Fig. 4.

SARS-CoV-2 3CL selectively targets STING function and inhibits K63-Ub modification of STING. a SARS-CoV-2 3CL specifically antagonizes cGAS-STING-mediated NF-κB signaling. HEK293T cells were co-transfection with NF-κB-Luc and expression plasmids for cGAS and STING, IKKβ, IKKα, TBK1, or p65 with or without the expression vector for SARS-CoV-2 3CL. Luciferase activity stimulated by cGAS and STING, IKKβ, IKKα, TBK1, or p65 in the absence of SARS-CoV-2 3CL was set to 100%. b, c SARS-CoV-2 3CL inhibits STING R284M- (b) or V155M (c)-stimulated NF-κB signaling. HEK293T cells were transfected with NF-κB-Luc, pRL-TK Renilla, and the STING R284M (b) or V155M (c) expression vectors with increasing amounts of 3CL. d 3CL inhibits K63-Ub modification of STING. HEK293T cells were transfected with Ub K63 alone, STING-cGAS alone, or co-transfected with STING, cGAS, and Ub K63 in the presence or absence of 3CL. MG132 (20 µM) was added 12 h later. Cell lysates were prepared and immunoprecipitated with anti-Flag beads 24 h after transfection. Precipitated samples were prepared and reacted with anti-Flag antibody to detect Flag-STING and anti-HA antibody to detect HA-Ub-K63 and 3CL-HA. Histone was used as the loading control. e 3CL disrupts the interaction between STING and endogenous TBK1 or IKKβ. HEK293T cells were transfected with STING-cGAS in the presence or absence of 3CL. Cell lysates were prepared and immunoprecipitated using anti-Flag beads 24 h after transfection. Precipitated samples were prepared and reacted with anti-Flag antibody to detect STING-Flag and anti-TBK1 and anti-IKKβ antibody to detect endogenous proteins. Histone was used as the loading control. f The 3CL mutant HC/AA has a reduced ability to inhibit cGAS-STING-stimulated NF-κB response element activation. HEK293T cells were transfected with NF-κB-Luc, pRL-TK Renilla, and the cGAS-STING expression vector with increasing amounts of 3CL or 3CL HC/AA. cGAS-STING alone served as a positive control and was set to 100%. Transactivation of the luciferase reporter was determined 24 h after transfection (n ≥ 3 independent biological experiments) (a–c, f). g The binding ability of IKKβ or TBK1 with STING was downregulated in the presence of 3CL in (e) and was quantified using the ImageJ software. Means and standard deviations are presented. Statistical significance was determined by two-sided unpaired t test, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (a–c, f–g)