Resistance to 2',3'-dideoxycytidine conferred by a mutation in codon 65 of the human immunodeficiency virus type 1 reverse transcriptase

Abstract

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%N inhibitory concentration, as well as for resistance to didanosine (2',3'-dideoxyinosine [ddl]). Lys-65-*Arg and virus resistance to ddC and ddl 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 ii 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.

Selection of Resistance-Conferring Mutations in HIV-1 by the Nucleoside Reverse Transcriptase Inhibitors (+/-)dOTC and (+/-)dOTFC

“…The K65R mutation has been shown to confer resistance to ddC, as well as low-level cross-resistance to 3TC, adefovir (PMEA) and ddI. As mentioned above, this mutation can be detected in some patients undergoing ddC therapy Zhang et al, 1994). Similar findings were observed with the (+) enantiomer of dOTFC, where the D67G mutation was selected in tissue culture, but the M184V mutation conferred a higher degree of resistance in MT-2 cells (Richard et al, 2000).…”

Primer Unblocking and Rescue of DNA Synthesis by Azidothymidine (AZT)-resistant HIV-1 Reverse Transcriptase

“…Indeed, AZTresistant RT incorporates AZT as efficiently as the wild type (wt) polymerase (9 -12). This situation contrasts with the enzymes that bear the mutations K65R (13,14), L74V (15), and M184V (16 -18) and that confer resistance toward ddC, ddI, and 3TC, respectively.…”

Interactions Between Drug Resistance Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase

“…A. Larder, unpublished results;Schinazi et al, 1993;St. Clair et al, 1991 ;Tisdale et al, 1993 ;Zhang et al, 1994); NNRTI (Balzarini et al, 1993 ;Byrnes et al, 1993;Dueweke et al, 1993;Kleim et al, 1993;Larder, 1992;Mellors et al, 1993;Nunberg et al, 1991 ;Richman et al, 1991). The lower line shows the position of RT mutations known to suppress AZT resistance.…”