Fig 2: The cGAS-STING pathway is required for increased ISG expression in ASCC3-deficient cells(A) Schematic of relevant innate immunity signaling pathways.(B) Western blot analysis of IRF3 and TBK1, and their phosphorylated forms (IRF3ser396 or TBK1ser172), in cells depleted of ASCC3.(C) qRT-PCR analysis of relative ISG expression in MRC5VA cells treated with siRNAs. Error bars represent standard deviation (SD) of three technical replicates and are representative of three biological replicates.(D) Western blot analysis of IRF3p (ser396), IRF3, TBK1p (ser172), TBK1, RSAD2, IFIT2, STAT1p (tyr701), STAT1, ASCC3, STING, and MAVS in the same cells as in (C).(E) Western blot analysis of RSAD2, IFIT2, STAT1p (tyr701), STAT1, ASCC3, and cGAS in parental MRC5VA and two different CGAS knockout cell lines (KO-7 and -12) after ASCC3 knockdown. Asterisks denote non-specific bands.(F) As in (E), but in U2OS cells.(G) qRT-PCR analysis of relative ISG expression in U2OS cells transfected with the indicated siRNAs. Error bars represent SD of three technical replicates and are representative of three biological replicates.(H) As in (G), but also using U2OS CGAS KO-16 cells.See also Figures S1–S4.

Fig 3: Defective induction of the type I IFN dependent antiviral state. (A) Immunoblot analysis of whole cell lysates prepared from patient II:3 and control fibroblasts following overnight treatment with IFNα2b (1000 IU/ml) demonstrating the failure to induce the ISG products of MX1, RSAD2, and IFIT1 in patient fibroblasts. Stars represent non-specific bands. Representative of n=3 repeat experiments. (B) Encephalomyocarditis virus (EMCV) cytopathic effect reduction bioassay demonstrating the failure of IFNα2b pre-treatment (1000 IU/ml) to induce the antiviral state in patient but not control fibroblasts. Displayed are mean ± SEM of n=3 repeat experiments ****P<0.001 ANOVA with Sidak’s post-test.

Fig 4: Type I IFN response is blunted in virus‐infected IRGM knockdown cells A–DControl and si‐IRGM transfected THP‐1 IFN reporter cells were kept uninfected (Mock) or infected with (A) JEV (MOI 5) or (B) CHIKV (MOI 5) or (C) HSV‐1 (MOI 2.5) or (D) VSV‐eGFP (MOI 1) and the supernatant collected 8 hpi were subjected to luciferase assay. The graphs depict fold change in interferon response. (n = 3, mean ± SD, *P < 0.05, **P < 0.005, ***P < 0.0005, Student’s unpaired t‐test).E–JControl and si‐IRGM transfected HT‐29 cells uninfected or infected with CHIKV and qRT–PCR analysis were performed with several ISG’s (E) SAMHD1 (F) HERC5 (G) ISG15 (H) viperin/RSAD2 (I) MX1 (J) APOBEC3G. (n = 3, mean ± SE, **P < 0.005, ***P < 0.0005, ****P < 0.00005, Student’s unpaired t‐test).KWestern blot analysis with cell lysates of mock and CHIKV (MOI 5, 24 h) infected control and si‐IRGM transfected THP‐1 cells and probed with the indicated antibodies. S.E, short exposure; L.E, long exposure.LWestern blot analysis with cell lysates of mock and CHIKV (MOI 5, 24 h) infected control and IRGM knockdown HT‐29 cells and probed with the indicated antibodies.MWestern blot analysis with cell lysates of mock and JEV (MOI 5, 24 h) infected control and si‐IRGM transfected HT‐29 cells and probed with the indicated antibodies.NWestern blot analysis with cell lysates of THP‐1 control or IRGM knockdown cells, untransfected or transfected with, heat‐killed whole CHIKV or CHIKV viral RNA and probed with the indicated antibodies.OqRT–PCR analysis to determine the knockdown efficiencies of PRR’s and adaptor proteins as indicated (n = 3, mean ± SE, ***P < 0.0005, Student’s unpaired t‐test).PqRT–PCR analysis with total RNA isolated from control and IRGM knockdown HT‐29 cells transfected with siRNA combinations as indicated that were infected with CHIKV (MOI 5, 24 h). (n = 3, mean ± SE, ***P < 0.0005, ****P < 0.00005, Student’s unpaired t‐test).QWestern blot analysis with cell lysates of mock and CHIKV (MOI 5, 24 h) infected control and IRGM knockdown HT‐29 cells transfected with indicated siRNA and probed with the indicated antibodies. Source data are available online for this figure.

Fig 5: Analysis of differentially expressed genes in HSAECs following RVFV infection. (a) Venn diagrams depict the upregulated (top panel) and downregulated (bottom panel) genes changed in MP12 only (left), ZH548 only (right), or both (center, gray) at 3, 9, and 18 hours post infection. These genes were changed by 1.5-fold or more and had a p-value ≤ 0.05. (b) HSAECs were mock-infected or infected with MP12 or ZH548 at MOI 5 for one hour. Lysates were collected in Trizol LS, RNA was extracted and prepared for RNA sequencing. RNA-sequencing reads were normalized to the total reads, then fold changes were calculated against the uninfected, mock samples at the specified time point. (c) RT-qPCR confirmation of some of the top changed transcripts during all time points post infection. HSAECs were mock-infected or infected with MP12 or ZH548 at MOI 5 for one hour. Lysates were collected in Trizol LS, RNA was extracted using the Direct-zol™ RNA MiniPrep, and analyzed for RT-qPCR with TaqMan Gene Expression Assays against IFIT1, IFIT2, IFIT3, and RSAD2. Fold changes were calculated relative to 18 S ribosomal RNA and normalized to mock samples using the ΔΔCt method. Data are expressed as the Mean ± SD (n = 3).