IFN-inducing activity correlates with virus replication of NS1 mutants in mice. (a) Virus-induced activation of the IFN-β promoter in transgenic reporter mice. Six- to eight-week-old mice expressing firefly luciferase (FF-luc) under the control of the IFN-β promoter (Lienenklaus et al., 2009) were infected intranasally with either 5×10⁴ f.f.u. (•) or 2×10⁵ f.f.u. (○) of the indicated viruses. At 24 h post-infection, luciferase activity was determined in the lung homogenates of the reporter mice. (b) Virus replication in mouse lungs. Virus titres in lung homogenates of IFN-β reporter mice were determined as described previously (Grimm et al., 2007). Mean values and data points of individual animals are shown. Representative data from one of two independent experiments are shown. The dotted line marks the detection limit.

IFN-inducing capacity of NS1 mutants in cell culture. (a) Activation of the IFN-β promoter in mouse embryo fibroblasts expressing firefly luciferase under the control of the IFN-β promoter. Luciferase activity was determined in lysates of cells infected at an m.o.i. of 1 for 18 h. Data from three experiments are shown; error bars indicate variations of the mean. (b) Induction of type I IFN in human A549 cells. Cells were infected (m.o.i. of 1) for 18 h and culture supernatants were then dialysed against low-pH buffer to inactivate virus, as described previously (Kochs et al., 2007b). After the pH was brought back to neutral, these supernatants were added to 293T cells carrying firefly luciferase under the control of the Mx1 promoter (Jorns et al., 2006). Eighteen hours later, luciferase activity of lysates from indicator cells was determined. Data from three experiments are shown; error bars indicate variations of the mean. (c) Activation of IRF3 in 293 cells. Cells were infected (m.o.i. of 1) for 18 h and lysed, and dimeric IRF3 in cell lysates was separated from monomeric IRF3 by non-denaturing gel electrophoresis (Iwamura et al., 2001). Western blot analysis was performed to detect IRF3, using a polyclonal rabbit antiserum (FL-425; Santa Cruz). IRF3, viral nucleoprotein (NP) and β-actin were also visualized in unfractionated cell lysates by standard Western blotting. (d) Western blot analysis of mouse embryo fibroblasts infected with the indicated viruses at an m.o.i. of 1. NS1, NP and β-actin were detected by using specific rabbit antisera (Solorzano et al., 2005).