Purification, determination of specific activity and TFV susceptibility of recombinant subtype C and B HIV-1 RTs. (A) Coomassie-Brilliant Blue staining of purified heterodimer RTs after 8% SDS-PAGE. MW (molecular mass standards in kilo daltons are shown on the left); b/cWT, (subtype B/C HIV-1 RT wild-type); b/cK65R, (subtype B/C HIV-1 RT harboring K65R); b/cK65R+M184V, (subtype B/C HIV-1 RT harboring K65R+M184V). The positions of purified recombinant RT heterodimers are indicated on the right. (B) Specific activity of recombinant RT enzymes as assessed using poly(rA)/oligo(dT) template/primer as described in Materials and Methods. All specific activities are expressed as a percentage of subtype B wild-type RT specific activity. (C) Incorporation efficiency of TFV-DP by subtype C WT and mutant RTs was monitored by gel-based assay and a representative image is shown in the panel on the left. Primer 19D was 5'-end labeled and annealed to template 57D. Reactions were performed with increasing concentrations of TFV-DP. P indicates the position of 5'-end labeled primer. Fifty percent inhibitory concentration (IC50) and fold resistance are shown on the right. Values are means of at least three independent experiments ± standard deviation. *P ≤ 0.05 compared to the IC50 of wild-type, by two-tailed Student's t-test.