FIG. 2.

Inhibition of HIV-1 RT-catalyzed pyrophosphorolysis by UC781. All reaction mixtures contained 65 nM pHIV-PBS RNA and 5′-[³²P]prPBS T-P (prepared as described in Materials and Methods) and 26 nM p51-p66 recombinant wt or AZT-resistant RT. Other components of the individual reaction mixtures are indicated by a plus sign. The final concentrations of these components were dNTPs (dATP, dCTP, dGTP, and TTP at 50 μM each), sodium PP_i (NaPPi; 1 mM), and UC781 (1 μM). RT DNA synthesis reactions are those containing dNTPs but no sodium PP_i. Pyrophosphorolysis reactions are those containing sodium PP_i but no dNTPs. The extent of inhibition of these reactions is readily discerned by the intensity of the starting 18-nt [³²P]prPBS primer (denoted as RTPBS+0). (A and B) Two different exposures of the same gel provided to facilitate comparison of the different reactions. The exposure for panel A emphasizes pyrophosphorolytic products, while that for panel B emphasizes the forward reaction DNA polymerization products. WT, reactions catalyzed by recombinant wild-type RT; AZT-R, reactions catalyzed by recombinant RT with the D67N, K70R, T215F, and K219Q mutations; Fp, full-length DNA product (primer extended by 173 nt).