FIG 2.

Effects of nucleotide substitutions at the 3′-end positions 13 to 15 and 5′-end positions 14′ to 16′ on RNA synthesis in an RNP reconstitution system. (A, C, E, and G) Primer extension analysis to assess the RNA levels derived from wild-type and mutant templates as showed in Fig. 1 in a PR8-derived RNP reconstitution system. The wild-type and mutant RNPs were reconstituted in 293T cells, and the levels of mRNA, cRNA, and vRNA were detected by a primer extension assay at 24 h posttransfection. The vRNA levels in negative-control (NC) lanes represent the viral RNA template transcribed from the input pPolI plasmid. (B, D, F and H) Quantification of the levels of mRNA, cRNA, and vRNA in panels A, C, E, and G by phosphorimager analysis. Values were normalized against cellular 5S RNA. The data are presented as means ± standard deviations of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, nonsignificant.