FIG. 2.

Analysis of the in vivo RNA synthesis activity of polymerase mutants used in the study. Plasmids expressing viral proteins (+) or empty plasmid vector (−), as indicated, were transfected into 293T cells together with a plasmid (pPOLI-NA-RT) (9) directing polymerase I-driven transcription of NA vRNA as a template for RNA synthesis. Total RNA was isolated at 15 h posttransfection and analyzed by NA gene-specific primer extension. vRNA detected in the absence of PB1 (lane 1) represents polymerase I-derived template RNA. wt, wild type; PB1a, PB1-D445A/D446A; PB1r, PB1-Y559A; PB1ar, PB1-D445A/D446A/Y559A; PB2c, PB2-F404A.