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 the presence of increasing concentrations of ddC in peripheral blood mononuclear cell cultures. A mutation causing a lysine-to-arginine substitution was noted in reverse transcriptase (RT) codon 65 of this ddC-selected virus. A cloned mutant virus with this codon 65 mutation was constructed by using a novel PCR approach for site-directed mutagenesis. Characterization of this virus confirmed that the RT Lys-65-->Arg substitution was necessary and sufficient for a fourfold increase in the ddC 50% inhibitory concentration, as well as for resistance to didanosine (2',3'-dideoxyinosine ddI). Lys-65-->Arg and virus resistance to ddC and ddI also developed during therapy in isolates from one ddC-treated patient and two ddI-treated patients. Recombinant-expressed codon 65 mutant RT enzyme was resistant to ddCTP and ddATP in cell-free polymerase assays. Results of mutant enzyme studies are consistent with Lys-65-->Arg leading to changes in binding of the triphosphate forms of these nucleoside analogs to the RT. These data have implications for future studies of ddC resistance, particularly those aimed at defining its clinical relevance.