Fig 2: SARS-CoV-2 nsp12 suppresses the nuclear translocation of IRF3. a Effect of SARS-CoV-2 nsp12 on SeV-induced nuclear translocation of IRF3. HEK293T cells were transfected with a control plasmid or an nsp12 expression plasmid. At 24 h post transfection, the cells were infected with SeV for 4 h and then immunostained with the indicated antibodies. Merge 1 and Merge 2 indicate the merged red and green channels and the merged red, green, and blue channels, respectively. Scale bar, 10 μM. The enlarged image is a magnified view of the corresponding box. b Quantitation of the nuclear translocation of IRF3. The percentage of nuclear IRF3-positive transfected cells was determined in three independent experiments. The two-tailed unpaired t-test was used for two-group comparisons, ***P < 0.001. c Immunoblot analysis of cytoplasmic and nuclear IRF3 after SeV infection. HEK293T cells were transfected with a control plasmid or an nsp12 expression plasmid. After 24 h, the cells were infected with SeV for 4 h and fractionated into cytoplasmic and nuclear fractions. The fractions were analyzed by western blotting for IRF3 and nsp12 detection. β-Tubulin and Lamin A were used as a cytoplasmic and a nuclear marker, respectively. The numbers under the lanes indicate the IRF3 band intensity, which was normalized to that of Lamin A. d Effect of SARS-CoV-2 nsp12 on RIG-IN-induced nuclear translocation of IRF3. HEK293T cells were transfected with RIG-IN or RIG-IN and nsp12 expression plasmids. At 24 h post transfection, the cells were immunostained with the indicated antibodies

Fig 3: (A) The expression levels of TBK, phosphorylated TBK (pTBK), IKKϵ, phosphorylated IKKϵ (pIKKϵ), IRF3, and phosphorylated IRF3 (pIRF3) in non-treated epithelial cells (cont), in epithelial cells treated with ephrinA1 (10 ng/ml), in rhinovirus-infected cells, and in ephrinA1-pretreated cell followed by rhinoviral infection for 24 h. (B) The expression levels of TBK, phosphorylated TBK (pTBK), IKKϵ, phosphorylated IKKϵ (pIKKϵ), IRF3, and phosphorylated IRF3 (pIRF3) in non-treated epithelial cells (cont), in epithelial cells treated with ephrinA1 (10 ng/ml), in poly(I:C)-treated cells, and in ephrinA1-pretreated cell followed by poly(I:C) treatment for 24 h. (C) The expression levels of TBK, phosphorylated TBK (pTBK), IKKϵ, phosphorylated IKKϵ (pIKKϵ), IRF3, and phosphorylated IRF3 (pIRF3) in non-treated epithelial cells (cont), in rhinovirus-infected cells, and in cells transfected with scr siRNA, transfected with ephA2 siRNA, and treated with ephA2 inhibitor followed by rhinoviral infection. (D) The expression levels of TBK, phosphorylated TBK (pTBK), IKKϵ, phosphorylated IKKϵ (pIKKϵ), IRF3, and phosphorylated IRF3 (pIRF3) in non-treated epithelial cells (cont), in poly(I:C)-treated cells, and in cells transfected with scr siRNA, transfected with ephA2 siRNA, and treated with ephA2 inhibitor followed by poly(I:C) treatment. Upper panels located in each figure show representative protein bands evaluated with western blot. All data derived from seven different subjects are presented as dot plots showing median levels. (* indicates P < 0.05).

Fig 4: Translocation of the cGAS downstream effectors. (A) Nuclear translocation of p65 was determined by subcellular fractionation in arginine-deprived CWR22Rv1 cells. (B) Nuclear p65 was quantified and normalized to lamin A/C. (n = 3, ***p < 0.001) (C) Immunostaining of p65 in CWR22Rv1 cells. Scale bars, 10 μm. (D) ChIP-qPCR analysis of IRF3 binding on the promoter regions of IFNA14 and IFNB1 in CWR22Rv1. (n = 3, *p < 0.05) (E) CWR22Rv1 cells overexpressing STING-V5 were subjected to arginine deprivation and stained with V5, calnexin and RCAS1 antibodies. Scale bars, 10 μm.

Fig 5: IKKε stimulates SBP enzyme gene transcription via ATF4 ARepresentative Western blot showing the level of ATF4 in ATF4‐silenced Flp‐In 293 HA‐IKKε cells treated with doxycycline (Dox) for 16 h.BqRT–PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in Flp‐In 293 HA‐GFP or Flp‐In 293 HA‐IKKε cells treated with doxycycline (Dox) for 16 h. Data are expressed as fold changes, relative to levels in non‐treated Flp‐In 293 HA‐GFP cells and normalised to β‐Actin (n = 4 biological replicates)CRepresentative Western blot showing levels of IKKε, IRF3, phosphorylated IRF3 (S396) and SBP enzymes in Flp‐In 293 HA‐GFP or Flp‐In 293 HA‐IKKε cells treated with doxycycline (Dox) for 16 h.DqRT–PCR analysis of PHGDH, PSAT1 and PSPH mRNA levels in ATF4‐silenced Flp‐In 293 HA‐IKKε cells treated with doxycycline (Dox) for 16 h. Data are expressed as fold changes, relative to levels in a non‐silenced, non‐treated control and normalised to β‐Actin (n = 5 biological replicates).ERepresentative Western blot showing the levels of IKKε, PHGDH, PSAT1 and PSPH in ATF4‐silenced Flp‐In 293 HA‐IKKε cells treated with doxycycline (Dox) for 16 h.Data Information: In (B, D), data are presented as mean ± SEM, statistics were performed using log‐transformed fold change values. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, measured using two‐way ANOVA with Bonferroni post hoc tests. Source data are available online for this figure.