Figure 2: The ESIE-motif within PB1 and PA mediates type I IFN response.

(a) A549 cells or NHBEs infected with the indicated viruses (multiplicity of infection (MOI)=5) were prepared for WB analysis 8 h.p.i. Enhanced type I IFN response following disruption of the ESIE-motif was analysed (pSTAT1 Y701). (b) Polymerase activity was measured in A549 cells. Cells not expressing PB1 served as control (Ctrl). (c) A549 cells treated with cycloheximide (CHX) or left untreated were infected with the indicated viruses (MOI=5). Elevated IFNβ-mRNA levels were measured following disruption of the ESIE-motif 8 h.p.i. independent of CHX treatment; CHX treatment was controlled by WB analysis of NS1. (d) A549 cells treated with actinomycin D (ActD) or left untreated were infected with the indicated viruses (MOI=5). Formation of IRF3 oligomers was measured following the disruption of the ESIE-motif 8 h.p.i. independent of ActD treatment; ActD treatment was controlled by WB analysis of NS1. (e–j) Mice (n=5 per group) were infected i.n. with 10^2.5 plaque-forming units (PFU) (BALB/c) and 10² PFU or 10³ PFU (C57Bl/6 and Ifnar1^−/−Ifnlr1^−/−). Mice were examined daily for (e,h) survival and (i) body weight loss for 14–15 days p.i. (f,j) Virus lung titres were determined 3 days p.i. (g) Elevated mRNA levels of IFNβ were measured 2 days p.i. Depicted are fold changes compared with PBS-inoculated mice. (k) A549 cells were infected with the indicated viruses (MOI=5) and prepared for WB analysis 8 h.p.i. Conservation of type I IFN regulatory properties of the ESIE-signature among IAV subtypes was analysed (pSTAT1 Y701) upon infection with highly pathogenic H5N1 and H7N7 viruses. (a,d,k) Shown are representative results from three independent experiments. (b,c) Bars show mean±s.d. of three independent experiments. (f,g,j) Each dot represents an individual mouse; bars show mean. For statistical analysis, (b,f,j) Student’s t-test, (c,g) analysis of variance with Tukey’s test, (e,h) log-rank (χ²) test for statistical analysis of Kaplan–Meier survival data and (i) Student’s t-test analysis (Holm–Sidak method) were performed; *P<0.05, **P<0.01, ***P<0.001, NS=not statistically significant. Full western blots are shown in Supplementary Fig. 4a–f.