Fig 2: IFN-? promoted apoptosis activated by RNase L.a, b Western blot of OAS1, OAS2, OAS3, RNase L, cytochrome C, and Caspase-9 in the mitochondrial or cytosolic fraction, co-immunoprecipitation of Bax and Bak, and cleavage activity of RNase L in NCl-H157 cells after the interference of RNase L. Prohibitin and GAPDH were regarded as the internal reference of mitochondria or cytosolic fractions, respectively. c Mitochondrial or cytosolic efflux of cytochrome C at different time points of IFN-? stimulation (n = 3). d Western blot of Caspase-9, Caspase-3, and PARP in BEAS-2B and NCI-H157 cells stimulated with IFN-? at different time points (n = 3). e, f Western blot of Caspase-9, Caspase-3, and PARP in BEAS-2B (e) and NCl-H157 (f) cells stimulated with IFN-? and Caspase-9 inhibitor (z-LEHD-fmk) or Caspase-3 inhibitor (z-DEVD-fmk) (n = 3). g Accumulated DNA ladder after IFN-? stimulation in BEAS-2B and NCI-H157 cells (n = 3)

Fig 3: Effect of avSG formation on IFN signaling. (A and B) HT1080 WT and G3BP1 KO cells were treated with IFN-ß (1,000 U/ml) for 24 h, and ISG15 and ISG56 mRNA levels were measured and normalized to those of GAPDH by qRT-PCR (A), or the cells were transfected with ISG15-luc or ISG56-luc reporter constructs, along with ß-galactosidase plasmids, and 24 h later treated with IFN-ß (1,000 U/ml), and luciferase activity was measured and normalized to ß-galactosidase levels (B). (C) WT, G3BP1 KO, and RNase L KO cells were treated with IFN-ß (1,000 U/ml) for the indicated times, and cell lysates were analyzed for phosphorylation of STAT1 and induction of OAS2, OAS3, and ISG56 in immunoblots. ß-Actin was used to normalize loading. (D) WT, G3BP1 KO, and RNase L KO cells were treated with IFN-ß (1,000 U/ml) for 16 h, and nuclear translocation of P-STAT1 was determined by immunofluorescence; nuclei were stained with DAPI. (Right) Quantification of P-STAT1 nuclear translocation from five random fields. The data represent means + SE for the results of three independent experiments. n.s, not significant.

Fig 4: EBV-encoded miRNAs regulate the expression of cellular genes and proteins involved in type I IFN activation. (A to E) 293T cells were cotransfected with the indicated wild-type luciferase reporter plasmids (WT) for RIG-I/DDX58 (A), IRAK2 (B), Viperin/RSAD2 (C), FYN (D), and OAS2 (E) or reporter plasmid in which the predicted seed sequence was mutated (mut) together with or without an miRNA-encoding plasmid. The luciferase expression in these cells was assessed and normalized to lysates from cells cotransfected with the wild-type 3'-UTR reporter and empty miRNA expression plasmid (Ø). P values were calculated using the one-way analysis of variance (ANOVA) test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (F) Human primary B lymphocytes were infected with wild-type EBV (r_wt/B95.8) or EBV devoid of its miRNAs (r_?miR). Five days postinfection, EBV-infected cells were counted and seeded at the same density. Four days later, the cells were lysed and the lysates were subjected to quantitative Western blot analysis using the Western blot stain-free TGX Bio-Rad normalization approach (Bio-Rad). Blots were probed with antibodies against IPO7, Viperin/RSAD2, TLR9, TLR7, FYN, RIG-I/DDX58, and OAS2. Protein levels were quantified and used to normalize the levels of the specific protein signals. Ratios of protein levels in cells infected with wild-type virus versus cells infected with mutant virus are shown. Reported are the results of four independent biological replicates.