FIG 1.

Effects of PB2 K627E mutation on polymerase assembly and activity in cultured cells and in vitro. (A) HEK-293T (human) or DF1 (chicken) cells were cotransfected with plasmids expressing PA, PB1, PB2 627K or 627E, NP, and a segment 6 vRNA. Accumulation of mRNA and vRNA was assessed by primer extension 48 h posttransfection (22). The RNA levels detected for the E627K polymerase were set to 100. The error bars indicate standard errors of the mean (SEM), and the asterisks indicate a significant difference from 100 (one-sample t test; ***, P < 0.001). Expression of PB2 was analyzed by Western blotting, with RanBP5 serving as a loading control. (B) HEK-293T cells were cotransfected with plasmids expressing PA, PB1-TAP, and PB2 627K or 627E, and polymerase was purified using IgG Sepharose chromatography 48 h posttransfection (26). The purified polymerase was analyzed by SDS-PAGE, and PB2 levels were assessed by using AIDA software (Raytest). PB2 627K levels were set to 100. The error bar indicates SEM of three experiments. (C) The activity of the purified polymerase from panel B was assessed in an in vitro transcription assay using ApG primer or β-globin mRNA as a cap donor (26, 27). The RNA levels detected for the E627K polymerase were set to 100. The error bars indicate SEM of three experiments.