IFN-Λ4 Attenuates Antiviral Responses by Enhancing Negative Regulation of IFN Signaling

IFN-λ4 Attenuates Antiviral Responses by Enhancing Negative Regulation of IFN Signaling

This information is current as Adeola A. Obajemu, Nina Rao, Kari A. Dilley, Joselin M. of September 29, 2021. Vargas, Faruk Sheikh, Raymond P. Donnelly, Reed S. Shabman, Eric G. Meissner, Ludmila Prokunina-Olsson and Olusegun O. Onabajo J Immunol published online 25 October 2017

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published October 25, 2017, doi:10.4049/jimmunol.1700807 The Journal of Immunology

IFN-l4 Attenuates Antiviral Responses by Enhancing Negative Regulation of IFN Signaling

Adeola A. Obajemu,* Nina Rao,* Kari A. Dilley,† Joselin M. Vargas,* Faruk Sheikh,‡ Raymond P. Donnelly,‡ Reed S. Shabman,† Eric G. Meissner,x Ludmila Prokunina-Olsson,*,1 and Olusegun O. Onabajo*,1

Type III IFNs are important mediators of antiviral immunity. IFN-l4 is a unique type III IFN because it is produced only in individuals who carry a dG allele of a genetic variant rs368234815-dG/TT. Counterintuitively, those individuals who can produce IFN-l4, an antiviral cytokine, are also less likely to clear hepatitis C virus infection. In this study, we searched for unique functional properties of IFN-l4 that might explain its negative effect on hepatitis C virus clearance. We used fresh primary human hepatocytes (PHHs) treated with recombinant type III IFNs or infected with Sendai virus to model acute viral infection and subsequently validated our ﬁndings in HepG2 cell line models. Endogenous IFN-l4 protein was detectable only in Sendai Downloaded from virus–infected PHHs from individuals with the dG allele, where it was poorly secreted but highly functional, even at concentrations < 50 pg/ml. IFN-l4 acted faster than other type III IFNs in inducing antiviral genes, as well as negative regulators of the IFN response, such as USP18 and SOCS1. Transient treatment of PHHs with IFN-l4, but not IFN-l3, caused a strong and sustained induction of SOCS1 and refractoriness to further stimulation with IFN-l3. Our results suggest unique functional properties of IFN-l4 that can be important in viral clearance and other clinical conditions. The Journal of Immunology, 2017, 199: 000–000. http://www.jimmunol.org/

nterferon-l4 is a novel type III IFN that was discovered as a the TT allele introduces a frame-shift and eliminates this IFN (1). factor interfering with clearance of hepatitis C virus (HCV) The dG allele is very common in individuals of African ancestry I infection (1). A decreased ability to clear HCV is associated (up to 78% allele frequency) but is less common in Europeans with the dG allele of a genetic variant (rs368234815-dG/TT); this (∼30%) and Asians (0–10%) (1). This strong variation between allele creates an open reading frame for IFN-l4 protein, whereas individuals in the ability to produce IFN-l4 might be important for health disparity in the immune response to viral infections and other relevant conditions.

*Laboratory of Translational Genomics, Division of Cancer Epidemiology and Ge- Before the discovery of rs368234815 (1), its association was by guest on September 29, 2021 netics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; captured by either of the two single nucleotide polymorphisms †Virology Group, J. Craig Venter Institute, Rockville, MD 20850; ‡Office of Bio- technology Products, Center for Drug Evaluation and Research, U.S. Food and Drug (SNPs) used in genome-wide association studies: rs12979860 (2, Administration, Silver Spring, MD 20993; and xDivision of Infectious Diseases, 3) and rs8099917 (4, 5). SNP rs12979860 is located within the Department of Microbiology and Immunology, Medical University of South Caro- lina, Charleston, SC 29425 first intron of IFNL4, with the dG and TT alleles of rs368234815 corresponding to the T and C alleles, respectively, of rs12979860 1L.P.-O. and O.O.O. contributed equally to this work. (1). SNP rs8099917 is located upstream of IFNL4 and captures the ORCIDs: 0000-0002-0695-5276 (R.P.D.); 0000-0002-5240-7115 (E.G.M.); 0000- 0002-9622-2091 (L.P.-O.); 0000-0001-6342-2380 (O.O.O.). difference additionally contributed by a missense IFNL4 variant, Received for publication June 5, 2017. Accepted for publication September 27, 2017. rs117648444-A/G (Pro70Ser), which affects the activity of IFN-l4(1, This work was supported by the Intramural Research Programs of the Division of 6). Thus, rs8099917-G allele tags IFNL4 haplotype (rs368234815-dG/ Cancer Epidemiology and Genetics, National Cancer Institute (to A.A.O., N.R., J.M.V., rs117648444-G) that produces a more active protein (IFN-l4–70Pro), L.P.-O., and O.O.O.) and the National Institute of Allergy and Infectious Diseases (to whereas rs8099917-T allele captures two other IFNL4 haplotypes E.G.M.), the Critical Care Medicine Department and the Clinical Research Center of the National Institutes of Health (to E.G.M.), intramural research funds from the U.S. Food corresponding to lack of IFN-l4 (rs368234815-TT/rs117648444-G) or and Drug Administration (to F.S. and R.P.D.), and internal funding from the J. Craig alessactiveIFN-l4–70Ser protein (rs368234815-dG/rs117648444-A) Venter Institute (project code 9260 to K.A.D. and R.S.S.). (1, 4–6). A.A.O., N.R., O.O.O., K.A.D., J.M.V., F.S., R.P.D., and R.S.S. performed experi- The ability to produce IFN-l4, with or without further modi- ments; E.G.M. contributed data; L.P.-O. and O.O.O. designed the experiments and supervised the research; A.A.O., O.O.O., and L.P.-O. wrote the manuscript; and all fication by P70S, was linked with differential pretreatment blood authors critically reviewed and revised the manuscript. HCV load and hepatic IFN-stimulated gene (ISG) expression (1, Address correspondence and reprint requests to Dr. Olusegun O. Onabajo, Laboratory 6–10), variable clearance of HCV infection spontaneously or after of Translational Genomics, Division of Cancer Epidemiology and Genetics, National Cancer Institute, National Institutes of Health, 8717 Grovemont Circle, Bethesda, treatment with IFN-a–based therapies (1, 2, 9, 11), and slower MD 20892-4605. E-mail address: [email protected] kinetics of HCV clearance in patients treated with IFN-free direct- The online version of this article contains supplemental material. acting antivirals (DAAs) (10). Growing literature demonstrates Abbreviations used in this article: Ct, PCR cycle at detection threshold; DAA, direct- genetic associations between rs368234815 or its linked variants acting antiviral; HCV, hepatitis C virus; HepG2–IFN-l3–GFP cell, stable HepG2 cell with other clinical phenotypes, such as relapse on DAA treatment line expressing doxycycline-inducible GFP-tagged IFN-l3 protein; HepG2–ISRE– Luc cell, stable HepG2 cell line carrying the ISRE-Luc reporter; ISG, IFN-stimulated of HCV (12), liver fibrosis (13, 14), hepatic metallothionein ex- gene; PHH, primary human hepatocyte; PNGaseF, peptide–N-glycosidase F; qRT- pression (15), postpartum immune activation (16, 17), and risk for PCR, quantitative RT-PCR; SeV, Sendai virus; siRNA, small interfering RNA; SNP, mucinous ovarian cancer (18), among others. Although other single nucleotide polymorphism. invariantly expressed type III IFNs might also be important for Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 these phenotypes, only IFN-l4 is directly and most dramatically

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700807 2 IFN-l4 ENHANCES NEGATIVE REGULATION OF IFN SIGNALING affected by the associated variant, rs368234815, which either Expression of IFN-l3–GFP and IFN-l4–GFP was induced by 0.5 mg/ml creates or eliminates IFN-l4. Thus, IFN-l4 might be the primary doxycycline for the indicated times and monitored by flow cytometry cause of further functional effects, including on other type III analysis on a FACSAria III (BD Biosciences). Intracellular IFN-l3–GFP was detectable only after blocking protein secretion with 6 mM GolgiStop IFNs, that have been attributed to these genetic associations. (BD Biosciences) for 4 h, whereas IFN-l4–GFP was detectable even Although genetic association studies provide strong support for without GolgiStop. All cell lines were regularly tested for mycoplasma the role of IFN-l4 in diverse clinical phenotypes, functional using the MycoAlert Mycoplasma Detection Kit (Lonza). studies on IFN-l4 are still limited. Mouse models have been rIFNs successfully used to study other type III IFNs (19–21), but IFN-l4 is missing in the mouse genome, making it difficult to perform Commercially available IFN-l1, IFN-l2, and IFN-l4 (all from R&D Systems) were generated in mouse myeloma cells, human HEK293 cells, comprehensive comparisons between type III IFNs in the same and Escherichia coli, respectively. Custom IFN-l3 was generated in a experimental models. baculoviral system (1), and custom IFN-l4 was generated in E. coli,as All IFN-ls, including IFN-l4, activate the JAK–STAT signaling described below. The custom IFN-l4 represents wild-type protein that pathway through a receptor complex consisting of IFNLR1 and lacks the natural nonsynonymous polymorphisms in the IFNL4 gene IL-10R2 (22, 23), leading to induction of ISGs and an antiviral (rs73555604-C17Y, rs142981501-R60P, and rs117648444-P70S) that may affect the activity of IFN-l4 (1, 6). An open reading frame for IFN-l4 was response (1, 22, 23). Although IFN-l4 and its closest family cloned in the pET28a vector (Novagen), introducing an N-terminal His tag, member, IFN-l3, share only 29% amino acid identity (1), they induce and expressed in E. coli after induction with 0.5 mM IPTG for 4 h at 37˚C. the same set of ISGs (1, 6, 24–26), making the specific contribution The bacterial culture was centrifuged, and the cells were resuspended in of IFN-l4 to the antiviral response difficult to evaluate. 5 ml of lysis buffer (50 mM Tris [pH 7.9], 0.5 M NaCl, 2% Triton X-100, and 1 mM PMSF) and sonicated three times (15 s on/15 s off) at 4˚C. Downloaded from In this study, we searched for functional properties of IFN-l4 Solution was centrifuged at 15,000 rpm for 15 min at 4˚C, and the pellet that differentiate it from other type III IFNs. We used fresh pri- (inclusion bodies) was resuspended in 6 M guanidine with rotation at 4˚C mary human hepatocytes (PHHs) that were treated with for 1 h. The protein was purified on a nickel Sepharose bead column (GE recombinant type III IFNs or infected with Sendai virus (SeV) to Life Sciences) with 40 ml of 6 M guanidine and eluted with a buffer model acute viral infection. We extended these findings by per- containing 8 M urea and 300 mM imidazole. Protein refolding was done for 12 h in an optimized buffer containing 440 mM arginine, 300 mM forming comparative studies between type III IFNs using several NaCl, 1 mM reduced glutathione, 0.2 mM oxidized glutathione, 0.05%

