Recombinant subtype C HIV-1 RTs containing K65R or K65R+M184V were purified from Escherichia coli. Enzyme activities and tenofovir (TFV) incorporation efficiency by wild-type (WT) and mutant RTs of both subtypes were determined in cell-free assays. Efficiency of (-) ssDNA synthesis and initiation by subtype C RTs was measured using gel-based assays with HIV-1 PBS RNA template and tRNA3^Lys as primer. Single-cycle processivity was assayed under variable dNTP concentrations. Steady-state analysis was performed to measure the relative inhibitory capacity (ki/km) of TFV-disphosphate (TFV-DP). ATP-dependent excision and rescue of TFV-or ZDV-terminated DNA synthesis was monitored in time-course experiments.

The efficiency of tRNA-primed (-)ssDNA synthesis by subtype C RTs was: WT > K65R > K65R+M184V RT. At low dNTP concentration, K65R RT exhibited lower activity in single-cycle processivity assays while the K65R+M184V mutant showed diminished processivity independent of dNTP concentration. ATP-mediated excision of TFV-or ZDV-terminated primer was decreased for K65R and for K65R+M184V RT compared to WT RT. K65R and K65R+M184V displayed 9.8-and 5-fold increases in IC50 for TFV-DP compared to WT RT. The Ki/Km of TFV was increased by 4.1-and 7.2-fold, respectively, for K65R and K65R+M184V compared to WT RT.