Fig. 5

Influenza virus stimulates DDX41-dependent IFN-β gene expression. a cGAS-293FT cells transfected with siRNA targeting DDX41 or control siRNA were infected with influenza virus for 24 h. Cell lysates were collected and blotted using the indicated antibodies (left panel). IFN-β mRNA levels were assessed by quantitative PCR with β-actin as an internal control (right panel). b, c cGAS-293FT cells were infected with WT (left panel) or ΔNS1 influenza virus (right panel) for 24 h in the presence or absence of LFM-A13 (100 μM) (b). WT or DDX41-deficient STING-A549 cells were infected with PR8 (left panel), or EMCV (right panel) for 24 h (c). IFN-β mRNA levels were assessed by quantitative PCR with β-actin as an internal control. d Pure cytosolic fraction prepared from digitonin extracts of mock- or ΔNS1 influenza virus-infected cGAS-293FT cells were treated with DNase I or RNase H. Cytosolic mtDNA was assessed by quantitative PCR. e HEK293FT cells were transfected with DNA extracted from DNase I- or RNase H-treated pure cytosolic fraction for 24 h. IFN-β mRNA levels were assessed by quantitative PCR with β-actin as an internal control. f STING-A549 cells transfected with siRNA targeting DDX41 or control siRNA were infected with PR8 virus for 24 h. Cell lysates were collected at 24 h post infection and blotted using the indicated antibodies (left panel). Pure cytosolic extracts were collected at 24 h post infection and analyzed for cGAMP by ELISA (right panel). g, h HEK293FT cells were transfected with siRNA targeting DDX41. Two days later, cells were transfected with the expression plasmid encoding Flag-tagged DDX41 or DDX41 (Y414F) mutant. Twenty-four hours after DNA transfection, the cells were infected with WT (g) ΔNS1 influenza virus (h) for 24 h. Cell lysates were collected at 24 h post infection and blotted using the indicated antibodies (left panel). IFN-β mRNA levels were assessed by quantitative PCR with β-actin as an internal control (right panel). These data are from three independent experiments (mean ± s.e.m.). **P < 0.01, ***P < 0.001; n.s., not significant (one-way ANOVA and Tukey’s test). Source data are provided as a Source Data file