HepG2 cell line models. Our results uncover important differences PEG 3350, 2 mM MgCl2, and 50 mM MES buffer (pH 6). Salts and buffers http://www.jimmunol.org/ in the regulation of the antiviral response by IFN-l4 and other were removed by multiple rounds of dialysis, and IFN-l4 was concen- type III IFNs; these ﬁndings might be important for understanding trated to 0.2 mg/ml. The endotoxin level in the custom IFN-l4 preparation was measured using a Limulus Amebocyte Lysate test (Pierce) and found the diverse clinical phenotypes that have been directly or indi- to be ,0.2 EU/1 mg, which is comparable to the level in the commercial rectly linked with the genetic ability to produce IFN-l4. IFN-l4(, 0.1 EU/1 mg; based on product data sheet). The identity of IFN-l4 was veriﬁed by Western blotting with primary monoclonal anti– Materials and Methods IFN-l4 Abs, rabbit (1:1500, ab196984; Abcam) and mouse (1:1000, MABF227; Millipore), and with secondary HRP-linked Abs, anti-rabbit Human hepatic cells and cell lines (1:5000, #7074; Cell Signaling) or anti-mouse (1:5000, sc-2031; Santa Cruz Biotechnology). Protein purity was evaluated by quantitative densi-

Fresh PHHs from 14 donors (Supplemental Table I) were purchased from by guest on September 29, 2021 BioreclamationIVT. Samples were received within 3 d after the donors’ tometry based on Coomassie staining and was determined to be .90% for death; no samples were obtained from executed prisoners or other insti- custom IFN-l4comparedwith.85% for commercial IFN-l4(basedonthe tutionalized persons. PHHs were received attached in 24-well plates and product data sheet). IFN-l4 activity was initially confirmed by its ability to maintained in InVitroGRO HI culture medium with Torpedo antibiotic mix induce STAT1 phosphorylation that was attenuated by 10 mg/ml blocking (BioreclamationIVT). DNA from PHHs was extracted with a DNeasy Blood anti–IL-10R2 Abs: mouse monoclonal (MAB874) or goat polyclonal and Tissue Kit (QIAGEN) and used for genotyping of IFNL4 rs368234815- (AF874; both from R&D Systems). Protein concentrations were estimated dG/TT with a custom TaqMan assay, as previously described (1). Paired pre- using a BCA Protein Assay Kit (Pierce). Recombinant proteins from the and posttreatment liver biopsies were obtained from 17 patients with chronic same batch were used at the indicated concentrations for all experiments. genotype-1 HCV treated with DAAs: 8 patients were treated for 24 wk with ISRE-Luc assays sofosbuvir and ribavirin (clinical trial NCT01441180), and 9 patients were treated for 12 wk with sofosbuvir and ledipasvir or for 6 wk with sofosbuvir HepG2-ISRE-Luc cells (1) were seeded in 96-well plates (1 3 104 cells per and ledipasvir in combination with an additional investigational DAA (clinical well) and treated with IFNs. Stable HepG2 cells were induced with 0.5 mg/ml trial NCT01805882) (9, 10). Two of seventeen patients experienced treatment doxycycline and then lysed and assayed for ISRE-Luc at the indicated time relapse, but posttreatment biopsies were obtained prior to virologic relapse. points with a GloMax-Multi Detection System (Promega). All ISRE-Luc Both studies were approved by the National Institute for Allergy and Infectious experiments were independently conducted at least three times with six bi- Diseases/National Institutes of Health Institutional Review Board, and all ological replicates per sample each time, unless otherwise specified. patients provided written informed consent (9, 10). The liver biopsies were initially screened with microarray analysis in a subset of samples to identify Coculture assays transcripts with the largest changes in expression caused by DAA treatment (9, 3 3 10). Expression of select transcripts was validated with individual TaqMan For the first coculture model, 5 10 HepG2–IFN-l3–GFP or HepG2– IFN-l4–GFP cells were seeded at a 1:1 ratio with 5 3 103 HepG2–ISRE– assays and analyzed in groups of patients stratified by their ability to produce 3 4 IFN-l4 protein, based on the presence of the rs368234815-dG allele (9, 10). Luc cells in a 96-well plate (total of 1 10 cells per well). For the second model, 5 3 103 HepG2–IFN-l4–GFP–ISRE–Luc cells were seeded at a The human hepatoma HepG2 cell line was purchased from the American 3 3 Type Culture Collection. HepG2 and all derived cell lines were maintained 1:1 ratio with 5 10 HepG2 cells; HepG2 cells were included to achieve a total of 1 3 104 cells per well while keeping the counts of the IFN-l4– in DMEM (Cellgro) supplemented with 10% FBS (Invitrogen), penicillin, 3 3 and streptomycin. A stable HepG2 cell line carrying the ISRE-Luc reporter producing and ISRE-Luc reporter cells (5 10 cells) comparable to the (HepG2–ISRE–Luc cells) (1) was maintained in media with 1 mg/ml pu- first model. Cells were incubated for 24 h, induced with 0.5 mg/ml romycin. A stable HepG2 cell line expressing doxycycline-inducible GFP- doxycycline, lysed, and assayed for ISRE-Luc at the indicated time tagged IFN-l3 protein (HepG2–IFN-l3–GFP cells) was developed as points. For blocking experiments, 20 mg/ml rabbit Abs (monoclonal anti– described for HepG2–IFN-l4–GFP cells (25); these cell lines were IFN-l4; ab196984; Abcam) or polyclonal IgG control (R&D Systems) was maintained in media with 5 mg/ml blasticidin and 1 mg/ml neomycin. A added immediately after induction with doxycycline. stable HepG2 cell line expressing both IFN-l4–GFP and the ISRE-Luc Western blotting reporter was generated by transducing HepG2–IFN-l4–GFP cells with the Luciferase Cignal Lenti ISRE reporter construct (QIAGEN) and selecting PHHs or HepG2 cells were lysed with RIPA buffer (Sigma) supplemented for positive clones, as previously described (1); these cells were main- with protease inhibitor mixture (Promega) and PhosSTOP (Roche). Equal tained in media with 5 mg/ml blasticidin, 1 mg/ml neomycin, and 1 mg/ml amounts of proteins were resolved on 4–12% Bis-Tris Bolt gels and puromycin. transferred using an iBlot 2 (Thermo Fisher). Primary Abs were rabbit The Journal of Immunology 3 anti-SOCS1 (1:200 dilution, ab62584; Abcam) and a panel of Abs from scrambled negative control siRNA (Silencer Select, 4390843) using Lipo- Cell Signaling Technology: rabbit anti-USP18 (1:500 dilution, #4813), fectamine RNAiMAX transfection reagent (all from Thermo Fisher). After anti-STAT1 (1:1000 dilution, #9172), anti–phospho-STAT1 (1:1000, Tyr701, 48 h of transfection, medium was replaced, and PHHs were treated for 24 h #7649), anti-STAT2 (1:1000, #4594), and anti–phospho-STAT2 (1:100, with 20 ng/ml IFN-l3 or 50 ng/ml IFN-l4. To test for refractoriness, Tyr690, #88410). The secondary Ab was a goat anti-rabbit, HRP linked transfected PHHs were treated with 20 ng/ml IFN-l3 for an additional 8 h (1:5000, #7074; Cell Signaling Technology). The results were detected with before harvesting. Based on analysis of mRNA expression in PHHs HyGLO Quick Spray (Denville Scientific) and viewed on a ChemiDoc transfected with SOCS1 and scrambled siRNA, and normalized by the Touch Imager with Image Lab 5.2 software (Bio-Rad). Due to the rapid expression of endogenous control (GAPDH), siRNA knockdown resulted SOCS1 turnover (27), 50 mM proteasome inhibitor MG132 (Sigma) was in a 2.3-fold decrease in SOCS1 expression. The siRNA effect was also added to PHHs 2 h before harvesting. visualized by Western blotting. Because of the fast turnover of SOCS1 For detection of endogenous IFN-l4, ∼3.5 3 105 PHHs per liver donor (27), 50 nM proteasome inhibitor MG132 was added to PHHs 2 h before were infected with SeV for 6 or 24 h. At harvesting, culture medium was harvesting. Positive and negative Western blot controls (OriGene) were collected separately and then cells were lysed with RIPA buffer and pulse represented by lysates of HEK293 cells transfected with a SOCS1 plasmid sonicated for 30 s, with 10-s burst-cooling cycles, at 4˚C. Lysates were or left untransfected, respectively. centrifuged at 10,000 3 g for 5 min to separate lysates from insoluble debris. All fractions (media, lysates, and insoluble debris) were immedi- Statistical analysis ately boiled in reducing sample buffer for 5 min at 100˚C, resolved on a gel Unless specified, data plotting and statistical analyses were performed with as described above, and transferred using the XCell II Blot Module Prism 7 (GraphPad). The p values are for the two-sided unpaired Student (Thermo Fisher). The blot was blocked with 0.5% milk for 1 h and then t tests, with p , 0.05 being considered significant. Means are presented incubated with rabbit anti–IFN-l4 (1:500, ab196984; Abcam) or mouse with SEs. anti–IFN-l4 (1:200, MABF227; EMD Millipore) in 0.5% milk at 4˚C overnight. Secondary Abs were HRP-linked goat anti-rabbit (1:5000, #7074; Cell Signaling Technology) or donkey anti-mouse (1:5000, sc2314; Santa Results Downloaded from Cruz Biotechnology), and detection was done with SuperSignal West Femto SeV infection induces expression of endogenous IFN-l4in Maximum Sensitivity Substrate (Thermo Fisher). Glycosylation of IFN-l4 human hepatic cells was tested by treating PHH lysates with detectable levels of IFN-l4 with peptide–N-glycosidase F (PNGaseF; NEB) for 1 h at 37˚C, followed by Expression of endogenous IFNL4 mRNA in virally infected Western blotting as described above. Prestained Western blot protein markers samples has been reported in several studies (1, 8, 28, 29), how- were Novex Sharp and SeeBluePlus2 (both from Thermo Fisher). ever, expression of endogenous IFN-l4 protein has been clearly http://www.jimmunol.org/ ELISA demonstrated only by confocal microscopy in PHHs treated with Poly I:C (1). To explore this further, we infected PHHs from 11 IFN-l4–GFP and IFN-l3–GFP were detected with a GFP ELISA Kit (Cell Biolabs) using purified GFP as a standard, with a lower limit of detection liver donors with SeV, because this virus has been shown to induce of 50 pg/ml. IFNL4 mRNA expression in hepatic cell lines (28). We detected strong induction of all type III IFNs, including IFNL4 (Fig. 1A), SeV infection with similar kinetic profiles (Supplemental Fig. 1A). Stocks of SeV Cantell strain were purchased from Charles River Labo- We then performed Western blotting for IFN-l4 in SeV-infected ratories. PHHs or HepG2 cells were infected in triplicates with SeV (7.5 3 5 50 PHHs from four donors (two with dG/dG genotype and one each 10 chicken embryo ID50 [CEID ] per milliliter) for 1 h, washed with PBS, and collected at the indicated time points. with a dG/TT or TT/TT genotype). In samples with dG/dG ge- by guest on September 29, 2021 notype, we detected a distinct band in cell lysates and insoluble RNA extraction and cDNA generation cell debris but not in media (Fig. 1B, Supplemental Fig. 1B, 1C). Total RNA from PHHs and HepG2 cells was extracted using an RNeasy Kit A faint band could be observed in PHHs from the donor with with on-column DNase I digestion (QIAGEN). RNA quantity and quality dG/TT genotype, but only after overexposing the original blot were evaluated using a NanoDrop 8000 (Thermo Fisher). cDNA was (Supplemental Fig. 1B). As expected, IFN-l4 was not detected in generated starting from 200 ng of total RNA from PHHs or 500 ng of RNA from HepG2 cells, using the RT2 First Strand Kit (QIAGEN) with an PHHs from the donor with TT/TT genotype (Fig. 1B). Expression additional DNA-removal step. of IFN-l4 protein followed the pattern of IFNL4 mRNA expression (Fig. 1C). The efficiency of SeV infection was comparable in Gene-expression analysis all samples, based on viral RNA loads, and could not explain the Expression of ISGs was measured in duplicates with an RT2 Profiler qRT- IFN-l4 expression pattern (Fig. 1C). PCR Human Antiviral Response Array (QIAGEN) that included 88 ex- Because IFN-l4 was shown to be N-glycosylated at amino acid pression assays for antiviral genes, as well as endogenous controls (ACTB, B2M, GAPDH, HPRT1, RPLP0) and positive and negative controls. Ex- N61 (26), we hypothesized that the larger than expected size of pression of select genes was measured with individual TaqMan expression IFN-l4 detected by Western blotting could correspond to the assays, in quadruplicates (Supplemental Table I), with GAPDH and ACTB glycosylated form. To test this, we treated one of the PHH lysates used as endogenous controls. SeV loads were measured with an expression where IFN-l4 expression was detectable (dG/dG genotype) with assay for SeV defective-interfering RNA, in quadruplicates (Supplemental PNGaseF and observed a shift from the initial band to a new ∼19- Table I), with GAPDH and ACTB used as endogenous controls. Expression was measured on a QuantStudio 7 (Thermo Fisher) using SYBR Green kDa band, which matches the expected size of IFN-l4 without any qPCR Master Mix (QIAGEN) for the antiviral response array and SeV load protein modifications (Fig. 1B). These results demonstrate that analysis and using TaqMan expression buffer (Thermo Fisher) for indi- IFN-l4 is expressed in PHHs in response to SeV infection, is vidual assays. Total RNA input for TaqMan assays was 2.5 ng per reaction glycosylated, and by Western blotting can be detected in cell ly- for PHHs and 5 ng per reaction for HepG2 cells; RNA input for antiviral response arrays was 2.5 ng per reaction. sates and in insoluble cell debris but not in the media. Expression was measured in PCR cycle at detection threshold (Ct) values, IFN-l4 activity requires secretion but at very low levels whicharedistributedonalog-2scale.Expression of target genes was normalized by geometric means of corresponding endogenous controls as DCt (target) = Ct The amount of IFN-l4 presumably secreted by PHHs into media in (control) – Ct (target). Differences in expression between groups of samples were response to SeV infection was below the detection limits of Western calculated, using the relative quantification method, as DDCt = DCt (group 1) blotting. This could be because the activity of IFN-l4doesnotre- – Ct (group 2). Fold differences can be calculated as fold = 2DDCt. quire secretion or that even the low amounts of IFN-l4 secreted into Small interfering RNA knockdown of SOCS1 expression media are sufficient for this function. To distinguish between these Fresh PHHs cultured in a 24-well plate were transfected in triplicates with possibilities, we developed stable doxycycline-inducible HepG2 cell 50 nM SOCS1 small interfering RNA (siRNA; Silencer Select, s16470) or lines expressing GFP-tagged IFN-l4 (HepG2–IFN-l4–GFP) (25) or 4 IFN-l4 ENHANCES NEGATIVE REGULATION OF IFN SIGNALING Downloaded from http://www.jimmunol.org/

