FIG. 6.

Inhibitory effect of l-FMAU-TP on the synthesis of the viral DNA primer for DHBV reverse transcription. The sequence of the short DNA primer covalently linked to the DHBV polymerase is 5′-GTAA-3′. (A) The DHBV polymerase was incubated with different concentrations of thymidine-TP analogs (TTP or ddTTP or l-FMAU-TP) together with dGTP and [α-³²P]dATP (3,000 Ci/mmol; final concentration, 0.3 μM) for 30 min. The incorporation of [α-³²P]dAMP was also analyzed in control reactions without TTP, without dGTP and TTP, or without DHBV polymerase. Reactions were analyzed with a 0.1% SDS–10% polyacrylamide gel and autoradiographed. The figure shows the results obtained with the TTP analog at a concentration of 50 μM. (B) Kinetics of [α-³²P]dAMP incorporation into primer DNA by the DHBV polymerase. The DHBV polymerase was incubated with thymidine-TP analogs at a concentration of 50 μM (⧫, TTP; ▪, ddTTP; ▴, l-FMAU-TP) together with dGTP (100 μM) and [α-³²P]dATP (3,000 Ci/mmol; final concentration, 0.3 μM). The incorporation of [α-³²P]dAMP in the viral primer was measured at different time points, as indicated. The activity at 30 min in the presence of TTP was arbitrarily defined as 100%.