Influenza A virus M2 protein triggers mitochondrial DNA-mediated antiviral immune responses

Cytosolic mitochondrial DNA (mtDNA) activates cGAS-mediated antiviral immune responses, but the mechanism by which RNA viruses stimulate mtDNA release remains unknown. Here we show that viroporin activity of influenza virus M2 or encephalomyocarditis virus (EMCV) 2B protein triggers translocation of mtDNA into the cytosol in a MAVS-dependent manner. Although influenza virus-induced cytosolic mtDNA stimulates cGAS- and DDX41-dependent innate immune responses, the nonstructural protein 1 (NS1) of influenza virus associates with mtDNA to evade the STING-dependent antiviral immunity. The STING-dependent antiviral signaling is amplified in neighboring cells through gap junctions. In addition, we find that STING-dependent recognition of influenza virus is essential for limiting virus replication in vivo. Our results show a mechanism by which influenza virus stimulates mtDNA release and highlight the importance of DNA sensing pathway in limiting influenza virus replication.


Supplementary Figure 3
Influenza virus induces phosphorylation of IRF3 in a MAVS-dependent manner. a,b, WT or MAVS-deficient HEK293FT cells were transfected with the plasmid encoding HA-tagged IRF3. Twenty-four hours after transfection, the cells were infected with WT or ΔNS1 influenza virus. Cell lysates were collected at 12 h post infection and blotted using the indicated antibodies. Data was representative of three independent experiments.

Supplementary Figure 5
GSDMD does not stimulate cytosolic mtDNA release. a,b, HEK293FT cells were transfected with the expression plasmid encoding EGFP, influenza virus M2, or GSDMD residues 1-275 (GSDMD1-275). LDH activity was measured at 24 h post transfection (a). Cytosolic mtDNA was assessed by quantitative PCR at 24 h post transfection (b). c, HEK293FT cells were infected with PR8 or EMCV. Cell lysates were collected at 24 h post transfection and blotted using an anti-GSDMD antibody (64-Y). Flag-tagged GSDMD-transfected cell lysates were used as a positive control. These data are from two independent experiments (mean ± s.e.m.). ***P < 0.001; (one-way ANOVA and Tukey's test). Source data are provided as a Source Data file.

Supplementary Figure 6
Adenovirus stimulates cGAS-and STING-dependent IFN-β gene expression in mouse lung fibroblast.
Primary lung fibroblast prepared from WT, cGAS-, and STING-deficient mice were infected with adenovirus. IFN-β mRNA levels were assessed by quantitative PCR with GAPDH as an internal control. Source data are provided as a Source Data file.
Primary lung fibroblast prepared from WT, cGAS-, STING-, and MAVS-deficient mice were infected with rgPR8/M2del29-31 virus. IFN-β mRNA levels were assessed by quantitative PCR with GAPDH as an internal control. Source data are provided as a Source Data file.

Supplementary Figure 8
EMCV stimulates cGAS/STING-dependent IFN-β mRNA expression in MEFs. a, WT MEFs were infected with EMCV. Cytosolic mtDNA was assessed by quantitative PCR at 24 h post infection. b, MEFs prepared from WT, cGAS-, and STING-deficient mice were infected with adenovirus or EMCV for 24 h. IFN-β mRNA levels were assessed by quantitative PCR with GAPDH as an internal control. These data are from three independent experiments (mean ± s.e.m.). ***P < 0.001; (one-way ANOVA and Tukey's test). Source data are provided as a Source Data file.

Supplementary Figure 9
Overexpression of cGAS reduced the expression levels of STING.
EGFP-293FT cells were transfected with indicated amounts of the expression plasmid encoding EGFP or HA-tagged cGAS. Cell lysates were collected at 24 h post transfection and blotted using anti-HA or indicated antibodies. Data was representative of three independent experiments.

Supplementary Figure 10
TRIM32 is required for influenza virus-induced IFN-β gene expression. a, Samples from WT or TRIM32 KO STING-A549 cells were blotted using the indicated antibodies. b, WT or TRIM32 KO STING-A549 cells were infected with 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 two independent experiments (b; mean ± s.e.m.). ***P < 0.001; (oneway ANOVA and Tukey's test). Source data are provided as a Source Data file.

Supplementary Figure 11
ΔNS1 influenza virus stimulates DDX41-dependent IFN-β mRNA expression in HEK293FT cells. a, Samples from HEK293FT cells transfected with siRNA targeting MAVS, STING, or control siRNA were blotted using the indicated antibodies. b, HEK293FT cells transfected with siRNA targeting MAVS, STING, or control siRNA were infected with ΔNS1 influenza virus for 24 h. IFN-β mRNA levels were assessed by quantitative PCR with β-actin as an internal control. c, Samples from HEK293FT cells transfected with siRNA targeting MAVS, STING, DDX41, or control siRNA were blotted using the indicated antibodies. d, HEK293FT cells transfected with siRNA targeting MAVS, STING, DDX41, or control siRNA were infected with ΔNS1 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 (b,d; mean ± s.e.m.). ***P < 0.001; (one-way ANOVA and Tukey's test). Source data are provided as a Source Data file.

Supplementary Figure 12
mtDNA release is intact in DDX41 KO STING-A549 cells after influenza virus infection. a, Samples from WT or DDX41 KO STING-A549 cells were blotted using the indicated antibodies. b, WT or DDX41 KO STING-A549 cells were infected with influenza virus for 24 h. Cytosolic mtDNA was assessed by quantitative PCR. These data are from two independent experiments (b; mean ± s.e.m.). n.s., not significant. Source data are provided as a Source Data file.

Supplementary Figure 13
ρ0 HEK293FT cells reduces IFN-β gene expression after ΔNS1 influenza virus infection. a, Total mtDNA copy number was measured by quantitative PCR. b, Control or ρ0 HEK293FT cells were infected ΔNS1 influenza virus for 24 h. Cytosolic mtDNA was assessed by quantitative PCR. c, Samples from Control or ρ0 HEK293FT cells were blotted using the indicated antibodies. d, Control or ρ0 HEK293FT cells were infected ΔNS1 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 (a,b,d; mean ± s.e.m.). **P < 0.01, ***P < 0.001; (one-way ANOVA and Tukey's test). Source data are provided as a Source Data file. Figure 14 ρ0 cGAS-293FT cells reduces IFN-β gene expression after WT or ΔNS1 influenza virus infection. a, Total mtDNA copy number was measured by quantitative PCR. b, Samples from Control or ρ0 cGAS-293FT cells were blotted using the indicated antibodies. c,d, Control or ρ0 cGAS-293FT cells were infected with WT A/PR8 (c) or ΔNS1 influenza virus (d) 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 (a,c,d; mean ± s.e.m.). ***P < 0.001; (one-way ANOVA and Tukey's test). Source data are provided as a Source Data file.