FIG. 9.

Effects of mutations within HCV NS5B on de novo initiation and primer extension. (A) At top is shown a schematic of the β-loop within the HCV RdRp that has been reported to affect de novo initiation and primer extension (9). The three amino acids examined by mutational analysis are numbered after the one-letter amino acid code. At bottom is shown a schematic of the relative order of the domains within the primary sequence of HCV RdRp (not to scale). The finger, palm, and thumb subdomains are identified in the figure. Deletions of the region C-terminal to the thumb subdomain are shown as vertical lines to the left of the central bar representing the HCV RdRp. (B) RNA synthesis by HCV Δ21 or select β-loop mutants using template LE21 in the presence of Mg²⁺, or both Mg²⁺ and Mn²⁺. All reaction mixtures contained 80 nM RdRp. (C) Ratio of the de novo initiation and primer extension (PE) products made by Δ21 and mutant proteins in the presence of 2 mM Mg²⁺ or 2 mM (each) Mg²⁺ and Mn²⁺. (D) Autoradiogram demonstrating the effects of C-terminal deletions in the HCV RdRp on the ratio of the de novo-initiated or primer-extended products.