Fig 2: HeLa cells were infected with EGFP-CHIKV (MOI 10) for 24 h.Subsequently, permeabilized cells were stained for IFITM3, ISG15, MX1, or BST-2. Shown are dot plots from one representative experiment.

Fig 3: Defective signaling and ISG induction downstream of IFNAR2 p.Ser53Pro. (A) PBMCs from P1 and three healthy controls (C1–C3) were treated with IFNß 100 IU/ml for 5, 15, or 30 min. Whole-cell lysates were harvested for Western blotting for the visualization of pSTAT1, STAT1, and GAPDH, which was used as the loading control. Not repeated due to limited patient material. (B) Primary dermal fibroblasts from P1, two healthy controls (C1 and C2), and the heterozygous mother of P1 (Het) were treated with IFNß 100 IU/ml for 30 min. Whole-cell lysates were harvested for immunoblotting and the visualization of IFNAR2, STAT1, pSTAT1, and GAPDH as the loading control. One representative immunoblot of n = 3 independent experiments is shown. (C) Primary IFNAR2-/- dermal fibroblasts reconstituted with lentiviruses expressing GFP, WT, or p.Ser53Pro IFNAR2 were treated with IFNa2b or IFN? 1,000 IU/ml for 30 min. Whole-cell lysates were harvested for immunoblotting and visualization of pJAK1, JAK1, pSTAT2, STAT2, STAT1, pSTAT1, and GAPDH as the loading control. One representative immunoblot of n = 3 independent experiments is shown. (D) PBMCs from P1 and three healthy controls (C1–C3) were treated with IFNß 100 IU/ml for 6 h. Total RNA was purified for RT-qPCR of IFIT1 and IRF7 relative to TBP (mean ± SD of n = 3 independent experiments; ****, P < 0.0001, ns, non-significant; two-way ANOVA with Šidák’s test for multiple comparisons). (E) Primary dermal fibroblasts from P1, two healthy controls (C1 and C2), and the mother of P1 (Het) were treated with IFNß 100 IU/ml for 6–24 h. Total RNA was purified for RT-qPCR of MX1 and IFIT1 relative to TBP (mean ± SD of n = 3 independent experiments; ****, P < 0.0001; ns, non-significant; two-way ANOVA with Dunnett’s test for multiple comparisons). (F) Primary dermal fibroblasts from P1 or a healthy control (C1) were treated with IFNa2b 1,000 IU/ml overnight. Whole-cell lysates were prepared for immunoblotting and visualization of MX1, USP18, RSAD2, ISG15, and a-tubulin (ATUB) as the loading control. One representative immunoblot of n = 3 independent experiments is shown. Source data are available for this figure: SourceData F2.

Fig 4: Expression of STAT1, OAS1, Mx1 and CXCL10 proteins in ovine spleens analyzed by western blot. Note: DN16 = Day 16 of the estrous cycle; DP13 = Day 13 of pregnancy; DP16 = Day 16 of pregnancy; DP25 = Day 25 of pregnancy. Significant differences (p < 0.05) are indicated by different superscript letters within the same color column.

Fig 5: Reconstitution of patient fibroblasts with WT IFNAR2 restores IFNAR2 signaling, ISG induction and viral control. (A and B) Primary dermal fibroblasts from P1, two healthy controls (C1 and C2), and the heterozygous mother of P1 (Het) were transduced with lentiviral vectors encoding EGFP, IFNAR2 WT, or IFNAR2 p.Ser53Pro. 2 d after transduction, cells were pretreated with IFNß 100 IU/ml for either (A) 30 min before being lysed and harvested for whole cell lysates for immunoblotting or (B) 6 h before being lysed and harvested for total RNA for RT-qPCR of MX1 and IFIT1 relative to GAPDH (mean ± SD of n = 3 independent repeats; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, non-significant; two-way ANOVA with Tukey’s test for multiple comparisons). For A, one representative immunoblot of three independent experiments is shown. (C and D) SV40-immortalized dermal fibroblasts from P1 and a healthy control were transduced with lentiviral vectors encoding EGFP or IFNAR2 WT. 2 d after transduction, cells were pretreated with IFNß 100 IU/ml for 24 h before infection with HSV1 (KOS strain) at an MOI of 1. At 24 h p.i., supernatants were harvested and titrated for (C) TCID50 (geometric mean ± SD of n = 3 independent replicates; ****, P < 0.0001; ns, non-significant; two-way ANOVA with Tukey’s test for multiple comparisons) and the cells were lysed for Western blotting for the HSV1 protein VP5, IFNAR2, and GAPDH as loading control (D). Source data are available for this figure: SourceData F5.