Figure 7: Effect of PB2-526R in interaction between PB2 and NEP.

(a) Co-immunoprecipitation of NEP with different versions of PB2 polymerase derived from H7N9 virus. Enhanced GFP (eGFP)-NEP and FLAG-PB2 were co-expressed in HEK293T cells. Whole-cell extracts were used for immunoprecipitation with an anti-GFP antibody and analysed by western blot analysis, using specific antibodies against eGFP and FLAG. The experiment was repeated three times. (b) Effect of different levels of NEP on RNP polymerase activity. RNP complexes containing different versions of PB2 were co-transfected with increasing amounts of NEP (H7N9) expression vector or empty vector, 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 37 °C, and the effect of different amounts of NEP on the polymerase activity of RNP with and without PB2-526R was compared. 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 the Student’s t-test: ***P<0.001, **P<0.01. (c) Western blot analysis of NEP expression in MDCK and A549 cells infected with H7N9 viruses carrying different versions of the PB2 segment. Confluent MDCK or A549 cells were infected with various reverse genetic H7N9 viruses, at an MOI of 1. Cells were collected at 5 and 7 h post infection and whole-cell lysates were analysed for NEP and NP expression by western blot using antibodies specific for NEP and NP, respectively. Blotting with antibody against β-tubulin, which served as a loading control, was also performed. Numbers indicate the ratio of NEP expression between viruses carrying 526R in the PB2 and those without 526R. The experiment was repeated twice. Full-size western blots are provided in Supplementary Fig. 6.