Fig. 6.

pS273R affects IRF3 activation independently of its cysteine protease activity. A Schematic of full-length pS273R and its truncated mutants. B cGAS (10 ​ng), STING (40 ​ng), pRL-TK (2 ​ng) plasmid, and IFNβ-Luc (20 ​ng), ISRE-Luc (20 ​ng), or IRF3-Luc (20 ​ng) reporter plasmid were co-transfected into HEK293T cells with empty vector, pS273R wild type (WT) (100 ​ng), Arm, Core, S273R-3M, or Core-3M expression plasmid. At 24 hpt, cells were harvested to analyze the luciferase activity. The expression of Flag-cGAS, Flag-STING and various S273R mutants were determined by Western blotting. C Flag-cGAS and Flag-STING were co-transfected with empty vector, pS273R WT, Arm, Core, S273R-3M, or Core-3M expression plasmid into HEK293T cells. At 24 hpt, the mRNA levels of IFN-β, ISG56, and ISG54 were analyzed by qRT-PCR. D Flag-TBK1 (800 ​ng) and HA-IRF3 (1 ​μg) were co-transfected with pS273R WT (1 ​μg), Core, S273R-3M, or Core-3M expression plasmid in HEK293T cells for 24 ​h. Cells were harvested and the lysates were immunoprecipitated with anti-Flag antibody and subjected to Western blotting analysis. E HEK293T cells were transfected with Flag-TBK1 (500 ​ng) and Myc-S273R (1 ​μg) for 12 ​h and then treated with E-64 (4 ​mmol/L) for 36 ​h. The cells were harvested for Western blotting analysis. F HEK293T cells were transfected with Flag-TBK1 (800 ​ng), HA-IRF3 (1 ​μg), and Myc-S273R (1 ​μg) for 12 ​h and then treated with E-64 (4 ​mmol/L) for 36 ​h. Cells were then harvested for co-immunoprecipitation analysis. Data are presented as mean ​± ​SD of three independent experiments. Statistical significance between groups was determined using a t-test with GraphPad Prism software. ∗P ​< ​0.05; ∗∗P ​< ​0.01; ∗∗∗P ​< ​0.001; ns, not significant.