Fig 2: STAT3 inhibits the transcriptional activation of IRF1.(A) qRT-PCR analysis of IRF1, IRF3, NFKB1 and RELA in HepG2 cells first treated with mimics or siRNAs, and then transfected with or without JFH1 RNA for 24 hr. (B) Analysis of IRF1 and IRF3 protein expression in HepG2 cells treated with siRNAs and then JFH1 RNA. (C) qRT-PCR analysis of IRF1 and IFNs in HepG2 cells transfected with vectors expressing IRF1 or RFP (after 2 days). (D) Analysis of p-STAT1 and MDA5 in HepG2 cells transfected with IRF1 or RFP plasmids for 2 days, and then treated with poly(I:C) for 3–24 hr. (E) Analysis of p-STAT1 and MDA5 in HepG2 cells transfected with the indicated doses of IRF1 plasmids (0.05–1 μg/well in a 24-well-plate) for 2 days. (F) Analysis of IRF1, p-STAT1 and MDA5 in HepG2 cells transfected with plasmids expressing 7 HA-tagged transcription factors (after 2 days). HA-GFP was used as a negative control. (G) Analysis of IRF1 and p-STAT1 in HepG2 cells first transfected with STAT3 siRNA for 2 days, and then treated with IFN-β or IL-29 for 5–360 min. qRT-PCR data are from one experiment that was representative of three experiments (mean ± SEM of technical triplicates). *p<0.05, **p<0.01 and ***p<0.001. 10.7554/eLife.41159.031Figure 4—source data 1.qRT-PCR analysis of transcription factors in HepG2 cells. 10.7554/eLife.41159.032Figure 4—source data 2.qRT-PCR analysis of IRF1 and IFN in HepG2 cells transfected with IRF1 plasmid.

Fig 3: miR-122 enhances IFN responses to various viral nucleic acids.(A) qRT-PCR analysis of type III (IL-29 and IL-28) and type I (IFN-β) IFN mRNAs in HepG2 cells transfected with the indicated nucleic acids for 24 hr. Except for HSV-60 and ssRNA40, which were transfected at 5 μg/ml, all other RNAs were transfected at 1 μg/ml. The expression of IFNs was normalized to GAPDH and then compared with the levels in cells without stimulation (mock). (B) qRT-PCR analysis of miR-122 expression in hepatoma cell lines and in normal liver tissue, as well as of HepG2 cells transfected with miR-122 or negative control (miR-NC) mimics. miR-122 expression was normalized to U6 and then compared with the levels in HepG2 cells. (C, D) qRT-PCR analysis of IFN mRNAs in HepG2 cells first treated with miR-122 or control mimics for 2 days, and then transfected with the indicated nucleic acids for 24 hr. Opti-MEM (medium) was used as the negative control. (E, F) ELISA analysis of IL-29/IL-28B and IFN-β proteins in HepG2 cells treated as in panel C. IL-29 and IL-28B were detected by the same set of antibodies. N.D., lower than the minimum concentration (3.9 pg/ml) that can be accurately detected. qRT-PCR data are from one experiment that was representative of three independent experiments (mean ±SEM of technical triplicates). ELISA data are from two experiments (mean +SD). *p<0.05, **p<0.01 and ***p<0.001. 10.7554/eLife.41159.004Figure 1—source data 1.qRT-PCR analysis of IFN expression in HepG2 and Huh7 cells transfected with JFH1 RNA. 10.7554/eLife.41159.005Figure 1—source data 2.qRT-PCR analysis of HBV pgRNA levels in indicated samples. 10.7554/eLife.41159.006Figure 1—source data 3.qRT-PCR analysis of IFN mRNAs in HepG2 cells transfected with different nucleic acids. 10.7554/eLife.41159.007Figure 1—source data 4.qRT-PCR analysis of miR-122 expression in the indicated samples. 10.7554/eLife.41159.008Figure 1—source data 5.qRT-PCR analysis of IFN mRNAs in HepG2 cells transfected with miR-122 and then treated with different nucleic acids. 10.7554/eLife.41159.009Figure 1—source data 6.ELISA analysis of IFNs in HepG2 cells transfected with miR-122 and then treated with different nucleic acids. 10.7554/eLife.41159.010Figure 1—source data 7.qRT-PCR analysis of ISGs in HepG2 cells transfected with miR-122 and then treated with JFH1. 10.7554/eLife.41159.011Figure 1—source data 8.Analysis of the IFN mRNAs in Huh7 cells transfected with miR-122 and then treated with JFH1.

Fig 4: Innate responses to SNV in Huh7 and A549 are specifically due to Vero E6-derived IFNλ.Total cellular RNA was isolated from Huh7 or A549 cells and used for ISG quantitation by qRT-PCR. (A) Huh7 were treated with SNV (50 µL of virus) or the indicated amount of recombinant human IL29 or IL28A for 3 h prior to MxA mRNA quantitation by qRT-PCR. (B) Huh7 were infected with SNV at an MOI = 1 (50 µL) with or without prior incubation with anti-IL29 or anti-IL28A antibodies at the indicated final concentrations for 1 h. Three hours post infection, ISG56 and MxA mRNA expression was measured by qRT-PCR. (C) SNV or IFNβ was preincubated with the indicated amount of anti-IFNβ antibody for 1 h. Virus or IFNβ was then added to Huh7 cells for 3 h and ISG56 and MxA was measured 3 h post exposure by qRT-PCR. (D) A549 were infected with SNV at an MOI = 1, with or without prior incubation with anti-IL29 blocking antibodies (10 µg/mL). Three hours post infection ISG56 and MxA levels were measured by qRT-PCR. Real-time qRT-PCR results for all experiments (parts A–D) are reported as the mean ± SEM from triplicate biological experiments.

Fig 5: Vero E6-derived SNV stocks induce innate responses via a Jak/STAT pathway and contain IFNλ.(A) Hec 1b Jak/STAT knockout cells were treated with poly (I∶C) (50 µg/mL) or the indicated stock of virus from Figure 1 at an MOI = 1 for 3 h. Total cellular RNA was isolated and used for qRT-PCR for ISG56 and MxA. Real-time qRT-PCR results are reported as the mean ± SEM from triplicate biological experiments. (B) Medium supplemented with 50 µg/mL of rhIL29, and SNV derived from Vero E6 8 dpi and 20 dpi were applied to a 100-kDa filter and centrifuged for 10 min. The filtrate was then applied to a 3-kDa filter and centrifuged for 30 min. Equal volumes of the retentate were then used for SDS-PAGE. Western blotting was then performed using an anti-IL29 antibody.