Fig 4.
Examination of RNA synthesis by mutant HCV RdRps. (A) Products made by the WT and mutant polymerases, with LE19 and PE46 as the templates. (B) Results from DSF spectroscopy of 1bΔ21 and the three mutant polymerases. Between 2.5 and 5 μg of the four proteins was used in the assay, and the temperature observed for the greatest change in fluorescence [Tm(app)] is shown. (C) RNA synthesis by the mutant polymerase in the presence of ANA-598, filibuvir, or VX-222. Two primer extension templates were used, and the resulting IC50s were compared to those from 1bΔ21 (reported as the fold change). The IC50s for 1bΔ21 with ANA-598, filibuvir, and VX-222 were 5 nM, 50 nM, and 11 nM with the duplex of poly(rA)-oligo(dT)18. The IC50s of filibuvir and VX-222 with 1bΔ21 were 73 nM and 31 nM with PE46, as documented in Table 1.