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1 Supporting Information Sung et al /pnas SI Materials and Methods Cell Lines, Primary Human Hepatocytes, and Reagents. Huh-7 cells, Huh-7.5 cells (Apath, LLC), HepG2 cells (KCLB), and 293TN cells were maintained at 37 C with 5% (vol/vol) CO 2 in Dulbecco s modified Eagle medium (DMEM) supplemented with 10% (vol/vol) FBS (WelGENE), 4.5 g/l glucose, L-glutamine, and 1% (wt/vol) penicillin/streptomycin (Invitrogen). Huh-7 TLR3 cells and IRF9, Y701F-STAT1, and STAT2-transduced Huh- 7.5 cells were maintained in complete DMEM supplemented with 1 μg/ml puromycin. Huh-7.5 cells harboring the fulllength H77 (genotype 1a) replicon were maintained in complete DMEM supplemented with 1 g/l G418 (A.G. Scientific). HepaRG cells were differentiated and maintained as previously described (34). Primary human hepatocytes were purchased from Invitrogen and shipped frozen. After thawing, the cells were centrifuged at 150 g for5minandincubatedin24-well plates overnight. Primary human hepatocytes were maintained in Williams E Medium containing cell maintenance supplement reagents from Invitrogen. Highly HCVcc-permissive primary human hepatocytes were selected and used for this study. SiRNAs against STAT1, STAT2, and IRF9 were obtained from Santa Cruz Biotechnology. All of the DNA transfections were performed using Turbofect (Thermo Scientific/Affinity BioReagents) via a forward transfection method for 293TN cells and a reverse transfection method for Huh-7 cells. SiRNA transfections were performed using lipofectamine RNAi MAX (Invitrogen). Recombinant human IFN-β was obtained from Peptrotech, peg IFN-α2b was obtained from MSD, and recombinant human IFN-λs were obtained from R&D Systems. Neutralizing antibody against IFN-β was purchased from PBL Assay Science and used at a concentration of 2,000 IU/mL for 72 h. Neutralizing antibody for IFN-λ was purchased from R&D Systems. This antibody was originally targeted against IFN-λ 1,butit also has complete neutralizing activity against IFN-λ 3 and partial activity against IFN-λ 2. This antibody was used at a concentration of 20 μg/ml, which is sufficient to neutralize all three IFN-λs. IL-28A, IL-29 ELISA kits were purchased from RayBiotech. An IFN-β ELISA kit was purchased from Fujirebio. Generation and Infection of JFH-1 HCVcc. The Japanese Fulminant Hepatitis-1 (JFH-1) strain (genotype 2a) of HCVcc was generated and propagated as previously described (35). Five percent (vol/vol) human serum-supplemented, high glucose DMEM was used to culture Huh-7.5 cells to produce highly infectious JFH1 HCVcc (36). For the titration of HCV infectivity, a colorimetric focus-forming assay was performed as previously described (35). Huh-7 TLR3 cells were infected with JFH-1 HCVcc at a multiplicity of infection (MOI) of Lentiviral Particle Generation and Gene Transduction. Plasmids encoding CMV promoter-driven cdna clones of human STAT1, STAT2, IRF9, and Y701F-STAT1 have been described previously (24). To produce lentiviruses, VSV-G plasmid, gag-pol plasmid, and plasmids containing ORF clones were cotransfected into 293TN cells using Turbofect reagent (Thermo). Seventy-two hours after transfection, the culture supernatant of transfected 293TN cells was harvested and passed through a 0.45-μM syringe filter. The filtered supernatant was more than 30-fold concentrated with WELPROT virus concentration reagent (WelGENE). Huh-7.5 cells were transduced with concentrated lentiviral particles in medium containing 8 μg/ml polybrene (Sigma-Aldrich). The transduced cells were selected in complete DMEM containing 1 μg/ml puromycin. Liver Tissues. Hepatitis virus-infected, nontumorous liver tissues were obtained from eight patients with HCV-associated hepatocellular carcinoma (HCC) and eight patients with HBV-associated HCC through the National Biobank of Korea (PNUH, Busan, Korea). During surgical resection of HCC, nontumorous tissues were obtained and frozen at 70 C. A portion of the liver tissue was dissected, formalin-fixed, and paraffin-embedded for immunohistochemistry. The frozen tissues were lysed with radioimmunoprecipitation assay (RIPA) buffer for immunoblotting, or lysed with TRIzol reagent (Invitrogen) for total RNA extraction. Six liver tissues without viral hepatitis were also obtained during surgical procedures, such as cholecystectomy, adrenalectomy, and partial liver resection for intrahepatic duct stones at Daejeon St. Mary s Hospital (Daejeon, Korea). Paraffin-embedded tissues were used for immunohistochemistry to evaluate the expression of STAT1 and PY-STAT1. Mouse monoclonal anti-stat1 (42/Stat1; BD Transduction Laboratories) and rabbit monoclonal anti-py-stat1 (58D6; Cell Signaling Technology) were used for immunohistochemistry. Immunoblotting. RIPA buffer was used to lyse cells and liver tissues. Subcellular fractionation into nuclear and cytoplasmic fractions was performed using the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo). Ten micrograms of each cell lysate or nuclear/cytoplasmic lysate was loaded onto SDS/PAGE gels. For the lysis of human liver tissue, tissue homogenization preceded lysis using BioMasher (LMS). The antibodies used for immunoblotting were as follows: mouse monoclonal anti-stat1 (42/Stat1; BD Transduction Laboratories), rabbit monoclonal anti-py-stat1 (58D6; Cell Signaling Technology), rabbit polyclonal anti-stat2 (C-20; Santa Cruz Biotechnology), rabbit polyclonal anti-py- STAT2 (Cell Signaling Technology), rabbit polyclonal anti-irf9 (C-20; Santa Cruz Biotechnology), mouse monoclonal anti-isg15 (3E5; Santa Cruz Biotechnology), rabbit monoclonal anti-usp18 (D4E7; Cell Signaling Technology), mouse monoclonal anti-hcv core (C7-50; Thermo), rabbit polyclonal anti-lamin A/C (Santa Cruz Biotechnology), mouse monoclonal anti-tubulin (B-5-1-2; Sigma-Aldrich), rabbit polyclonal anti-actin (Sigma-Aldrich), and horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories). The detailed immunoblotting method has been described previously (37). RNA Extraction, cdna Synthesis, and Real-Time Quantitative PCR. Total RNA isolation, cdna synthesis, and TaqMan real-time quantitative PCR were performed as previously described (37). TaqMan Gene Expression Assays (Applied Biosystems) were used to determine the mrna levels of the target genes. Quantification of intracellular HCV RNA copies was performed as previously described (37). When the expression of a gene was undetectable, a Ct value of 40 was assigned. The results were standardized to the mrna level of β-actin, and the data are presented as the mean ± SEM. ChIP Assay. ChIP assays for cell lines were performed using a Pierce Agarose ChIP kit (Pierce), and ChIP assays for liver tissue were performed using a SimpleChIP Plus Enzymatic Chromatin IP Kit (Cell Signaling Technology) according to the manufacturers instructions. Briefly, whole-cell lysates from cells were immunoprecipitated using 2 μg of rabbit polyclonal anti- STAT1 (42/Stat1; BD Transduction Laboratories), rabbit polyclonal anti-stat2 (C-20; Santa Cruz Biotechnology), rabbit 1of6

