G10-Mediated IRF3 Phosphorylation, ISG mRNA Induction, and Anti-Alphaviral Activity are not Detectable in Cells Lacking STING.

(A) Immunoblot of lysates from THF-ΔSTING following 7h exposure to 1% DMSO, 0.1μg/mL poly(I:C), SeV, UV-CMV, 1μg/mL 2’3’-cGAMP, or 100uM G10 as indicated showing phosphorylation status of IRF3 S386, total IRF3, IPS1, STING, and GAPDH. (B) Expression of IRF3/IFN-dependent LUC in THF-ISRE-ΔIPS1 and THF-ISRE-ΔSTING following 7h exposure to 1% DMSO, indicated concentrations of G10, SeV, UV-CMV, or 1μg/mL LPS. Values are presented as average fold change in quadruplicate measurements ±SD relative to cells treated with 1% DMSO. (C) Average fold changes ±SD from duplicate experiments of ISG54, ISG15, and Viperin mRNA relative to cells treated with 1% DMSO in THF-ISRE-ΔIPS1 (black bars) or THF-ISRE-ΔSTING (gray bars) following exposure to UV-CMV, SeV, or 100μM G10. (D) Media titers of CHIKV and VEEV at 24h (VEEV) or 48h (CHIKV) obtained from THF-ISRE-ΔSTING cells treated with 1% DMSO, 100μM G10, or 1000U/mL IFNβ as indicated. Infections were performed in triplicate.