Fig 2: (A, B) The effects of p17 on the interactions and cellular co-localizations between STING and TBK1/IKKϵ. (A) 293T cells in 6-well plate were co-transfected with pcGAS-HA (0.5 μg) and pGFP-STING (0.5 μg) with or without pdsRed-p17 (0.5 μg and 1 μg) for 24 h (B) 293T cells were co-transfected with pGFP-STING (0.5 μg) and pMyc-IKKϵ (0.5 μg) with or without pdsRed-p17 (0.5 μg and 1 μg) for 24 (h) The cell lysates were subjected for immuno-precipitation and Western blotting using the indicated antibodies. (C, D) The effect of p17 on the interaction between endogenous STING and TBK1. (C) 3D4/21 cells were stimulated by transfection of poly I:C (1 μg/mL) or 2’3’-cGAMP (1 μg/mL), respectively, for 12h. The cell lysates were subjected for immune-precipitation with rabbit anti-STING pAb, and subsequent Western blotting using the rabbit anti-TBK1 mAb and anti-STING pAb. (D) The pCAGGS-p17-HA (2 μg) were transfected into 3D4/21 cells for 12h and then stimulated by transfection of 2’3’-cGAMP (1 μg/mL) for another 12h. The cell lysates were subjected for immune-precipitation and subsequent Western blotting. (E, F) The effect of p17 on the cellular co-localization between STING and TBK1/IKKε was analyzed by IFA. GFP and dsRed expression in cells was directly visualized by confocal microscopy. The endogenous TBK1 and Myc-IKK were stained by rabbit anti-TBK1 mAb and anti-Myc, respectively, followed by Alexa Fluor plus 647 anti-rabbit secondary antibody. (G, H) The effects of p17 on the activations of IFN promoter (G) and NF-κB promoter (H) triggered by transfected STING (20 ng), TBK1 (20 ng), IKKϵ (20 ng) and IRF3-5D (20 ng). The fold changes are relative to vector transfected controls. Values represent the mean ± S.D. *P < 0.05, **P < 0.01, ns not significant.

Fig 3: (A-D)The effects of ASFV p17 on nuclear translocations of IRF3 and NF-κB p65. The 3D4/21 cells grown on the coverslips in 24-well plates (1×105 cells/well) were transfected with IRF3-GFP (0.5 μg) and pCAGGS-p17-2HA (0.5 μg) or control vector (0.5 μg) (A, B), p65-GFP (0.5 μg) and pCAGGS-p17-2HA (0.5 μg) or control vector (0.5 μg) (C, D) using Lipofectamine 2000. At 18 h post translation, the cells were stimulated with transfection of 2’3’-cGAMP (1μg/ml) for another 6 (h) The cells on coverslips were sequentially probed with rabbit anti-HA mAb, goat anti-rabbit IgG H&L Alexa Fluor 594, counter-stained with nucleus marker DAPI. (A) The effects of ASFV p17 on nuclear translocations of IRF3 was observed by fluorescence microscope with the enlarged images shown on the right. (B) Statistical analysis of the ratios of IRF3-GFP in nuclear fractions vs cytoplasmic fractions. (C) The effects of ASFV p17 on nuclear translocations of p65 was observed by fluorescence microscope with the enlarged images shown on the right. (D) Statistical analysis of the ratios of p65-GFP in nuclear fractions vs cytoplasmic fractions. (E, F) 3D4/21 cells in 24-well plates were transfected with the STING siRNA or Control siRNA (50 nM) by lipofectamine 2000 for 24 h, followed by co-transfected p17, ISRE-Fluc and Rluc for 12 h, and then stimulated by cGAMP for another 12 h. (E) Effects of the activation of ISRE promoter were detected by luciferase measurement. (F) Effects of the phosphorylation of TBK1 (p-TBK1) were detected by Western blot. Fold change values represent the mean ± S.D. *P < 0.05, **P < 0.01, ns not significant.

Fig 4: The apoptosis and autophagy induced by STING signaling are both IFN independent. (A) The alignment of human, mouse and porcine STING with CTT domain enlarged, IRF and TBK1 recruitment motifs box marked. The pSTING S365 in IRF recruitment motif and L373 in TBK1 recruitment motif are also indicated. (B, D) 293T cells were co-transfected with pcDNA-pcGAS-2HA (0.5 μg) plus pcDNA-pSTING-2HA, pSTING S365A, pSTING L373A or pSTING ΔCTT (each 0.5 μg) for 24 h, followed by Western blotting (B) and flow cytometry (C, D). **p < 0.01.

Fig 5: Upregulation of dsRNA sensors in 293FT TMEM41B KO and VMP1 KO cells upon DENV infection.(A) Western blotting analysis to detect RIG-I, MDA5, TBK1, and p-TBK1 protein levels in 293FT WT cells, TMEM41B KO clone #1, and VMP1 KO clone #1, upon DENV infection. Cells were infected with DENV1 at MOI of 1 and lysates were harvested at 48 hours post-infection. (B) Western blotting analysis to detect RIG-I and MDA5 protein levels in 293FT WT, TMEM41B KO clone #1, and VMP1 KO clone #1 cells, upon HCoV-OC43 infection. Cells were infected with HCoV-OC43 at MOI of 0.5 and lysates were harvested at 48 hours post-infection. (C,D) IFN-ß mRNA (C) and DENV RNA (D) abundance in uninfected and DENV1 infected (MOI of 1, 48 hours post-infection) WT and KO cells. Values demonstrated are relative to the expression levels in uninfected WT cells (C), or DENV infected WT cells (D). (E,F) DENV viral protein accumulation and infectious viral particles produced in 293FT WT, TMEM41B KO clone #1, and VMP1 KO clone #1 cells, upon further knocking out of RIG-I and MDA5. Infection assays were carried out at MOI of 1 and (E) the amount of infectious virus in the supernatant was determined at 48 hours post-infection by plaque assay, and (F) protein levels of the indicated proteins were determined by western blotting analysis. GAPDH was used as a loading control for all blots. 18S rRNA was used as the control for the RT-qPCR experiments. All data shown represent results from at least two independent experiments. Single-dashed lines divide blots from two gels loaded with the same set of lysates. Double-dashed lines divide blots from gels belonging to separate experiments using the same experimental conditions. Error bars represent mean +/- SEM, n = 3. * indicates p-value < 0.05 as determined by one-way ANOVA. LOD indicates the limit of detection.