Fig. 7.
NS1 binding to mtDNA attenuates its immunostimulatory potential. a cGAS-293FT cells co-transfected with expression plasmids encoding EGFP, Flag-tagged NS1, or NS138/41 mutant, together with IFN-β reporter plasmids and poly(dA:dT) (left panel) or mtDNA (right panel). Twenty-four hours after transfection, cell lysates were collected and analyzed for luciferase activity. b Samples from HEK293FT cells transfected with siRNA targeting TFAM or control siRNA were blotted using the indicated antibodies. c HEK293FT cells transfected with siRNA targeting TFAM or control siRNA were infected with PR8 virus for 24 h. Cytosolic mtDNA (left panel) and IFN-β mRNA levels (right panel) were assessed by quantitative PCR. d Pure cytosolic fraction prepared from digitonin extracts of mock- or PR8-infected HEK293FT cells were treated with proteinase K. Proteinase K-treated pure cytosolic extracts were analyzed by immunoblotting with indicated antibodies. e DNA was extracted from proteinase K-treated pure cytosolic fraction using QIAquick Nucleotide Removal kit (QIAGEN). Cytosolic mtDNA was assessed by quantitative PCR. f cGAS-293FT cells were transfected with DNA extracted from proteinase K-treated pure cytosolic fraction for 6 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, e, f; 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