Figure 7

Mapping of the RNA synthesis initiation site on the 83-nt RNA template. The RdRp assay was performed with the 83-nt RNA template. (A) An autoradiogram showing the major RNA product synthesized by JEV NS5 using the 83-nt RNA template. Products were resolved on a 5% polyacrylamide sequencing gel (20 × 40 cm) containing 8 M urea. The RNA size markers, 5'-end labeled RNA template (End), and a set of labeled RNA fragments generated by alkaline hydrolysis of the 5'-end labeled RNA template (End/OH), were resolved on the same gel. Arrowhead indicates the internally initiated 81-nt RNA product. (B) The close-up autoradiogram of the same gel shown in (A). (C) Secondary structure of the 83-nt RNA template predicted by the Mfold program. Bent arrow denotes the predicted RNA synthesis initiation site.