Figure 2: Polymerase activity of H7N9 PB2 variants in vitro.

(a) HEK293T cells were transfected with H7N9 RNP complexes composed of NP, PB1 and PA from the A/Zhejiang/DTID-ZJU01/2013 strain and PB2 from different strains containing different adaptation markers, as shown in Table 1, together with the firefly luciferase reporter pYH-Luci and a Renilla luciferase reporter (internal control). Luciferase activity was measured at 24 h post transfection, following incubation at 33 or 37 °C. Comparison of RNP polymerase activity with PB2 derived from human and avian H7N9 isolates; PB2 protein levels estimated by western blot for the minigenome assay and FLAG-tagged pCMV clones are shown at the bottom. (b) Comparison of RNP polymerase activity for H3N2 (A/Hong Kong/1/68) RNP containing PB2 with or without the 526R substitution in HEK293T cells at 24 h post transfection. (c) Comparison of RNP activity of H3N2 (A/Guangdong/ST798/2008) RNP containing PB2 with or without the 526K reverse mutation in HEK293T cells at 24 h post transfection. (d) Comparison of RNP activity with H5N1 (A/Indonesia/5/2005) RNP containing PB2-526R or PB2-526K reverse mutation in HEK293T cells at 24 h post transfection. (e) DF-1 cells were transfected with the same set of RNP complexes as in a. Luciferase activity was measured at 24 h post transfection, following incubation at 39 °C. Data represent mean luciferase activity from three separate experiments, calculated after normalization with Renilla luciferase activity,±s.d. ‘—’ represents blank control; RNP without the PB2 gene. Statistical significance was analysed by one-way analysis of variance, corrected by the Bonferroni post-test: ***P<0.001, **P<0.01 and *P<0.05. Full-size uncropped western blots showing PB2 protein levels are displayed in Supplementary Fig. 6.