Molecular mechanism of HIV-1 resistance to 3′-azido-2′,3′-dideoxyguanosine

•The mechanism of HIV-1 resistance to 3′-azido-ddG is defined.•Kinetic characterization of HIV-1 RT containing L74V, F77L, V106I, L214F, R277K, and K476N.•The L74V mutation allows RT to discriminate between dGTP and 3′-azido-ddGTP.•K476N partially restored RT’s ability to excise 3′-azido-ddGMP on an RNA/DNA T/P. We reported that 3′-azido-2′,3′-dideoxyguanosine (3′-azido-ddG) selected for the L74V, F77L, and L214F mutations in the polymerase domain and K476N and V518I mutations in the RNase H domain of HIV-1 reverse transcriptase (RT). In this study, we have defined the molecular mechanisms of 3′-azido-ddG resistance by performing in-depth biochemical analyses of HIV-1 RT containing mutations L74V, F77L, V106I, L214F, R277K, and K476N (SGS3). The SGS3 HIV-1 RT was from a single-genome-derived full-length RT sequence obtained from 3′-azido-ddG resistant HIV-1 selected in vitro. We also analyzed two additional constructs that either lacked the L74V mutation (SGS3-L74V) or the K476N mutation (SGS3-K476N). Pre-steady-state kinetic experiments revealed that the L74V mutation allows RT to effectively discriminate between the natural nucleotide (dGTP) and 3′-azido-ddG-triphosphate (3′-azido-ddGTP). 3′-azido-ddGTP discrimination was primarily driven by a decrease in 3′-azido-ddGTP binding affinity (Kd) and not by a decreased rate of incorporation (kpol). The L74V mutation was found to severely impair RT’s ability to excise the chain-terminating 3′-azido-ddG-monophosphate (3′-azido-ddGMP) moiety. However, the K476N mutation partially restored the enzyme’s ability to excise 3′-azido-ddGMP on an RNA/DNA, but not on a DNA/DNA, template/primer by selectively decreasing the frequency of secondary RNase H cleavage events. Collectively, these data provide strong additional evidence that the nucleoside base structure is major determinant of HIV-1 resistance to the 3′-azido-2′,3′-dideoxynucleosides.