FIG. 5.

The E18R mutant can stimulate de novo initiation by Δ21. (A) A surface and ribbon representation of the X-ray crystal structure of HCV RdRp (PDB identifier 1QUV) to highlight the location of E18 (red spheres) and its interaction with R401 (blue spheres). The complete thumb domain (T), a part of the palm (P) domain, and a part of the finger (F) domain are marked. The active site metal coordinating residues in the active site are in yellow. The Δ1 loop that connects the fingers and thumb domain is in ribbon representation and is colored cyan, while the side chains of its residues are in purple. (B) SDS-PAGE of proteins used in the reactions and their behavior in a DSF reaction. (C) Representative image of the products of the RdRp assay with templates LE19P and PE46 with increasing concentrations of the Δ21 or E18R protein. The ratio of increase in the de novo-initiated product has been quantified below the gel image. (D) Effects of amending an RNA synthesis where Δ21 was kept constant at 20 nM while the E18R protein was at 10 or 60 nM. The two enzymes were preincubated on ice for 30 min before the RNAs were added. (E) R401A mutation does not affect wild-type activity of NS5B. The template LE19 was used in this assay, and the gel image of a representative reaction is shown, indicating the 19-nt de novo product and the 32-nt primer extension product.