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.
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.
Domaoal
Robert A
RA
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Bambara
Robert A
RA
Demeter
Lisa M
LM
D001483:Base Sequence; D017931:DNA Primers; D004261:DNA Replication / Q000187:drug effects*; D024882:Drug Resistance, Viral / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D007700:Kinetics; D009154:Mutation; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*
D017931:DNA Primers; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase