RNA synthesis from linear and circular templates by full-length and an active-site mutant, D225A, of the HCV RdRp. (A) Huh7 cells transformed by the wild-type subgenomic replicon (designated as NS5B) or one with the mutation D225A. Transfections were performed as described in Materials and Methods and selected for cells resistant to G418 treatment. Cell colonies were visualized 3 wk after transfection by Coomassie blue staining. The results show that D225A is a critical mutation for HCV RNA replication in cells. (B) RNA synthesis by D225A (expressed in the context of Δ21) and by the full-length NS5B proteins from 13-nt +1C-L, +1C-C, +1U-L, and +1U-C templates and (C) 21-nt LE21-L and LE21-C templates. The templates used in each reaction are shown above the gel image.
