Fig. 6
From: Influenza A virus M2 protein triggers mitochondrial DNA-mediated antiviral immune responses
Influenza virus NS1 protein associates with mtDNA. a HEK293FT cells were transfected with the expression plasmid encoding EGFP, Flag-tagged M2, NS1, or NS138/41 mutant. Twenty-four hours after transfection, the cells were infected with ΔNS1 influenza virus for 24 h. Pure cytosolic extracts prepared from digitonin extracts of ΔNS1 influenza virus-infected cells were immunoprecipitated with mouse monoclonal antibody against Flag, followed by immunoblotting of immunoprecipitates with rabbit polyclonal antibody against Flag (left panel). DNA was extracted from immunoprecipitated samples using QIAquick Nucleotide Removal kit (QIAGEN). NS1-bound mtDNA was assessed by quantitative PCR (right panel). b HEK293FT cells were transfected with the expression plasmid encoding EGFP, Flag-tagged NS1, or NS1 38/41 mutant. Twenty-four hours after transfection, the cells were infected with ΔNS1 influenza virus for 24 h. Cell lysates were collected and blotted using the indicated antibodies (left panel). Cytosolic mtDNA was assessed by quantitative PCR (middle panel). IFN-β mRNA levels were assessed by quantitative PCR with β-actin as an internal control (right panel). c HEK293FT cells were infected with WT (rgPR8) or rgPR8/NS138/41A influenza virus at MOI of 1 for 24 h. Cell lysates were collected and blotted using the indicated antibodies (left panel). Cytosolic mtDNA (middle panel) and IFN-β mRNA levels (right panel) were assessed by quantitative PCR. d cGAS-293FT cells transfected with siRNA targeting STING or control siRNA were infected with rgPR8/NS138/41A influenza virus for 24 h. IFN-β mRNA levels were assessed by quantitative PCR with β-actin as an internal control. These data are from three independent experiments (mean ± s.e.m.). *P < 0.05, **P < 0.01, ***P < 0.001; (one-way ANOVA and Tukey’s test). Source data are provided as a Source Data file
