Linear and circular 13-nt templates for RNA-dependent RNA synthesis. (A) Sequences of the linear and circular templates are shown with the arrow indicating the direction of RNA synthesis. RNAs +1C-L and +1U-L differ only at the 3′-most nucleotide (underlined). (B) Circularization of the RNAs. Both +1C and +1U were circularized with T4 RNA ligase to result in a conformation that migrated at a lower position in a 7.5 M urea–20% polyacrylamide gel. Each lane contains between 30 and 50 pmol of RNA. The RNA bands were visualized by staining with toluidine blue. Treatment with the T4 ligase is indicated by +. The faint band at the bottom of the gel image is bromophenol blue (BB). (C) A demonstration that the ligated RNAs exist in a covalently closed form. The purified RNAs were adjusted to 0.1 μM and then incubated in poly(A) polymerase buffer, 0.1 units of poly(A) polymerase (Ambion Inc.), and [α-32P]ATP. The reaction products were then precipitated with ethanol, centrifuged, and electrophoresed in a 7.5 M urea–15% polyacrylamide gel. (D) An image from a denaturing polyacrylamide gel containing products made by Δ21 with increasing concentrations of linear or circular template. Flaviviral RdRps can synthesize one product molecule from two or more linear molecules of the template RNA. These template-switch products are therefore multiples of the length of the template (Kim and Kao 2001). The names and amounts of the RNAs are listed above the appropriate lanes of the gel image. The lengths of the product RNAs from the linear template, in nucleotides, are shown to the left of the gel image. The bar to the right of the gel signifies a monomer template-sized product, two bars correspond to dimer, and the bar within parentheses with subscript “n” corresponds to multimeric forms. (E) Initiation of RNA synthesis from linear and circular templates. The 3′-terminal cytidylate is the primary initiation nucleotide for RNA synthesis by the HCV Δ21 from linear, but not circular, templates. All four reactions were performed with 0.1 pmol of linear and 10 pmol of circular template RNA and 0.04 μM Δ21.
