FIG 3.

EP364R and C129R target intracellular 2′,3′-cGAMP for degradation. (A and B) HEK293T cells were transfected with Flag-EP364R or Flag-C129R plasmids as indicated along with a firefly luciferase reporter plasmid encoding the IFN-β promoter, the TK-Renilla plasmid as a transfection control to normalize firefly luciferase activity, and expression plasmids of STING, TBK1, and IKKε for 24 h. STING-overexpressing 293-Dual hSTING-A162 cells were used for poly(dA-dT)-, cGAS-, and cGAMP-induced luciferase assays. 293-Dual hSTING-A162 cells were transfected by both Flag-EP364R and Flag-C129R plasmids as indicated along with 3×Flag-cGAS expression plasmids or transfected 2′,3′-cGAMP or poly(dA-dT) for 12 h. After 36 h of transfection, luciferase activity of each sample was measured. Results are expressed relative to those with Renilla luciferase alone (internal control). Data represent at least two independent experiments, each with similar results. (C) 293-Dual hSTING-A162 cells were cotransfected with Flag-EP364R or Flag-C129R dose dependently with 3×Flag-cGAS plasmid with vector as a transfection control. (D and E) PK-15 cells and PAMs were transfected with Flag-EP364R and Flag-C129R at increasing doses with Flag control vector as a transfection control and then infected with ADV-GFP (MOI = 1) (D) and HSV-GFP (E) at 24 hpt. 293-Dual hSTING-A162 cells were harvested 24 hpi, and PK-15 cells and PAMs were harvested 4 hpi; cells were analyzed for intracellular 2′,3′-cGAMP by 2′,3′-cGAMP ELISA. Data in panels A and B are representative of three independent experiments, each with similar results, and all the values are expressed as means and SD for two biological replicates. All the ELISA and GFP absorbance data are representative of at least two independent experiments, each with similar results, and the values are expressed as means and SD for three biological replicates. Student's t test: **, P < 0.01; ***, P < 0.001; ns, not significant.