2 polyclonal anti-irf9 (C-20; Santa Cruz Biotechnology), or rabbit IgG. Real-time quantitative PCR using SYBR green mastermix (kappa) was used to amplify the promoter regions near the MyD88, IRF1, IFI27, and Mx1 genes. Sequences of the primer pairs have been described in a previous study (24). Reactions were performed in triplicate, and the means were normalized to 1% (vol/vol) of the chromatin input. Statistical Analyses. Data from experiments with cell lines and primary human hepatocytes are presented as the mean ± SEM. Data from human liver tissues are presented as the mean ± SD. Unpaired t tests or two-tailed Mann Whitney U tests were performed for statistical analysis. All of the analyses were performed using GraphPad Prism version 5.01 (GraphPad Software). A P value of less than 0.05 was considered to be statistically significant. Fig. S1. Up-regulation of ISGs in HCV-infected Huh-7 TLR3 cells by exogenous IFN-β treatment. HCV-infected Huh-7 TLR3 cells were treated with IFN-β for 6 h, and real-time qpcr was performed to quantify Mx1, OAS-1, MyD88, and IRF1 mrna levels. Bar graphs represent the means ± SEM (n = 3). **P < 0.01, ***P < compared with control. Fig. S2. Binding of PY-STAT1 to the promoter of MyD88 and Mx1 4 h after IFN-β stimulation. HCV-infected Huh-7 TLR3 cells were treated with IFN-β for 4 h, and a ChIP assay was performed using anti-py-stat1 antibody. Bar graphs represent the means ± SEM (n = 3). *P < 0.05 compared with control. 2of6