FIGURE 1. IFN-l4 is produced in PHHs in response to SeV infection. ( ) Expression of IFNL4 and other type III IFNs in PHHs at 6 or 24 h post–SeV by guest on September 29, 2021 infection. Expression is presented in relation to IFNL4 genotypes with dG allele (dG/dG, n = 2 and dG/TT, n = 5) versus TT/TT (n = 4); results are plotted as DDCt values (log-2 scale) after normalization to endogenous controls (GAPDH and ACTB) and to uninfected samples (0 h). Shown are individual values and group means (horizontal lines). (B) Western blotting of lysates of PHHs (n = 4) infected with SeV for 6 or 24 h, showing a band corresponding in size to glycosylated IFN-l4. IFN-l4 (0.1 and 1 ng), produced in E. coli and, therefore, nonglycosylated, is used for comparison. Deglycosylation treatment with PNGaseF resulted in a band ∼ 19 kDa. Band sizes are indicated on the side. Donor IDs correspond to Supplemental Table I. (C) Expression of IFNL4 and SeV RNA in samples shown in (B). Gene expression is presented as DCt values (log-2 scale) after normalization to endogenous controls (GAPDH and ACTB). M, prestained protein size marker.

IFN-l3 (HepG2–IFN-l3–GFP). As reported previously (25), a sig- poor secretion, and it may be biologically active at concentrations nificant proportion of IFN-l4–GFP was retained intracellularly and , 50 pg/ml, which are not detectable with most assays, including was readily detectable, whereas most IFN-l3–GFP was secreted and Western blotting. could be detected intracellularly only after blocking protein secretion Next, we evaluated the possibility that IFN activity could be with GolgiStop for 4 h (Fig. 2A). contributed by intracellular accumulation of IFN-l4 because of its To test the role of IFN-l4 secretion in its activity, we cocultured poor secretion. In the first model, HepG2–IFN-l4–GFP cells were HepG2–IFN-l4–GFP cells or HepG2–IFN-l3–GFP cells, which cocultured in a 1:1 ratio with HepG2–ISRE–Luc cells; in this would produce these IFNs upon induction, in a 1:1 ratio with model, IFN-l4 must be secreted to activate ISRE-Luc in the re- HepG2–ISRE–Luc cells, which lack these IFNs but carry the reporter cells. For the second model, we developed a stable HepG2 porter that would detect the signal only when these IFNs are cell line expressing both IFN-l4–GFP and ISRE-Luc, thereby secreted from the producing cells. Time-course analysis of ISRE- combining the production and detection of IFN-l4 in the same Luc activation in the coculture system showed a response as early cells. This model would detect the signal induced by IFN-l4 se- as 6 h after inducing IFN-l4–GFP expression, whereas the re- creted from the producing and the surrounding cells, as well as sponse to IFN-l3–GFP was detectable only after 10 h (Fig. 2B). intracellularly retained IFN-l4. This observation was surprising because it contrasted with the IFN-l4 was induced in both models for 24 h, in the presence or concentrations of these IFNs in media, as measured by the GFP absence of anti–IFN-l4 blocking Ab (25), which is expected to tag (Fig. 2C, 2D). With the limit of detection ∼ 50 pg/ml, IFN-l4– block signaling induced by secreted, but not intracellular, IFN-l4, GFP was undetectable in media until 8 h (0.2 ng/ml), whereas and ISRE-Luc activation was measured. We observed that anti– IFN-l3–GFP was measurable by 6 h after induction (4 ng/ml). IFN-l4 blocking Ab was equally effective in blocking ISRE-Luc After 14 h, the amount of secreted IFN-l4–GFP was still very low activation in both models (Fig. 2E), indicating that, to induce IFN (1 ng/ml, Fig. 2C), yet ISRE-Luc activation had peaked. These signaling, IFN-l4 must be secreted, and the intracellular retention results suggest that IFN-l4 is an extremely potent IFN, despite its of IFN-l4 does not significantly contribute to its antiviral activity. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. IFN-l4 activity requires secretion but at very low levels. (A) Representative ﬂow cytometry plots for expression of IFN-l3–GFP and IFN- l4–GFP induced with doxycycline (Dox) for 24 h in corresponding stable HepG2 cell lines; expression of IFN-l4–GFP was readily detectable, whereas IFN-l3–GFP was detectable only after treatment with GolgiStop for 4 h to block protein secretion. (B) Kinetics of ISRE-Luc activation in stable HepG2– IFN-l4–GFP cells or HepG2–IFN-l3–GFP cells cocultured in a 1:1 ratio with HepG2–ISRE–Luc cells. (C) Concentration of GFP measured by ELISA (n = 2) in culture media of the experiment described in (B); shown is one of two independent experiments. (D) Western blotting for GFP in culture media of stable inducible HepG2–IFN-l4–GFP cells (not detectable) and HepG2–IFN-l3–GFP cells (detectable, marked by arrow) from the experiment described in (B). Band sizes are indicated on the side. Left and right panels represent separate blots that were run and imaged simultaneously. (E) The effect of secreted IFN-l4 is tested in stable HepG2–IFN-l4–GFP cells cocultured in a 1:1 ratio with HepG2–ISRE–Luc cells. The effect of secreted and intracellular IFN-l4istestedin stable HepG2–IFN-l4–GFP cells and HepG2–ISRE–Luc cells. Cells were induced with doxycycline (Dox) for 24 h to express IFN-l4–GFP in the presence of 20 mg/ml rabbit Abs, blocking anti–IFN-l4 or IgG control. Shown is one of two independent experiments. M, prestained protein size marker.

