FIG 4.

Binding of MmsA to STING inhibits type I IFN production via STING-TBK1-IRF3 pathway. (A) HEK293T cells were cotransfected with the interferon reporter plasmid IFN-β-Luc together with the plasmids expressing human STING and increasing amounts of M. tuberculosis MmsA, with a Renilla luciferase plasmid as an internal control. Luciferase activities were determined 24 h posttransfection. (B) RAW264.7 cells were infected with M. smegmatis::vector and M. smegmatis::MmsA for 0, 2, 4, and 6 h at an MOI of 10, and IFN-β mRNA was measured by qPCR. GAPDH was used as a loading control. (C) HEK293T cells were cotransfected with the interferon reporter plasmid IFN-β-Luc together with the plasmids expressing M. tuberculosis MmsA and STING, TBK1, or IRF3. A Renilla luciferase plasmid was an internal control. Luciferase activities were determined 24 h posttransfection. (D) RAW264.7 cells were transfected with empty vector or the plasmids expressing EGFP-MmsA in increasing amounts of 0.5 μg, 1.0 μg, and 2.0 μg. Forty-eight hours posttransfection, STING monomer and dimer were analyzed by immunoblotting. (E) HEK293T cells were transfected with pMyc-human-STING and Flag-TBK1, with or without pMyc-MmsA, for 48 h. Cell lysates were immunoprecipitated with the anti-Flag antibody and then immunoblotted with the indicated antibodies. (F) Raw264.7 cells were transfected with plasmid pMyc-murine-STING and vector or pFlag-MmsA for 20 h and treated with poly(dA:dT) (2 μg/ml) for 8 h. Cell lysates were analyzed for IRF3 dimerization by native PAGE. Total IRF3 expression levels and other indicated proteins were analyzed by immunoblotting. The results are shown as means ± SEM. NS, not significant; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; each by Student's t test.