3 Fig. S3. Induction of Mx1 and OAS1 by increased expression of U-STAT1, STAT2, and IRF9 without IFN stimulation. Huh-7.5 cells were transduced with lentiviruses carrying plv control vector or expression vectors for Y701F-STAT1, STAT2, or IRF9. The expression of Mx1 and OAS1 was examined by TaqMan realtime quantitative PCR. Bar graphs represent the means ± SEM (n = 3). *P < 0.05, **P < 0.01 compared with control. Fig. S4. Induction of endogenous IFN-λs and IFN-β in HCV-infected primary human hepatocytes and Huh-7 TLR3 cells. (A) Primary human hepatocytes were infected with JFH1 HCVcc (MOI = 2) and harvested 5 d later. The expression of IFN-β, IFN-λ 1, and IFN-λ 2 was examined at the mrna level by TaqMan real-time quantitative PCR and at the protein level by ELISA. (B) Huh-7 TLR3 cells were infected with JFH1 HCVcc (MOI = 10). The infected cells were harvested at the indicated time points, and the expression of IFN-β, IFN-λ 1, and IFN-λ 2 was examined at the mrna level by TaqMan real-time quantitative PCR and at the protein level by ELISA. The data represent the means ± SEM (n = 3). *P < 0.05, **P < 0.01 compared with control. 3of6

4 Fig. S5. Induction of U-ISGF3 and U-ISGs after treatment of IFN-β and IFN-λs in Huh-7.5 cells. (A D) Huh-7.5 cells were treated with 3 ng/ml IFN-β (A and C) or 100 ng/ml the indicated IFN-λ (B and D) and harvested at the indicated time points. Immunoblotting of STAT1, PY-STAT1, STAT2, PY-STAT2, and IRF9 was performed (A and B), and the expression of ISGs was examined by TaqMan real-time quantitative PCR (C and D). The data represent the means ± SEM (n = 3). *P < 0.05, **P < 0.01 compared with control. 4of6

5 Fig. S6. Induction of U-ISGF3 after treatment of IFN-β and IFN-λ 1 in PHHs, differentiated HepaRG cells, and HepG2 cells. PHHs, differentiated HepaRG cells, and HepG2 cells were treated with 1 ng/ml IFN-β or 100 ng/ml IFN-λ 1 and harvested at the indicated time points. Immunoblotting of STAT1, PY-STAT1, STAT2, PY- STAT2, and IRF9 was performed. Fig. S7. IFNAR1 and IFNLR1 mrna expression in various liver-derived cells. Real-time qpcr was performed to measure the level of IFNAR1 and IFNLR1 mrna. Bar graphs represent the means ± SEM (n = 3). 5of6

6 Fig. S8. Differential responses to exogenous IFN-α, IFN-β, and IFN-λ 1 in IFN-λ 3 pretreated cells. Huh-7.5 cells were pretreated with IFN-λ 3 for 5 d, and treated with IFN-α, IFN-β, and IFN-λ1. Immunoblotting was performed 30 min after IFN stimulation to evaluate the phosphorylation of STAT1 (A), and real-time qpcr was performed 6 h after IFN stimulation to measure the level of Mx1 mrna (B). Bar graphs represent the means ± SEM (n = 3). *P < 0.5. Fig. S9. Defects in inducing ISGs in U3A cells. U3A cells were transfected with WT STAT1, and treated with 50 IU/mL IFN-β for 4 h. Many representative ISGs, including IFIT3, Mx1, and Casp1, were not induced. These genes were significantly induced in IFN-β treated primary STAT1-null fibroblasts (8) transfected with WT STAT1. UT, untreated cells. The data represent the means ± SEM (n = 3). 6of6