Fig. 5.

Suppression mutation in transcription and cap-binding activities for the FluB PB2-363D mutant. (A and B) The levels of accumulation of viral mRNA (A) and cRNA (B) were measured by qPCR. (C) Cap-binding activities of mutants. Coprecipitated capped RNAs with 100 ng of recombinant RNA polymerase complexes (wild type [wt], lanes 1, 4, 7, and 10; 363D mutant, lanes 2, 5, 8, and 11; 325R-363D double mutant, lanes 3, 6, 9, and 12) were recapped before (lanes 1 to 3 and 7 to 9) and after (lanes 4 to 6 and 10 to 12) decapping by β-elimination. Recapped RNAs were treated without (lanes 1 to 6) or with (lanes 7 to 12) tobacco acid pyrophosphatase (TAP) and analyzed by TLC (PEI-CEL, 0.65 M LiCl), and radioactive nucleotides were determined by autoradiography. (D) The radioactivity of [³²P]m⁷Gp of TAP-treated products which were recapped after decapping was counted with a liquid scintillation counter. The cap-binding activity is represented as a ratio to the amount of [³²P]m⁷Gp derived from the wild type. These results are averages and SD from three independent experiments, and the level of significance was determined by Student's t test (unpaired) (*, P < 0.0025; **, P < 0.0005).