Fig 2: PICH deficiency induces type‐I IFN production. A) WT and Pich‐KO LSK cells were subjected to gene expression profiling through RNA sequencing. The enriched IFN‐β response gene set is shown. B) WT and Pich‐KO FL cells were subjected to mass spectrometry analysis, showing expression of BST2. C) mRNA expression of type‐I IFN genes in Pich f/f (Pich flox/flox) and Pich Δ/Δ (conditional knock out of Pich in hematopoietic cells using vav‐cre) fetal livers, as analyzed by RT‐PCR, n = 3 for each, n = 3 for each. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤ 0.001. D) IFN‐β levels were determined by FACS in LSKs of Pich f/f and Pich Δ/Δ mice, n = 7 for each. ∗∗∗p ≤ 0.001. E) Protein levels of IFIT1, IRF3, and IRF7 were determined by western blotting in Pich f/f and PichΔ/Δ FLs.

Fig 3: BTV replication is restricted by both cow antiviral ISGs and their sheep orthologues. (A) (Left) Western blot of KO-IFIT1 cells (clones 3B and 7A) and parental BovFibT (WT) showing expression of IFIT1, phosphorylated STAT1, and GAPDH after IFN treatment. (Right) Quantification of Western blots using Image Studio Lite software (LI-COR Biosciences). Data were obtained from 3 independent experiments. (B) (Left) Western blot of KO-RSAD2 cells (clones 3C and 5B) and parental BovFibT (WT) showing expression of RSAD2, phosphorylated STAT1, and GAPDH after IFN treatment. (Right) Quantification of Western blots using Image Studio Lite software (LI-COR Biosciences). Data were obtained from 3 independent experiments. (C) Graph showing infectivity of BTV-8 mGFP in KO-IFIT1 and parental BovFibT in cells pretreated with IFN (1,000 U), or carrier control, before infection with serial dilutions of BTV-8-mGFP for 6 h. Cells were then fixed, and mGFP expression was determined by flow cytometry. (D) Graph showing infectivity of BTV-8 mGFP in KO-RSAD2 and parental BovFibT in cells pretreated with IFN (1,000 U), or carrier control, before infection with serial dilutions of BTV-8-mGFP for 6 h. Cells were then fixed, and mGFP expression was determined by flow cytometry. (E) Virus replication curves of growth of BTV-8 in immortalized BovFibT, KO-IFIT1 cells, and KO-RSAD2 cells in the presence (dashed lines) or absence (solid lines) of 1,000 U uIFN pretreatment. Cells were infected at an MOI of 0.01, and supernatants were harvested at the indicated time points postinfection. Cell-free virus was titrated by endpoint dilution on BSR cells, and values are means and SEM from at least 3 independent experiments. Statistical significance between −uIFN and +uIFN conditions for each time point was calculated using t tests with Welch’s correction. (F and G) Relative infectivity of BTV-8-mGFP in 293T cells overexpressing bovine and ovine restriction factors at 16 (F) and 32 (G) hpi (n = 8). 293T cells were transduced with lentiviruses expressing the ovine or bovine orthologues for 48 h and infected with BTV-8-mGFP for 16 or 32 h, and mGFP expression was quantified by flow cytometry. BTV infectivity was normalized to the mean obtained from all the negative-control wells. Each dot represents an independent repeat. The mean and standard deviation are presented for each condition. (H and I) Virus titers of BTV-8 in CPT-Tert stably expressing either ovine or bovine IFIT1 (H) and ovine or bovine RSAD2 (I) at the times indicated. Cells stably transfected with an empty lentivirus were used as controls. Cells were infected with BTV-8 (MOI ≈ 0.01), and supernatants were harvested at the indicated time points postinfection. Cell-free virus titers were quantified by endpoint dilution and normalized to titers obtained from the control cell line. Data (n =4) are from 2 independent BTV-8 stocks and 2 independently generated stable cell lines for each gene tested. Multiple t tests were carried out following a Shapiro-Wilk normality test. ns, not significant (P > 0.05); *, P < 0.05; ***, P < 0.001; ****, P < 0.0001.

Fig 4: Virus-induced global host cell protein shutoff by BTV is faster and more pronounced in ovine than bovine endothelial cells. (A) Immunoblotting of phosphorylated STAT1, total STAT1, Mx1, IFIT1, and BTV NS1 and protein translation rates (puromycin labeling treatment for 1 h) in BovEC and OvEC infected with BTV-8. Cells were infected at 0.04 or 4 MOI equivalents (measured in the indicator cell line CPT-Tert) and harvested at the time points indicated. (B to F) Relative quantification of immunoblots as in panel A obtained from 3 independent experiments. (B) Quantification of relative protein shutoff over the course of BTV infection as determine by puromycin incorporation. All values were normalized to the mock (set to 1) for each time point. (C) Relative expression of BTV NS1 over time normalized to tubulin in OvEC and BovEC infected with BTV-8. (D) Expression of STAT1 (relative to mock infection) over time normalized to tubulin in OvEC and BovEC infected with BTV-8. (E) Relative expression of IFIT1 over time normalized to tubulin in OvEC and BovEC. Cells were infected with BTV-8 using 2 different MOI and harvested at 3 different time points. Data are from 3 independent experiments. (F) Relative expression of Mx1 over time normalized to tubulin in OvEC and BovEC. Cells were infected with BTV-8 using 2 different MOI and harvested at 3 different time points. Data are from 3 independent experiments. (G) Immunoblotting of phosphorylated STAT1, total STAT1, IFIT1, Mx1, and BTV NS1 and protein translation rates (puromycin labeling treatment for 1 h) in OvEC and BovEC pretreated with 4 μM Rux and infected with BTV-8. Cells were infected at an MOI of 0.04 or 4 and harvested at 24 hpi. (H to K) Relative quantification of immunoblots, as shown in panel G, obtained from 3 independent experiments. (H) Relative protein shutoff in OvEC and BovEC infected with BTV as determined by puromycin incorporation. All values were normalized to the mock-infected control. (I) Relative expression of BTV NS1 (relative to tubulin) in OvEC and BovEC infected with BTV-8. (J) Relative expression of IFIT1 (normalized to tubulin) in OvEC and BovEC. (K) Relative expression of Mx1 in OvEC and BovEC. For panels H to K, cells were pretreated with 4 μM Rux for 4 h prior to infection and maintained in the medium after infection. All data are from 3 independent experiments. Statistical significance was calculated using a two-way ANOVA performed using Tukey’s multiple-comparison test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.