Fig. 2

Ion channel activity of influenza virus M2 protein is essential for mtDNA release. a HEK293FT cells were transfected with the expression plasmid encoding EGFP or Flag-tagged influenza virus proteins. Cell lysates were collected at 24 h post transfection and analyzed by immunoblot with mouse monoclonal antibody against Flag or EGFP (left panel). Cytosolic mtDNA was assessed by quantitative PCR at 24 h post transfection (right panel). b HEK293FT cells were transfected with the expression plasmid encoding EGFP or Flag-tagged WT or mutant M2 protein. Cell lysates were collected at 24 h post transfection and analyzed by immunoblot using indicated antibodies (left panel). Cytosolic mtDNA was assessed by quantitative PCR at 24 h post transfection (right panel). c HEK293FT cells were transfected with the expression plasmid encoding EGFP or Flag-tagged M2 protein in the presence or absence of BAPTA-AM (20 μM) or Mito-TEMPO (500 μM). Cell lysates were collected at 24 h post transfection and blotted using the indicated antibodies (left panel). Cytosolic mtDNA was assessed by quantitative PCR at 24 h post transfection (right panel). d HEK293FT cells were transfected with siRNA targeting MAVS or control siRNA. Two days later, cells were transfected with the expression plasmid encoding EGFP or Flag-tagged M2 protein. Cell lysates were collected at 24 h post DNA transfection and blotted using the indicated antibodies (left panel). Cytosolic mtDNA was assessed by quantitative PCR at 24 h post DNA transfection (right panel). e, f HEK293FT cells were infected with WT (rgPR8), M2del29–31 virus (rgPR8/M2del29–31) (e), or amantadine sensitive-recombinant influenza virus (rgPR8/M2_N31S) in the presence or absence of amantadine (100 μM) (f). Cytosolic mtDNA was assessed by quantitative PCR at 24 h post infection. These data are from three independent experiments (a–f; mean ± s.e.m.). ***P < 0.001; n.s., not significant (one-way ANOVA and Tukey’s test). Source data are provided as a Source Data file