FIGURE 3.

In vitro template activity of sgRNAs containing WT or 3′-end deletions. sgRNAs containing WT or 3′-end deletions were analyzed for template activities in vitro using purified NS5. (A) sgRNAs containing Δ4+GC (lane 1), Δ8+GGC (lane 2), Δ10+GGC (lane 3), Δ14+GGC (lane 4), Δ17+GC (lane 5), and Δ22+GC (lane 6) were used as templates for negative-strand synthesis. (Lane 7) No RNA control. The products of the reaction were separated by electrophoresis and visualized by autoradiography. (1×) De novo product, (2×) double-stranded hairpin product. Ethidium bromide gel shows approximately equal amounts of sgRNAs used for RdRp assays. Experiments were repeated at least three times and the results of a representative experiment are shown. (B) Densitometric scan of gels were performed, and the sum of 1× and 2× RNAs was compared with WT. Error bars are based on three independent experiments. (C) Densitometric scan of 2× product only, compared with that of WT. (D) Densitometric scan of 1× RNA compared with the WT. (E) In vitro template activity of sgRNAs bearing adaptive mutations identified in recovered genomes. sgRNAs bearing 3′-end sequences, WT and 1–8, were used as templates in the in vitro assays. The RNA products were detected by autoradiography.