Fig 2: Activation of cGAS pathway in syncytia induced by SARS-CoV-2 infection and Spike-ACE2 expression. a and b Channeled images (a) and line scan analysis (b) showed cGAS and IRF3 were co-localized with micronuclei. Scale bars: 1 μm for the inserts in the lower left corner; 20 μm for full images. c Quantification of micronuclei positive in cGAS (+) and IRF3 (+) in Hela-ACE2 cells transfected with spike protein. d The relative mRNA level of IFIT2, IFNB1, CCL5 and CXCL10, respectively in Hela-ACE2 cells 24 h after transfection. *P < 0.05; **P < 0.01. e and f The expression of cGAS, STING, IRF3 and their phosphorylated form were increased upon expression of spike protein analyzed by Western blot (e) and quantification (f). All results were normalized by the expression of β-actin. Data are mean ± SD of triple quantification. *P < 0.05; **P < 0.01; ***P < 0.001

Fig 3: SARS-CoV-2 nsp12 does not inhibit the phosphorylation of IRF3. HEK293T cells were transfected with a control plasmid or nsp12 expression plasmid. After 24 h, the cells were infected with SeV and collected at the indicated times. Whole-cell lysates were analyzed by western blotting for p-IRF3, IRF3, TBK1, nsp12, and P protein of SeV. b HEK293T cells were transfected with a control plasmid or increasing amounts of plasmids expressing nsp12. After 24 h, the cells were infected with SeV for 8 h. Whole-cell lysates were analyzed by western blotting for p-IRF3, IRF3, nsp12, and P protein of SeV

Fig 4: IFN regulatory factor 5 (IRF5) is activated by TLR8 ligands and cytosolic dsDNA sensing, IRF3 is activated by TLR4 signaling and cytosolic dsDNA and dsRNA sensors, while only TLR8 is antagonized by TLR2 ligand. (A) Monocytes were stimulated with CL75 (1 μg/ml), LPS (K12, 100 ng/ml), pdA:dT (dsDNA, 1 μg/ml), or poly(I:C) (dsRNA, 1 μg/ml). The nucleic acid ligands were transfected using L2K. Fixation was done at 30, 60, and 120 min poststimulation and IF staining of IRF5 and IRF3 was performed. (B) Monocytes were stimulated with the TLR8 agonist polyuridylic acid (pU) in complex with poly-l-arginine (pLA) or with pdA:dT/L2K. The ligands (1 μg/ml) were added alone or together with FLS-1 (100 ng/ml), and cells were fixed and stained after 120 min of incubation. Quantification of nuclear accumulation was done by high-content screening (Scan^R, 20×). Significance was tested relative to no stimuli (NS). n = 4.

Fig 5: Binding of BPLF1 to 14-3-3 is required for inhibition of the IFN response.A. The BPLF1-D86-90R fails to inhibit the nuclear translocation of IRF3 in cells expressing constitutively active RIG-I-2CARD. Confocal images were obtained at 63x lens objective magnification. IRF3 is in green, RIG-I-2CARD in red, BPLF1 in grey. B. Quantification of the percentage of cells exhibiting nuclear IRF3. The mean ± SD of two independent experiments is shown. C. BPLF1-D86-90R fails to inhibit the transcription of IFN and IFN-regulated genes induced by treatment with poly(I:C). The levels of specific mRNAs were quantified by qRT-PCR. The results are shown as fold change relative to vector transfected poly(I:C) treated cells. The mean ± SD of two experiments each performed in triplicate is shown. D. Representative western blots illustrating the expression of phosphorylated IRF3 in the experiment shown in Fig 6C. Phospho-IRF3 is downregulated in cells expressing catalytically active BPLF1 but not in cells expressing BPLF1-D86-90R. One representative experiment out of two is shown in the figure.