FIG. 9.

RdRp reaction of the point mutants of X RNA with HCV NS5B. (A) Nucleotide sequences in stem I of the mutant RNAs. The characters in boldface represent the elements for rendering the bulge structure. The nucleotides in italics indicate the pyrimidine-rich region in stem I. (B) Products of RdRp reaction on wild-type X RNA and its point mutants on a 7 M urea-8% polyacrylamide gel. The length of the marker RNA is indicated at the left of the gel. Lane 1, template X RNA internally labeled with ³²P; lanes 2 to 7, RdRp products of X, Xmut (1), Xmut (2), Xmut (3), Xmut (4), and Xmut (5) RNAs, respectively. (C) An 8 M urea-5% polyacrylamide sequencing gel electrophoresis autoradiogram showing the de novo synthesis products of X RNA and the point mutant RNAs before (−) or after (+) treatment with NaIO₄. The template RNAs for directing RNA synthesis by NS5B are indicated at the top of the gel. RNase T₁, 5′-labeled X RNA partially digested by RNase T₁; OH, partial alkaline hydrolysis of the 5′-labeled X RNA; RNA, template X RNA labeled internally with [α-³²P]CTP as a marker. The numbers on the left give the length of the RNA, and the bar at the left of the gel indicates the pyrimidine-rich region of X RNA.