Minigenome assays were performed in 293T cells in 24 or 48-well plates.

For 24 well plates, Pol I–firefly luciferase minigenome reporter, at 0.08 μg and PCAGGS-Renilla, at 0.1 μg were transfected with PCAGGS plasmids coding for wildtype and mutated polymerase subunits (PB1, PB2 and PA) and NP at 0.08, 0.08, 0.04 and 0.12 μg respectively derived from A) Eng195 first wave and B) Eng687 third wave pH1N1 virus. Plasmid amounts were halved for 48-well plate experiments. Luciferase signal was read 24 hours post-transfection. Polymerase activity is given as a ratio Firefly to Renilla signals. One-way ANOVA with Dunnett’s multiple comparison test, *** p<0.001, **** p<0.0001, ns = not significant. N = 3. Error bars show s.d. Experiments were repeated 3 times and a representative experiment is shown.