FIG. 1.

Inhibition of DNA-dependent DNA polymerase RT activity by NNRTIs determined by a fixed-time gel-based RT assay. (A) Graphic representation of the primer-template system (ppt17D-ppt57D) used to monitor the inhibition of HIV-1 RT DNA polymerase activity by NNRTIs. The 17-mer DNA primer ppt17D was labeled with ³²P at the 5′ terminus and annealed to the 57-mer DNA template ppt57D. +1 and +4, positions of the first and the last nucleotides incorporated, respectively; *, position of the incorporated ddGTP. (B) Dose-dependent inhibition of DNA polymerase activity by NNRTIs. All reactions were resolved by denaturing 6% polyacrylamide gel electrophoresis, visualized by phosphorimaging, and quantified with ImageQuant software (GE Healthcare). The positions of the labeled primer (P) and the full-length extension product (+4) are indicated on the left. The concentrations of the NNRTIs used are as follows: for ETR, 0, 0.6, 1.7, 5, 15.2, 45.6, 137, and 411 nM and 1.23, 3.7, and 11.1 μM; for DAP, 0, 5, 15, 45.7, 137, and 410 nM; 1.23, 3.7, 11.1, 100, and 500 μM; and 1 mM; for EFV, 0, 0.56, 1.69, 5, 15, 45.7, 137, and 410 nM and 1.23, 3.7, 11.1, 33.3, and 100 μM; and for NVP, 0, 10, 31, 95, 285, 857 nM; 2.5, 7.7, 23, 69, 208, and 625 μM; and 1.87 mM.