Figure 2.

Biological activity of wild-type and PB2 mutated recombinant RNPs. HEK293T cells were co-transfected with plasmids expressing PB1, PA, NP and either WT or mutant PB2, a plasmid encoding Renilla luciferase and a plasmid encoding Firefly luciferase in negative polarity, flanked by the non-translated regions of influenza NP segment. Plasmid expressing PB2 was omitted as a negative control. (A) At 20 h post-transfection, luciferase accumulation was determined. Values were normalized to Renilla expression, and the activity of the WT RNP was set to 100% (mean ± SEM; N = 9–18; **** P < 0.0001 by Student's t test compared to WT RNP activity). At the bottom is shown confirmation of WT or mutant PB2 expression in human HEK cells by western blotting using β-actin as a loading control. (B) Representative gel of primer extension assay showing the different RNA levels produced by WT and mutant polymerases in the minigenome assay. (C-E). Viral RNA accumulation measured by primer extension assay. 24 h post-transfection, total RNA was isolated and analysed by luciferase gene-specific primer extension. A primer specific for 5S rRNA was used for normalization (mean ± SEM; N = 6; ***P < 0.001 by Student's t-test compared to WT RNP activity).