Fig. 3.
Effects of increasing concentrations of HPMPCpp or HPMPApp on HIV-1 RT activity. (A) Primer extension reactions using RNA or DNA templates directing the incorporation of a single HPMPC molecule. Each 10-μl reaction mixture contained FAM-labeled primer P1 annealed to template T9RNA (lanes 2 to 7) or T9 (lanes 9 to 14), 5 μM dCTP, 10 μM dATP, 10 μM dGTP, 10 μM dTTP, and 50 nM HIV-1 RT. Reactions were further supplemented with HPMPCpp at 0, 1, 3, 10, 30, or 100 μM as indicated. After incubation at 37°C for 5 min, each reaction was stopped by adding gel loading buffer and reaction products were separated on a 15% polyacrylamide gel and then analyzed by fluorescent imaging. Lanes marked “Primer” show the position of the labeled primer, and the positions of pause sites are indicated by arrows. (B) Primer extension reactions using RNA or DNA templates directing the incorporation of two consecutive HPMPC molecules. Reaction conditions were as described for panel A, except that templates T2RNA (lanes 2 to 7) and T2 (lanes 9 to 14) were used. (C) Primer extension reactions using RNA or DNA templates directing the incorporation of a single HPMPA molecule. The reaction mixtures contained a FAM-labeled primer P1 annealed to template T11RNA (lanes 2 to 7) or template T11 (lanes 9 to 14), 5 μM dATP, 10 μM dCTP, 10 μM dGTP, 10 μM dTTP, and 50 nM HIV-1 RT. HPMPApp was also added at the concentrations indicated. The reactions were processed and imaged as described for panel A. (D) Primer extension reactions using RNA or DNA templates directing the incorporation of two consecutive HPMPA molecules. Reaction conditions were as described for panel C, except that templates T12RNA (lanes 2 to 7) and T12 (lanes 9 to 14) were used.