'-DIDEOXYCYTIDINE CONFERRED BY A MUTATION IN CODON-65 OF THE HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 REVERSE-TRANSCRIPTASE

A human immunodeficiency virus type 1 variant resistant to zalcitabine (2',3'-dideoxycytidine [ddC]) was selected by sequential passage in t he presence of increasing concentrations of ddC in peripheral blood mo nonuclear cell cultures. A mutation causing a lysine-to-arginine subst itution was noted in reverse transcriptase (RT) codon 65 of this ddC-s elected virus. A cloned mutant virus with this codon 65 mutation was c onstructed by using a novel PCR approach for site-directed mutagenesis . Characterization of this virus confirmed that the RT Lys-65-->Arg su bstitution was necessary and sufficient for a fourfold increase in the ddC 50% inhibitory concentration, as well as for resistance to didano sine (2',3'-dideoxyinosine [ddI]). Lys-G-->Arg and virus resistance to ddC and ddI also developed during therapy in isolates from one ddC-tr eated patient and two ddI-treated patients. Recombinant-expressed codo n 65 mutant RT enzyme was resistant to ddCTP and ddATP in cell-free po lymerase assays. Results of mutant enzyme studies are consistent with Lys-65-->Arg leading to changes in binding of the triphosphate forms o f these nucleoside analogs to the RT. These data have implications for future studies of ddC resistance, particularly those aimed at definin g its clinical relevance.