HIV-1 reverse transcriptase mutants resistant to nonnucleoside reverse transcriptase inhibitors do not adversely affect DNA synthesis: pre-steady-state and steady-state kinetic studies

Abstract

We have previously demonstrated that nonnucleoside reverse transcriptase inhibitor (NNRTI)-resistant mutants have different levels of replication fitness relative to wild type; those with greater reductions in fitness are less likely to develop during therapy in patients. We have also found that reductions in rates of RNase H cleavage by mutant RTs correlate with reductions in fitness and that NNRTI-resistant RTs catalyze polymerization with a processivity similar to wild type. In this study, we evaluated the polymerase function of 3 clinically occurring NNRTI-resistant RTs (K103N, P236L, and V106A) in greater detail, under both pre-steady-state and steady-state conditions. The overall pathway of single-nucleotide incorporation was unchanged for the mutant RTs compared with wild type. In addition, the NNRTI-resistant mutants were each similar to wild type in rate of nucleotide incorporation (kpol), affinity for dGTP (Kd), and steady-state rate of polymerization (kss and kcat), using either RNA or DNA templates. These findings suggest that the close proximity of the NNRTI-resistance mutations to the polymerase active site does not affect the interactions of the enzyme with the incoming nucleotide or the primer-template sufficiently to affect polymerization and support the hypothesis that these reductions in RNase H activity contribute to reductions in replication fitness.