IFN-l4 is associated with an earlier antiviral response infection. Both genotype groups expressed similar levels of types I, Analysis of the acute antiviral response in relation to IFNL4 ge- II, and III IFNs (Figs. 1A, 3A) and had similar levels of viral in- notypes, which correspond to the ability to produce IFN-l4, may fection, monitored by measuring SeV RNA (Fig. 3B). PHHs from provide additional insights into IFN-l4 function. To explore this, donors with dG allele expressed signiﬁcantly higher levels of MX1, we measured the antiviral response to SeV in PHHs from 11 donors, ISG15,andOAS1 compared with donors with TT/TT genotype 6 h 7 of whom carry the dG allele and can produce IFN-l4(2with post–SeV infection (Fig. 3A). However, by 24 h, the expression dG/dGand5withdG/TTgenotypes)and4withtheTT/TTge- levels of these ISGs were similar between the genotype groups. notype (do not produce IFN-l4). We measured the expression of These results suggest that the presence of dG allele (i.e., the ability to three important ISGs (MX1, ISG15,andOAS1) 6 and 24 h post–SeV produce IFN-l4) is associated with a faster acute antiviral response. 6 IFN-l4 ENHANCES NEGATIVE REGULATION OF IFN SIGNALING Downloaded from

FIGURE 3. IFN-l4 is associated with earlier antiviral response to SeV infection. (A) Expression of select ISGs and IFNs in PHHs, 6 or 24 h post–SeV infection, presented in relation to IFNL4 genotypes, with dG allele (dG/dG, n = 2 and dG/TT, n = 5) versus TT/TT (n = 4). (B) Expression of SeV in http://www.jimmunol.org/ corresponding samples from (A). Gene expression is presented as DDCt values after normalization to endogenous controls (GAPDH and ACTB) and uninfected samples (0 h) (A), whereas expression is presented as DCt values after normalizing to endogenous controls only (B). DCt and DDCt values are presented on log-2 scale. Data are shown as individual values and group means (horizontal lines). *p , 0.05. LLOD, lower limit of detection.

Activity of IFN-l4 is rapid, but transient, compared with other IFN-l4 for 10 h (estimated peak antiviral activity, Fig. 4E), infected type III IFNs the cells with SeV, and measured SeV RNA after 12 h (Fig. 5A). To further evaluate the implications of IFN-l4 activity during viral Maximum inhibition of SeV infection was achieved by 50 ng/ml infection, we generated biologically active IFN-l4, which was IFN-l3 and 200 ng/ml IFN-l4 (Fig. 5B); further increases in pro- by guest on September 29, 2021 expressed in E. coli, refolded, and purified. IFN-l4 was charac- tein concentrations did not affect SeV RNA levels. This maximum terized by Coomassie staining and Western blotting (Fig. 4A, 4B) inhibition resulted in a 13-fold reduction in SeV RNA levels (Fig. 5B), and by detecting IL-10R2–dependent STAT1 phosphorylation in giving an estimate that 50% inhibition corresponded to a 6.5-fold HepG2 cells (Fig. 4C, 4D). reduction in SeV RNA levels, which was observed at 20 ng/ml We evaluated the comparative kinetics of all recombinant type III IFN-l3 and 50 ng/ml IFN-l4 (Fig. 5B). In HepG2–ISRE–Luc IFNs, including IFN-l4, by testing the range of 11 concentrations cells, these protein concentrations also provided similar peak antiviral (0.02–1000 ng/ml) of each IFN in HepG2–ISRE–Luc cells over activity by 8 h, but IFN-l4 showed significantly higher activity at 4 h 24 h. The induction of ISRE-Luc by IFN-l4 was rapid, reaching and much lower activity by 24 h compared with IFN-l3 (Fig. 5C). ∼50% of its peak activity (determined at 10 h) within the first 4 h The concentrations that provided similar peak IFN activity were of treatment, whereas other IFNs were at ∼20% of peak induction consideredequipotentandusedtoevaluate the antiviral kinetics of at 4 h (Fig. 4E, 4F). By 24 h, the activity of IFN-l4 had dropped to IFN-l3andIFN-l4 in subsequent experiments. These equipotent 20% of its peak (Fig. 4E, 4G), whereas the activity of other IFNs concentrations need to be experimentally estimated for specific con- was still .60% of their peaks. These results indicate that IFN-l4 ditions, assays, and batches of protein preparations. induces a rapid, but transient, response, whereas other type III IFNs To compare the effects of these IFNs on the kinetics of SeV demonstrated a slower, but more sustained, IFN activity. Similar infection, PHHs were treated with IFN-l3 or IFN-l4 for different rapid, but transient, activity was observed for a commercially periods of time and then infected with SeV; cells were harvested sourced IFN-l4 (Supplemental Fig. 2). Pretreatment incubation of after 12 h and analyzed for SeV RNA expression (Fig. 5D). Pre- IFN-l4 and IFN-l3 for 12 h at 37˚C resulted in similar declines in treatment for 12 h with either IFN inhibited SeV, but only IFN-l4 their activities (Fig. 4H), thus arguing against the selective loss of caused viral inhibition at earlier time points (4 h, Fig. 5E). HepG2– IFN-l4 stability as the reason for its shorter activity time. IFN-l4–GFP cells also showed an earlier antiviral response compared with HepG2–IFN-l3–GFP cells (Fig. 5F). Overall, IFN-l4– IFN-l4 shows faster antiviral activity than IFN-l3 in an SeV GFP was more potent than IFN-l3–GFP in inhibiting SeV infection infection model at all time points, showing that, despite its poor secretion (Fig. 2C), It is unclear whether differences in IFN kinetics reflect their an- IFN-l4 induced an earlier and more potent antiviral response tiviral properties. To evaluate this, we compared the antiviral ac- compared with IFN-l3. tivities of IFN-l4 and IFN-l3. We selected IFN-l3 for comparison because IFN-l3 is the most potent of IFN-l1–3. These three proteins Transcriptional analysis identifies ISGs differentially induced l l share .95% protein sequence similarity and antiviral profiles (30); by IFN- 3 and IFN- 4 with 29% protein identity, IFN-l3 is also most similar to IFN-l4(1). To determine which antiviral genes are differentially induced by We treated HepG2 cells with different concentrations of IFN-l3and IFN-l3 and IFN-l4 at early and late time points, we used antiviral The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. Activity of IFN-l4 is rapid, but transient, compared with other type III IFNs. Detection of the custom IFN-l4 (350 ng) with Coomassie staining (A) and Western blotting (B) using the monoclonal mouse and rabbit anti–IFN-l4 Abs. All methods detected a single band at ∼19 kDa, which corresponds to the estimated size of IFN-l4. Band sizes are indicated on the side. (C) Western blotting for p-Y701–STAT1 and STAT1 in HepG2 cells treated for 30 min with different concentrations of IFN-l4. (D) Western blotting as in (C), but in the presence of 10 mg/ml the blocking anti–IL-10R2 mouse mAb or goat polyclonal Ab (pAb) that is expected to block IFN-l4 signaling and decrease p-Y701–STAT1 expression. (E) ISRE-Luc activity in HepG2– ISRE–Luc cells treated with 11 concentrations of all four type III IFNs in a 24-h time course. Vertical dashed lines indicate the peak ISRE-Luc induction observed at 10 h. The ratio of average activity at 4 h (F) and at 24 h (G) to the peak at 10 h, shown for the top six concentrations of each type III IFN. (H) Similar stability of IFN-l3 and IFN-l4 was demonstrated by preincubation of IFNs at 37˚C for the indicated times, followed by treatment of HepG2–ISRE– Luc cells for 8 h. ***p , 0.001. M, prestained protein size marker. quantitative RT-PCR (qRT-PCR) arrays to measure mRNA ex- HepG2–IFN-l4–GFP or HepG2–IFN-l3–GFP. Consistent with pression of a panel of 88 ISGs in HepG2 cells treated with IFN-l4 our results in HepG2 cells and PHHs treated with IFNs exoge- and IFN-l3 (Fig. 6A). Within the first 1–2 h of treatment, we nously, we observed that endogenously expressed IFN-l4–GFP observed a strong increase in the expression of ISGs, such as MX1, also induced higher levels of OAS1, ISG15, and MX1 compared ISG15, OAS2, DDX58 (RIG-I), DHX58, and STAT1, in cells with IFN-l3–GFP at all time points (Fig. 6C). However, by 24 h, treated with IFN-l4 compared with IFN-l3 (Fig. 6A). However, at there was no decline in the response induced by endogenously 8 h, both IFNs induced these ISGs to a similar level (Fig. 6A); produced IFN-l4 compared with cells treated with IFN-l4 ex- expression of most ISGs induced by IFN-l4 declined by 24 h, ogenously (Fig. 6C), highlighting possible differences in the re- whereas the effect of IFN-l3 was more sustained and lasted be- sponse to continuous (chronic) versus transient exposure to IFN-l4. yond 24 h (Fig. 6A). These expression profiles for individual ISGs Overall, we show that IFN-l4 induces an earlier activation of key recapitulated our results for ISRE-Luc reporter assays, including antiviral effectors compared with IFN-l3. the determination of equipotent concentrations for these IFNs at IFN-l4 is a potent inducer of negative regulators of 8 h (Fig. 5C). IFN response Similarly, treatment of PHHs from three liver donors showed stronger effects of IFN-l4 compared with IFN-l3 on the expres- To understand the mechanisms for the faster, but more transient, sion of MX1, ISG15, and OAS1 at 2 h and similar effects at 8 h of activity of IFN-l4, we explored the kinetics of STAT phosphoryla- treatment; by 24 h, IFN-l4 activity had declined significantly, tion. In HepG2 cells and PHHs, IFN-l4 induced strong STAT1 and whereas IFN-l3 activity was still sustained or even increased STAT2 phosphorylation within the first 15 min to 1 h of treatment, (Fig. 6B). We then monitored the kinetics of ISG expression in buttheeffectstartedtodiminishby24h,whereasIFN-l3 caused a 8 IFN-l4 ENHANCES NEGATIVE REGULATION OF IFN SIGNALING Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 5. IFN-l4 shows faster activity than IFN-l3 in an SeV infection model. (A) SeV RNA quantified by qRT-PCR and presented as Ct values (log-2 scale) in HepG2 cells, 3, 6, 12, and 24 h post–SeV infection. (B) Change in SeV RNA in HepG2 cells infected with SeV after pretreatment with different concentrations of IFN-l3 or IFN-l4 for 10 h. SeV RNA values are normalized to the expression of endogenous controls (GAPDH and ACTB) and presented as the percentage of SeV RNA levels in untreated cells. Shown is one of two independent experiments, each with three biological replicates. (C) ISRE-Luc induction in HepG2–ISRE–Luc cells treated with IFN-l3 or IFN-l4 in a 24-h time course. (D) Schematic representation of SeV infection experiment that includes pretreatment with IFN-l3 or IFN-l4. SeV RNA in PHHs (E) or stable HepG2 cells (F) pretreated or induced to express IFN-l4–GFP or IFN-l3– GFP and then infected with SeV. Results are presented as in (B). *p , 0.05, **p , 0.01, ***p , 0.001. more gradual increase in p-STAT1 and p-STAT2 that was sustained observed for other ISGs (Fig. 6C); however, there was no atten- beyond 24 h (Fig. 7A, Supplemental Fig. 3A for an additional PHH uation of response by 24 h, possibly as the result of continuous donor). exposure to IFNs in these cells. In PHHs treated with IFN-l4, Blocking of STAT phosphorylation and inhibition of the JAK/ USP18 protein was already induced at 8 h; however, at 24 h, it was STAT pathway are modulated on several levels, including by similarly induced by IFN-l3 and IFN-l4 (Fig. 7C). Expression of negative regulators of IFN signaling, such as USP18 and SOCS SOCS1 protein was similar at 2 h, but a sustained induction at 8 proteins (31–35). Increased expression of USP18 and SOCS1 has and 24 h was observed only in PHHs treated with IFN-l4 also been associated with reduced HCV clearance (36–39). We (Fig. 7C). observed that, in HepG2 cells and PHHs, USP18 had an expres- Of all recombinant type III IFNs tested at different concentra- sion profile of a typical ISG (Fig. 6), with earlier, but more tions for treating PHHs for 8 h, IFN-l4 induced the highest levels transient, induction by IFN-l4 compared with IFN-l3 (Fig. 7B). of SOCS1 (Fig. 7D); the effects were comparable between our The expression profile of SOCS1 was more distinct in HepG2 custom and commercial IFN-l4 proteins (Supplemental Fig. 3B). cells, and even more so in PHHs; it was induced earlier and lasted We hypothesized that strong and sustained induction of SOCS1 in beyond 24 h following IFN-l4 treatment compared with IFN-l3 PHHs treated with IFN-l4 might induce refractoriness to addi- (Fig. 7B). In stable inducible HepG2 cells, the expression pro- tional IFN-l3 treatment. To test this, we treated PHHs with files of USP18 and SOCS1 (Fig. 7B) were comparable to those IFN-l4 or IFN-l3 for 24 h (the timing was determined based on The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021 FIGURE 6. Transcriptional profiling identifies ISGs differentially induced by IFN-l3 and IFN-l4. (A) Heat map for expression of select ISGs measured by an antiviral qRT-PCR array in HepG2 cells treated with IFN-l4 or IFN-l3 for the indicated times. Red and green colors indicate higher expression of ISG induced by IFN-l4 treatment and IFN-l3 treatment, respectively. (B) Expression of select ISGs in PHHs obtained from three liver donors and treated with IFN-l3 or IFN-l4. Donor IDs correspond to Supplemental Table I. (C) Expression of select ISGs in stable HepG2 cells induced to express IFN-l3– GFP or IFN-l4–GFP. Expression is presented as DDCt values (log-2 scale) that are normalized to endogenous controls (GAPDH and ACTB) and uninfected samples (0 h). Plots show individual biological replicates. *p , 0.05, **p , 0.01, ***p , 0.001.

SOCS1 expression profiles, Fig. 7C) and then treated them with 4 with TT/TT genotype. Expression of SOCS1 was significantly IFN-l3 for an additional 8 h. Pretreatment with IFN-l4 signifi- higher in carriers of the dG allele at 6 and 24 h post–SeV infection, cantly attenuated the subsequent response to IFN-l3 (Fig. 7E), whereas the increase in the expression of USP18 was more transient; whereas pretreatment with IFN-l3 had no effect. Because USP18 it was higher only at 6 h postinfection, whereas expression had de- protein levels were similar at 24 h of treatment with IFN-l3or creased at 24 h and was similar in both genotype groups (Fig. 8A). IFN-l4, we reasoned that an increase in SOCS1, rather than in We also analyzed the available data for expression of USP18 and USP18 expression, may be responsible for the refractoriness of several other ISGs (MX1, ISG15, and OAS2) in paired (pre- and IFN-l4–treated PHHs to subsequent IFN-l3 stimulation. To test posttreatment) liver biopsies from 17 HCV patients treated with this, we monitored refractoriness of IFN-l4–treated PHHs in the DAA, either with sofosbuvir and ribavirin for 24 wk (n =8)or presence or absence of siRNA knockdown of SOCS1 (Fig. 7F). with sofosbuvir combined with one or two additional DAAs for 6– The 2.3-fold knockdown of SOCS1 was sufficient to cause a sig- 12 wk (n = 9) (9, 10). It is known that expression of many ISGs, nificant increase in ISG expression (Fig. 7G). This indicates that including USP18, is increased in pretreatment liver biopsies of SOCS1 induction was contributing to the refractoriness of IFN- carriers of the dG allele (9); this conclusion could be made based l4–treated PHHs to additional IFN-l3 stimulation. on our liver biopsies as well (Fig. 8B). However, by analyzing changes in gene expression in paired liver biopsies, we could draw IFN-l4 is associated with higher expression of USP18 and additional conclusions. We observed that sustained high expres- SOCS1 during viral infection sion of USP18 and other ISGs in pretreatment liver biopsies oc- Our findings suggest that the antiviral response could be differ- curred primarily in carriers of the dG allele, in line with what we entially negatively regulated based on the genetic ability to produce observed in stable HepG2 cells continuously exposed to IFN-l4 IFN-l4, which is defined by the presence of the dG allele. We (Figs. 6C, 7B). In our HepG2 cell line models, IFN-l4 expression tested this hypothesis in the set of SeV-infected PHHs used in our is induced in vitro, whereas in liver biopsies it is induced by HCV previous analysis (Fig. 3) that included 11 samples: 7 with dG infection. DAA treatment resulted in a significant decrease in the allele (2 with dG/dG genotype and 5 with dG/TT genotype) versus expression of USP18 and other ISGs, but only in carriers of the dG 10 IFN-l4 ENHANCES NEGATIVE REGULATION OF IFN SIGNALING Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 7. IFN-l4 is a potent inducer of negative regulators of IFN response. (A) Western blot analysis for p-Y701–STAT1, STAT1, p-Y690–STAT2, and STAT2 in HepG2 cells and PHHs treated with IFN-l3 and IFN-l4, shown for one of three independent experiments. (B) Expression of select transcripts in HepG2 cells and PHHs treated with IFN-l3 or IFN-l4 and in stable HepG2 cells induced to produce IFN-l3–GFP or IFN-l4–GFP. Gene expression is presented as DDCt values (log-2 scale) that are normalized to endogenous controls (GAPDH and ACTB) and untreated samples. (C) Western blot analysis of USP18 and SOCS1 after treatment of PHHs with IFN-l3 or IFN-l4 in a 24-h time course. Positive (+) control represents lysate of HEK293 cells transfected with SOCS1 plasmid; negative (–) control represents untransfected HEK293 cells. (D) Expression of SOCS1 in PHHs treated with different concentrations of all four type III IFNs for 8 h. Gene expression was analyzed as described in (B). (E) Expression of select transcripts in PHHs obtained from one liver donor and treated with IFN-l3 or IFN-l4 in different combinations. X and Y represent PHHs pretreated with IFN-l3 and IFN-l4, respectively, before restimulating with IFN-l3. Gene expression was analyzed as described in (B). Results represent one of three independent experiments, each in biological duplicates. (F) Western blot analysis of SOCS1 in PHHs transfected with scrambled siRNA (Scr siRNA) or SOCS1 siRNA for 48 h and then stimulated with IFN-l4 for 24 h; 50 nM proteasome inhibitor MG132 was added to PHHs 2 h prior to harvesting. (G) Expression of SOCS1 and (Figure legend continues) The Journal of Immunology 11

allele. Individuals who are genetically unable to produce IFN-l4 (TT/TT genotype) may be less compromised by the expression of negative regulators, leading to faster viral clearance, in line with a previous study (10).

Discussion We demonstrate in this article that the kinetics of IFN-l4 differs signiﬁcantly from other type III IFNs and describe potential consequences of IFN-l4 expression during viral infection that could affect viral clearance in individuals with different genotypes of rs368234815. Antiviral activity of IFN-l4 was suggested to be comparable to the activities of other type III IFNs (24, 26, 28). We now show that IFN-l4 acts faster than other type III IFNs in inducing an acute antiviral response, but at the same time it induces the expression of negative regulators of the IFN response. Strong and prolonged induction of SOCS1 was achieved even after transient exposure of PHHs to IFN-l4, whereas the prolonged induction of USP18 required continuous exposure to IFN-l4. Hepatocytes from HCV-infected patients may present another Downloaded from example of this regulation: even low, but continuous, expression of IFN-l4 induced by HCV in carriers of the dG allele might sustain expression of ISGs, as well as negative regulators of the IFN response, making hepatocytes refractory to additional IFN stimulation and resistant to spontaneous or treatment-induced viral

clearance. http://www.jimmunol.org/ IFN-l4 is unique among IFNs because it is poorly secreted and undetectable in the media of virally infected cells (1, 25, 26, 28). We show IFN-l4 detection with Western blotting in lysates from SeV-infected PHHs, and specifically from donors with the dG allele, consistent with our previous observations showing IFN-l4 in PHHs treated with Poly I:C (1). Although we did not detect IFN-l4 in media of SeV-infected PHHs, we show that IFN-l4 acts as a secreted protein at media concentrations below the limit of detection of our assays (,50 pg/ml), indicating that IFN-l4isan by guest on September 29, 2021 extremely potent IFN. Interestingly, the antiviral response in stable HepG2 cells expressing IFN-l4 was significantly higher than in PHHs treated with IFN-l4. This could be due to biological differences between immortalized cancer cells and fresh primary hepatocytes, as well as the availability of biologically active IFN-l4. In stable HepG2 cells, induction provides continuous FIGURE 8. IFN-l4 production is associated with higher expression of production of endogenous IFN-l4, whereas PHHs are exposed to A USP18 and SOCS1 during viral infection. ( ) Expression of SOCS1 and a single dose of IFN-l4. Furthermore, even the limited amount of USP18 in PHHs, 6 or 24 h post–SeV infection, presented in relation to IFN-l4 secreted from mammalian cells is expected to be properly IFNL4 genotypes, with dG allele (dG/dG, n = 2 and dG/TT, n = 5) versus TT/TT (n = 4) and plotted as DDCt values (log-2 scale) that are normalized folded and biologically active, whereas only a fraction of IFN-l4 to endogenous controls (GAPDH and ACTB) and to uninfected samples produced in E. coli is properly refolded (usually estimated to be (0 h). (B) Pre- and posttreatment liver biopsies from 17 HCV patients were ,25% of total protein refolded from E. coli inclusion bodies) analyzed for expression of MX1, ISG15, OAS2, and USP18. The results are (40). presented as DCt values (log-2 scale) normalized by expression of en- IFN-l4 has only 29% amino acid similarity with IFN-l3 (1, dogenous control (GAPDH) and presented according to IFNL4 genotype 41), with the most conserved regions corresponding to the protein groups. The p values for pre- and posttreatment expression levels are based sequences that interact with IFNLR1, and the least conserved on paired t tests within genotype groups and unpaired t tests between region corresponding to the putative binding site of IL-10R2 (1, genotype groups. Shown are individual values and group means (horizontal 41). Thus, higher potency and faster antiviral kinetics of IFN-l4 lines). *p , 0.05. might be attributed to its stronger receptor affinity compared with other type III IFNs (42). Faster, but more transient, antiviral ki- allele (Fig. 8B). These results suggest that viral clearance, even netics has been demonstrated in PHHs treated with IFN-a com- after DAA treatment, depends on reduced expression of negative pared with type III IFNs (43, 44). IFN-a and IFN-ls activate the regulators of IFN signaling, such as USP18, in carriers of the dG same JAK/STAT pathway, but IFN-a induces stronger STAT1

select ISGs in PHHs transfected with scrambled or SOCS1 siRNA. At 48 h after transfection, cells were treated with IFN-l3 (20 ng/ml) or IFN-l4 (50 ng/ml) for 24 h. After 24 h, all samples were restimulated with IFN-l3 (20 ng/ml) for 8 h and analyzed for gene expression. Expression is presented as DDCt values (log-2 scale) that are normalized to endogenous control (GAPDH) and uninfected samples (0 h). siRNA knockdown resulted in a decrease in SOCS1 expression by a DDCt value of 1.2, which corresponds to a 2.3-fold decrease. Plots show individual biological replicates from one of two independent experiments. Band sizes for a protein marker are indicated on the side (A, C, and F). *p , 0.05, **p , 0.01, ***p , 0.001. 12 IFN-l4 ENHANCES NEGATIVE REGULATION OF IFN SIGNALING phosphorylation compared with similar amounts of IFN-ls (45). bacterial pathogens, as well as the role that it plays during these In our experiments, IFN-l4 also had faster and more transient infections, remain unknown; these topics warrant investigation. effects compared with equipotent concentrations of IFN-l3. This Although our studies demonstrate differential kinetics and potency suggests that IFN-l4 and IFN-a may be using similar mechanisms of IFN-l4 compared with other type III IFNs in PHHs using SeV to induce negative regulators of IFN signaling, which may con- infection as a model, additional studies are necessary before ex- tribute to the failure of IFN-a–based therapies in managing HCV trapolating these findings to other cell types and diseases, in- infection in patients expressing IFN-l4 (1, 46, 47). cluding HCV. Higher levels of ISGs in pretreatment liver biopsies were reported in HCV patients who can produce IFN-l4 (6, 8–10, 48), Disclosures and we observed the same pattern for several ISGs, including R.P.D. and L.P.-O. are coinventors on the National Cancer Institute patent USP18, in pretreatment biopsies of 17 HCV patients. USP18 was related to IFN-l4. The other authors have no financial conflicts of interest. proposed as a key inhibitor of anti-HCV activity of IFN-a (37, 49, 50), with attenuation of USP18 expression correlating with res- toration of intrahepatic IFN-a signaling and success of IFN-free References 1. Prokunina-Olsson, L., B. Muchmore, W. Tang, R. M. Pfeiffer, H. Park, HCV therapy (6, 9). Our analysis of ISG expression in liver H. Dickensheets, D. Hergott, P. Porter-Gill, A. Mumy, I. Kohaar, et al. 2013. A biopsies, pre- and posttreatment with DAAs, showed that an HCV- variant upstream of IFNL3 (IL28B) creating a new interferon gene IFNL4 is associated increase in USP18 expression occurred only in indi- associated with impaired clearance of hepatitis C virus. Nat. Genet. 45: 164–171. 2. Thomas, D. L., C. L. Thio, M. P. Martin, Y. Qi, D. Ge, C. O’Huigin, J. Kidd, viduals who can produce IFN-l4. This suggests that increased K. Kidd, S. I. Khakoo, G. Alexander, et al. 2009. Genetic variation in IL28B and negative regulation of IFN signaling may be contributing to a spontaneous clearance of hepatitis C virus. Nature 461: 798–801. Downloaded from higher risk for the development of chronic HCV in individuals 3. Ge, D., J. Fellay, A. J. Thompson, J. S. Simon, K. V. Shianna, T. J. Urban, E. L. Heinzen, P. Qiu, A. H. Bertelsen, A. J. Muir, et al. 2009. Genetic variation with the dG allele, whereas individuals not producing IFN-l4 are in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 461: less affected by negative regulation and have more robust viral 399–401. 4. Suppiah, V., M. Moldovan, G. Ahlenstiel, T. Berg, M. Weltman, M. L. Abate, clearance. In PHHs treated with IFN-l4 compared with IFN-l3, M. Bassendine, U. Spengler, G. J. Dore, E. Powell, et al. 2009. IL28B is asso- USP18 protein expression was induced more quickly (already at ciated with response to chronic hepatitis C interferon-alpha and ribavirin therapy. Nat. Genet. 41: 1100–1104. 8 h), but the consequences of this faster induction on the IFN http://www.jimmunol.org/ 5. Tanaka, Y., N. Nishida, M. Sugiyama, M. Kurosaki, K. Matsuura, N. Sakamoto, response are unclear. Treatment of a hepatic cell line (Huh7) with M. Nakagawa, M. Korenaga, K. Hino, S. Hige, et al. 2009. Genome-wide as- IFN-l4 led to a sustained induction of USP18, making cells re- sociation of IL28B with response to pegylated interferon-alpha and ribavirin fractory to IFNa stimulation, but the kinetics of USP18 expression therapy for chronic hepatitis C. Nat. Genet. 41: 1105–1109. 6. Terczynska-Dyla, E., S. Bibert, F. H. Duong, I. Krol, S. Jørgensen, E. Collinet, induced by IFN-l4 versus other IFNs was not explored (49). Z. Kutalik, V. Aubert, A. Cerny, L. Kaiser, et al; Swiss Hepatitis C Cohort Study Increased expression of SOCS1 has been shown to block anti- Group. 2014. Reduced IFNl4 activity is associated with improved HCV clearance and reduced expression of interferon-stimulated genes. Nat. Commun. 5: viral responses induced by type I and type III IFNs (32, 33, 38, 5699. 39). In HCV infection, downregulation of SOCS1 expression in- 7. Uccellini, L., F. C. Tseng, A. Monaco, F. M. Shebl, R. Pfeiffer, M. Dotrang, creases the anti-HCV activity of IFNs (39). Robust type III IFN D. Buckett, M. P. Busch, E. Wang, B. R. Edlin, et al. 2012. HCV RNA levels in a multiethnic cohort of injection drug users: human genetic, viral and demographic by guest on September 29, 2021 signaling is important during the acute phase of HCV infection in associations. Hepatology 56: 86–94. PHHs in vitro (51, 52), as well as in chimpanzees (51, 53), im- 8. Amanzada, A., W. Kopp, U. Spengler, G. Ramadori, and S. Mihm. 2013. plying that a defective type III IFN signaling could contribute to Interferon-l4 (IFNL4) transcript expression in human liver tissue samples. PLoS One 8: e84026. development of chronic HCV infection. We show that IFN-l4 9. Meissner, E. G., A. Kohli, K. Virtaneva, D. Sturdevant, C. Martens, induces a strong and sustained induction of SOCS1 expression, S. F. Porcella, J. G. McHutchison, H. Masur, and S. Kottilil. 2016. Achieving sustained virologic response after interferon-free hepatitis C virus treatment which may make PHHs refractory to subsequent type III IFN correlates with hepatic interferon gene expression changes independent of cir- signaling, although other negative regulators of IFN responses rhosis. J. Viral Hepat. 23: 496–505. cannot be ruled out. Increased expression of SOCS1 induced by 10. Meissner, E. G., D. Bon, L. Prokunina-Olsson, W. Tang, H. Masur, T. R. O’Brien, E. Herrmann, S. Kottilil, and A. Osinusi. 2014. IFNL4-DGge- IFN-l4 may also have an effect on the adaptive immune response notype is associated with slower viral clearance in hepatitis C, genotype-1 pa- to viral infections, because SOCS1 plays an important role in tients treated with sofosbuvir and ribavirin. J. Infect. Dis. 209: 1700–1704. regulating the activity of IL-6 and IFN-g (35), key cytokines in the 11. Aka, P. V., M. H. Kuniholm, R. M. Pfeiffer, A. S. Wang, W. Tang, S. Chen, J. Astemborski, M. Plankey, M. C. Villacres, M. G. Peters, et al. 2014. Asso- adaptive immune response. ciation of the IFNL4-DG allele with impaired spontaneous clearance of hepatitis Type III IFNs are considered first responders to pathogens that C virus. J. Infect. Dis. 209: 350–354. 12. O’Brien, T. R., S. Kottilil, J. J. Feld, T. R. Morgan, and R. M. Pfeiffer. 2017. enter the body through epithelial surfaces (54, 55). If efficient, an Race or genetic makeup for hepatitis C virus treatment decisions? Hepatology early innate antiviral response modulated by type III IFNs at the 65: 2124–2125. pathogen entry sites might limit the infection and prevent the 13. Eslam, M., A. M. Hashem, R. Leung, M. Romero-Gomez, T. Berg, G. J. Dore, H. L. Chan, W. L. Irving, D. Sheridan, M. L. Abate, et al. 2015. Interferon-l necessity of other defense mechanisms (56) and inflammation rs12979860 genotype and liver fibrosis in viral and non-viral chronic liver dis- (21). However, an earlier antiviral response can be detrimental if it ease. Nat. Commun. 6: 6422. is inefficient and impairs the activity of other IFNs (57) or if it 14. Eslam, M., D. McLeod, K. S. Kelaeng, A. Mangia, T. Berg, K. Thabet, W. L. Irving, G. J. Dore, D. Sheridan, H. Grønbæk, et al. 2017. IFN-l3, not inhibits the adaptive immune response (58). Rapid antiviral ki- IFN-l4, likely mediates IFNL3-IFNL4 haplotype-dependent hepatic inflammation netics of IFN-l4 might be beneficial for some infections that re- and fibrosis. Nat. Genet. 49: 795–800. 15. Read, S. A., K. S. O’Connor, V. Suppiah, C. L. Ahlenstiel, S. Obeid, K. M. Cook, quire an immediate, albeit short-lived, immune response, whereas A. Cunningham, M. W. Douglas, P. J. Hogg, D. Booth, et al. 2017. Zinc is a it is a liability in chronic infections, such as HCV. potent and specific inhibitor of IFN-l3 signalling. Nat. Commun. 8: 15245. Type III IFNs have been shown to control a range of clinically 16. Honegger, J. R., D. Tedesco, J. A. Kohout, M. R. Prasad, A. A. Price, T. Lindquist, S. Ohmer, M. Moore-Clingenpeel, A. Grakoui, and C. M. Walker. important infections, such as flu (21, 59), respiratory syncytial 2016. Influence of IFNL3 and HLA-DPB1 genotype on postpartum control of virus (60), norovirus (19, 20), rotavirus (61), Zika virus (62), West hepatitis C virus replication and T-cell recovery. Proc. Natl. Acad. Sci. USA 113: Nile virus (63), and dengue virus (64). Expression of type III IFNs 10684–10689. 17. Price, A. A., D. Tedesco, M. R. Prasad, K. A. Workowski, C. M. Walker, in cells of epithelial origin was also induced by clinically relevant M. S. Suthar, J. R. Honegger, and A. Grakoui. 2016. Prolonged activation of bacterial pathogens, such as Staphylococcus aureus and Listeria innate antiviral gene signature after childbirth is determined by IFNL3 genotype. Proc. Natl. Acad. Sci. USA 113: 10678–10683. monocytogenes,aswellasMycobacterium tuberculosis (65). 18. Kelemen, L. E., K. Lawrenson, J. Tyrer, Q. Li, J. M. Lee, J.-H. Seo, Whether IFN-l4 can be induced endogenously by these viral and C. M. Phelan, J. Beesley, X. Chen, T. J. Spindler, et al. 2015. Genome-wide The Journal of Immunology 13

significant risk associations for mucinous ovarian carcinoma. Nat. Genet. 47: 43. Jilg, N., W. Lin, J. Hong, E. A. Schaefer, D. Wolski, J. Meixong, K. Goto, 888–897. C. Brisac, P. Chusri, D. N. Fusco, et al. 2014. Kinetic differences in the induction 19. Nice, T. J., M. T. Baldridge, B. T. McCune, J. M. Norman, H. M. Lazear, of interferon stimulated genes by interferon-a and interleukin 28B are altered by M. Artyomov, M. S. Diamond, and H. W. Virgin. 2015. Interferon-l cures infection with hepatitis C virus. Hepatology 59: 1250–1261. persistent murine norovirus infection in the absence of adaptive immunity. 44. Marcello, T., A. Grakoui, G. Barba-Spaeth, E. S. Machlin, S. V. Kotenko, Science 347: 269–273. M. R. MacDonald, and C. M. Rice. 2006. Interferons alpha and lambda inhibit 20. Baldridge, M. T., T. J. Nice, B. T. McCune, C. C. Yokoyama, A. Kambal, hepatitis C virus replication with distinct signal transduction and gene regulation M. Wheadon, M. S. Diamond, Y. Ivanova, M. Artyomov, and H. W. Virgin. 2015. kinetics. Gastroenterology 131: 1887–1898. Commensal microbes and interferon-l determine persistence of enteric murine 45. Bolen, C. R., S. Ding, M. D. Robek, and S. H. Kleinstein. 2014. Dynamic ex- norovirus infection. Science 347: 266–269. pression profiling of type I and type III interferon-stimulated hepatocytes reveals 21. Galani, I. E., V. Triantafyllia, E. E. Eleminiadou, O. Koltsida, A. Stavropoulos, a stable hierarchy of gene expression. Hepatology 59: 1262–1272. M. Manioudaki, D. Thanos, S. E. Doyle, S. V. Kotenko, K. Thanopoulou, and 46. Dill, M. T., F. H. Duong, J. E. Vogt, S. Bibert, P. Y. Bochud, L. Terracciano, E. Andreakos. 2017. Interferon-lambda mediates non-redundant front-line anti- A. Papassotiropoulos, V. Roth, and M. H. Heim. 2011. Interferon-induced gene viral protection against influenza virus infection without compromising host expression is a stronger predictor of treatment response than IL28B genotype in fitness. Immunity. 46: 875–890.e876. patients with hepatitis C. Gastroenterology 140: 1021–1031. 22. Kotenko, S. V., G. Gallagher, V. V. Baurin, A. Lewis-Antes, M. Shen, 47. Rauch, A., Z. Kutalik, P. Descombes, T. Cai, J. Di Iulio, T. Mueller, M. Bochud, N. K. Shah, J. A. Langer, F. Sheikh, H. Dickensheets, and R. P. Donnelly. 2003. M. Battegay, E. Bernasconi, J. Borovicka, et al. 2010. Genetic variation in IL28B IFN-lambdas mediate antiviral protection through a distinct class II cytokine is associated with chronic hepatitis C and treatment failure: a genome-wide receptor complex. Nat. Immunol. 4: 69–77. association study. Gastroenterology 138: 1338–1345, 1345.e1–7. 23. Sheppard, P., W. Kindsvogel, W. Xu, K. Henderson, S. Schlutsmeyer, 48. McGilvray, I., J. J. Feld, L. Chen, V. Pattullo, M. Guindi, S. Fischer, I. Borozan, T. E. Whitmore, R. Kuestner, U. Garrigues, C. Birks, J. Roraback, et al. 2003. G. Xie, N. Selzner, E. J. Heathcote, and K. Siminovitch. 2012. Hepatic cell-type IL-28, IL-29 and their class II cytokine receptor IL-28R. Nat. Immunol. 4: 63–68. specific gene expression better predicts HCV treatment outcome than IL28B 24. Lauber, C., G. Vieyres, E. Terczynska-Dyla, R. Anggakusuma, R. Dijkman, genotype. Gastroenterology 142: 1122–1131.e1121. H. H. Gad, H. Akhtar, R. Geffers, F. W. Vondran, V. Thiel, et al. 2015. Tran- 49. Fan, W., S. Xie, X. Zhao, N. Li, C. Chang, L. Li, G. Yu, X. Chi, Y. Pan, J. Niu, scriptome analysis reveals a classical interferon signature induced by IFNl4in et al. 2016. IFN-lambda4 desensitizes the response to IFN-alpha treatment in human primary cells. Genes Immun. 16: 414–421. chronic hepatitis C through long-term induction of USP18. J. Gen. Virol. Downloaded from 25.Onabajo,O.O.,P.Porter-Gill,A.Paquin,N.Rao,L.Liu,W.Tang,N.Brand, 50. François-Newton, V., G. Magno de Freitas Almeida, B. Payelle-Brogard, and L. Prokunina-Olsson. 2015. Expression of interferon lambda 4 is associ- D. Monneron, L. Pichard-Garcia, J. Piehler, S. Pellegrini, and G. Uze´. 2011. ated with reduced proliferation and increased cell death in human hepatic cells. USP18-based negative feedback control is induced by type I and type III in- J. Interferon Cytokine Res. 35: 888–900. terferons and specifically inactivates interferon a response. PLoS One 6: e22200. 26. Hamming, O. J., E. Terczynska-Dyla, G. Vieyres, R. Dijkman, S. E. Jørgensen, 51. Thomas, E., V. D. Gonzalez, Q. Li, A. A. Modi, W. Chen, M. Noureddin, H. Akhtar, P. Siupka, T. Pietschmann, V. Thiel, and R. Hartmann. 2013. Inter- Y. Rotman, and T. J. Liang. 2012. HCV infection induces a unique hepatic innate feron lambda 4 signals via the IFNl receptor to regulate antiviral activity against immune response associated with robust production of type III interferons.

HCV and coronaviruses. EMBO J. 32: 3055–3065. Gastroenterology 142: 978–988. http://www.jimmunol.org/ 27. Ungureanu, D., P. Saharinen, I. Junttila, D. J. Hilton, and O. Silvennoinen. 2002. 52. Marukian, S., L. Andrus, T. P. Sheahan, C. T. Jones, E. D. Charles, A. Ploss, Regulation of Jak2 through the ubiquitin-proteasome pathway involves phos- C. M. Rice, and L. B. Dustin. 2011. Hepatitis C virus induces interferon-l and phorylation of Jak2 on Y1007 and interaction with SOCS-1. Mol. Cell. Biol. 22: interferon-stimulated genes in primary liver cultures. Hepatology 54: 1913– 3316–3326. 1923. 28. Hong, M., J. Schwerk, C. Lim, A. Kell, A. Jarret, J. Pangallo, Y. M. Loo, S. Liu, 53. Bigger, C. B., K. M. Brasky, and R. E. Lanford. 2001. DNA microarray analysis C. H. Hagedorn, M. Gale, Jr., and R. Savan. 2016. Interferon lambda 4 expression of chimpanzee liver during acute resolving hepatitis C virus infection. J. Virol. is suppressed by the host during viral infection. J. Exp. Med. 213: 2539–2552. 75: 7059–7066. 29. Lu, Y. F., D. B. Goldstein, T. J. Urban, and S. S. Bradrick. 2015. Interferon-l4is 54. Lazear, H. M., T. J. Nice, and M. S. Diamond. 2015. Interferon-l: immune a cell-autonomous type III interferon associated with pre-treatment hepatitis C functions at barrier surfaces and beyond. Immunity 43: 15–28. virus burden. Virology 476: 334–340. 55. Wack, A., E. Terczynska-Dyla, and R. Hartmann. 2015. Guarding the frontiers: 30. Dellgren, C., H. H. Gad, O. J. Hamming, J. Melchjorsen, and R. Hartmann. 2009. the biology of type III interferons. Nat. Immunol. 16: 802–809. ´ Human interferon-lambda3 is a potent member of the type III interferon family. 56. Costa, V. V., C. T. Fagundes, D. F. Valadaõ, T. V. Avila, D. Cisalpino, by guest on September 29, 2021 Genes Immun. 10: 125–131. R. F. Rocha, L. S. Ribeiro, F. R. Ascençaõ, L. M. Kangussu, M. Q. Celso, Jr., 31. Croker, B. A., H. Kiu, and S. E. Nicholson. 2008. SOCS regulation of the JAK/ et al. 2014. Subversion of early innate antiviral responses during antibody- STAT signalling pathway. Semin. Cell Dev. Biol. 19: 414–422. dependent enhancement of Dengue virus infection induces severe disease in 32. Piganis, R. A., N. A. De Weerd, J. A. Gould, C. W. Schindler, A. Mansell, immunocompetent mice. Med. Microbiol. Immunol. 203: 231–250. S. E. Nicholson, and P. J. Hertzog. 2011. Suppressor of cytokine signaling 57. Ferraris, P., P. K. Chandra, R. Panigrahi, F. Aboulnasr, S. Chava, R. Kurt, (SOCS) 1 inhibits type I interferon (IFN) signaling via the interferon alpha re- J. M. Pawlotsky, L. Wilkens, P. Osterlund, R. Hartmann, et al. 2016. Cellular ceptor (IFNAR1)-associated tyrosine kinase Tyk2. J. Biol. Chem. 286: 33811– mechanism for impaired hepatitis C virus clearance by interferon associated with 33818. IFNL3 gene polymorphisms relates to intrahepatic interferon-lambda expression. 33. Song, M. M., and K. Shuai. 1998. The suppressor of cytokine signaling (SOCS)1 Am. J. Pathol. 186: 938–951. and SOCS3 but not SOCS2 proteins inhibit interferon-mediated antiviral and 58. Hertzog, P. J. 2012. Overview. Type I interferons as primers, activators and in- antiproliferative activities. J. Biol. Chem. 273: 35056–35062. hibitors of innate and adaptive immune responses. Immunol. Cell Biol. 90: 471– 34. Yoshikawa, H., K. Matsubara, G. S. Qian, P. Jackson, J. D. Groopman, 473. J. E. Manning, C. C. Harris, and J. G. Herman. 2001. SOCS-1, a negative regulator of 59. Davidson, S., T. M. McCabe, S. Crotta, H. H. Gad, E. M. Hessel, S. Beinke, the JAK/STAT pathway, is silenced by methylation in human hepatocellular carci- R. Hartmann, and A. Wack. 2016. IFNl is a potent anti-influenza therapeutic noma and shows growth-suppression activity. Nat. Genet. 28: 29–35. without the inflammatory side effects of IFNa treatment. EMBO Mol. Med. 8: 35. Yoshimura, A., T. Naka, and M. Kubo. 2007. SOCS proteins, cytokine signalling 1099–1112. and immune regulation. Nat. Rev. Immunol. 7: 454–465. 60. Chi, B., H. L. Dickensheets, K. M. Spann, M. A. Alston, C. Luongo, 36. Miyoshi, H., H. Fujie, Y. Shintani, T. Tsutsumi, S. Shinzawa, M. Makuuchi, L. Dumoutier, J. Huang, J. C. Renauld, S. V. Kotenko, M. Roederer, et al. 2006. N. Kokudo, Y. Matsuura, T. Suzuki, T. Miyamura, et al. 2005. Hepatitis C virus Alpha and lambda interferon together mediate suppression of CD4 T cells in- core protein exerts an inhibitory effect on suppressor of cytokine signaling duced by respiratory syncytial virus. J. Virol. 80: 5032–5040. (SOCS)-1 gene expression. J. Hepatol. 43: 757–763. 61. Hernańdez, P. P., T. Mahlakoiv, I. Yang, V. Schwierzeck, N. Nguyen, F. Guendel, 37. Randall, G., L. Chen, M. Panis, A. K. Fischer, B. D. Lindenbach, J. Sun, K. Gronke, B. Ryffel, C. Hoelscher, L. Dumoutier, et al. 2015. Interferon-l and J. Heathcote, C. M. Rice, A. M. Edwards, and I. D. McGilvray. 2006. Silencing interleukin 22 act synergistically for the induction of interferon-stimulated genes of USP18 potentiates the antiviral activity of interferon against hepatitis C virus and control of rotavirus infection. Nat. Immunol. 16: 698–707. infection. Gastroenterology 131: 1584–1591. 62. Bayer, A., N. J. Lennemann, Y. Ouyang, J. C. Bramley, S. Morosky, 38. Shao, R. X., L. Zhang, Z. Hong, K. Goto, D. Cheng, W. C. Chen, N. Jilg, E. T. Marques, Jr., S. Cherry, Y. Sadovsky, and C. B. Coyne. 2016. Type III K. Kumthip, D. N. Fusco, L. F. Peng, and R. T. Chung. 2013. SOCS1 abrogates interferons produced by human placental trophoblasts confer protection against IFN’s antiviral effect on hepatitis C virus replication. Antiviral Res. 97: 101–107. Zika virus infection. Cell Host Microbe 19: 705–712. 39. Xu, G., F. Yang, C. L. Ding, J. Wang, P. Zhao, W. Wang, and H. Ren. 2014. MiR- 63. Lazear, H. M., B. P. Daniels, A. K. Pinto, A. C. Huang, S. C. Vick, S. E. Doyle, s anti-HCV effect by downregulating SOCS1 and SOCS3. M. Gale, Jr., R. S. Klein, and M. S. Diamond. 2015. Interferon-l restricts West׳accentuates IFN 221 Virology 462–463: 343–350. Nile virus neuroinvasion by tightening the blood-brain barrier. Sci. Transl. Med. 40. Palmer, I., and P. T. Wingfield. 2004. Preparation and extraction of insoluble 7: 284ra59. (inclusion-body) proteins from Escherichia coli. Curr. Protoc. Protein Sci. 64. Palma-Ocampo, H. K., J. C. Flores-Alonso, V. