Fig 2: Ubiquitination promotes TBK1 targeting to the Golgi apparatus for activation. a HEK293T cells were transfected with an empty vector (Ev) or with plasmids encoding myc-tagged WT TBK1 (WT), TBK1K38M (K38M), or TBK1K30R/K401R (K30R/K401R). After 16 h, TBK1 activation and exogenous TBK1 expression were assessed by immunoblotting with anti-p-TBK1S172 and anti-myc antibodies, respectively. GAPDH was used as a loading control. b HEK293T cells were transfected with either an IFNß promoter reporter or an NF-?B reporter, together with the Renilla luciferase gene as an internal control. In parallel, the cells were also transfected with an Ev or with plasmids encoding myc-tagged WT TBK1 (WT), TBK1K38M (K38M), or TBK1K30R/K401R (K30R/K401R). Luciferase assays were performed 24 h after transfection and the results were normalized against Renilla luciferase activity. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with WT TBK1 in Student’s t test). RLU, relative luminescence units. c Immunoblotting analysis of TBK1–/– MEFs reconstituted with WT TBK1, TBK1K38M (K38M), or TBK1K30R/K401R (K30R/K401R). As controls, TBK1+/+ and TBK1–/– MEFs are shown. d TBK1–/– MEFs reconstituted with WT TBK1 or mutants were either left untreated (MOCK) or transfected with HMW poly(I:C) (5 µg/mL) for 4 h (trPoly(I:C)). TBK1 aggregation was then assessed by immunofluorescence staining and counting of the aggregates. The data shown are means ± SD from three independent experiments (300 cells were counted per condition). **0.001 < P < 0.01 versus MEFs reconstituted with WT TBK1 (Student’s t test). e The reconstituted MEFs described in (c) and the initial TBK1–/– MEFs were transfected with HMW poly(I:C) (5 µg/mL) for 0, 2, and 4 h (trPoly(I:C)). IFNB1 mRNA levels were then assessed by RT-qPCR with normalization against GAPDH. The data shown are means ± SD from three independent experiments (analysis of variance and comparison with WT TBK1-reconstituted MEFs in Student’s t test). AU, arbitrary unit

Fig 3: The progesterone-PGR axis promotes innate antiviral response via activation of SRC. a Effects of progesterone or viral infection on PR activation. The PGR-overexpressed HEK293 cells were transfected with PR reporter for 24 h, and then left untreated or treated with increased doses of progesterone (0.01, 0.1, 1 µM) or infected with different doses of SeV for 10 h before luciferase assays. b Effects of progesterone and viral infection on transcription of nuclear PGR-targeted genes. T-47D cells were treated with DMSO or P4 (1 µM) for 1 h and then left uninfected or infected with SeV for 6 h before qPCR analysis of mRNA levels of the indicated genes. c Effects of progesterone on virus-induced transcription of antiviral genes in SRC-knockdown cells. The control and SRC-knockdown T-47D cells were treated with P4 (1 µM) for 1 h and then left uninfected or infected with SeV for 6 h before qPCR analysis of mRNA levels of the indicated genes. The knockdown efficiency of SRC was shown by qPCR analysis of mRNA level in the left panels. d Effects of progesterone on IFN-?-induced transcription of IRF1 genes in SRC-knockdown cells. The control and SRC-knockdown T-47D cells were treated with P4 (1 µM) for 1 h and then left untreated or treated with IFN-? (100 ng/ml) for 6 h before qPCR analysis of IRF1 mRNA level. e Effects of progesterone on virus-induced phosphorylation events in SRC-knockdown T-47D cells. The control and SRC-knockdown T-47D cells were treated with P4 (1 µM) for 1 h, and then left uninfected or infected with SeV for the indicated times before immunoblotting analysis with the indicated antibodies. The relative intensities of p-IRF3S386 and SRC (normalized to ß-actin) were analyzed by ImageJ. f Effects of SRC inhibitor on progesterone-potentiated transcription of antiviral genes. BMDCs were pretreated with DMSO or Dasatinib (SRC inhibitor, 10 µM) and together with the indicated doses of P4 for 1 h. The cells were then left uninfected or infected with SeV for 6 h before qPCR analysis of the mRNA levels of Ifnb1 and Cxcl10 genes. g Effects of progesterone on virus-induced activation of SRC, TBK1 and IRF3. T-47D cells were treated with P4 (1 µM) for 1 h, and then left un-infected or infected with SeV for the indicated times before immunoblotting analysis with the indicated antibodies. h Effects of PGR-deficiency on SeV-induced activation of SRC, TBK1, and IRF3. The control and PGR-KO T-47D cells were cultured in P4-containing medium, and then left uninfected or infected with SeV for the indicated times before immunoblotting analysis with the indicated antibodies. Data shown in a-d, f are mean ± SD (n = 3) from one representative experiment, which was repeated for at least two times with similar results. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant

Fig 4: Knockdown of DDX56 potentiates RNA virus-triggered signaling. (A) Effects of DDX56-RNAi plasmids on the expression of endogenous DDX56. (B–E) Effects of DDX56-RNAi plasmids on SeV or poly(I:C)-triggered activation of the IFN-ß promoter and ISRE. Stable DDX56-knockdown 293T cells (105) were transfected with the IFN-ß promoter or ISRE (100 ng). At 24 h after transfection, the cells were left uninfected or infected with SeV for 12 h, or were treated with poly(I:C) (1 µg/ml) or left untreated for 18 h before reporter assays were performed. Shown are representative experiments of three independent experiments with mean±s.d. of three technical replicates. **P<0.01. (F) Knockdown of DDX56 increases the SeV-induced phosphorylation of TBK1, IRF3 and I?Ba. The stable DDX56-knockdown 293T cells were infected with SeV for the indicated times. Cell lysates were analyzed by immunoblotting (WB) with the indicated antibodies. The degree of protein phosphorylation was calculated with ImageJ software and is presented under the blot. (G–K) Effects of DDX56-RNAi plasmids on SeV-triggered transcription of IFNB1, TNFa, Il8, Rantes and Isg56 genes. The stable DDX56-knockdown 293T cells (4×105) were left uninfected or infected with SeV for 12 h before qRT-PCR was performed. Shown are representative experiments of three independent experiments with mean±s.d. of three technical replicates. **P<0.01. Luc, luciferase; Coni, control siRNA.

Fig 5: Knockout of DDX56 potentiates RNA virus-triggered IFN-ß signaling. (A) DDX56 levels in the HeLa cells were analyzed by immunoblotting. (B) VSV replication in wild-type and DDX56- knockout (KO) HeLa cells. HeLa (5×104) were infected by VSV–GFP (MOI 0.1) for 2 h and imaged by microscopy. Scale bar: 400 µm. (C) Effects of DDX56 deficiency on secretion of IFN-ß induced by SeV in HeLa cells. DDX56-knockout HeLa cells were infected with SeV for 12 h. The culture medium was collected for quantification of the indicated cytokines by ELISA. The experiment shown is representative of three independent experiments with mean±s.d. of three technical replicates. **P<0.01. (D–G) Effects of DDX56 knockout on SeV- or poly(I:C)-induced activation of the IFN-ß promoter and ISRE. DDX56-knockout HeLa cells (105) were transfected with the IFN-ß promoter or ISRE (100 ng). At 24 h after transfection, the cells were left uninfected or infected with SeV for 12 h or were treated or untreated with poly(I:C) (1 µg/ml) for 18 h before reporter assays were performed. Shown are representative experiments of three independent experiments with mean±s.d. of three technical replicates. *P<0.05, **P<0.01. (H) Effects of DDX56 deficiency on SeV-induced phosphorylation of TBK1, IRF3, p65 and I?Ba. The DDX56-knockout HeLa cells were untreated or treated with SeV for the indicated times, and cell lysates were analyzed by immunoblotting with the indicated antibodies. The degree of protein phosphorylation was calculated by ImageJ software and is presented under the blot. (I–M) Effects of DDX56 deficiency on SeV-triggered transcription of IFNB1 and TNFa genes. The DDX56-knockout HeLa cells (4×105) were left uninfected or infected with SeV for 12 h before qRT-PCR was performed. Shown are representative experiments of three independent experiments with mean±s.d. of three technical replicates. *P<0.05, **P<0.01. WT, wild type; Luc, luciferase.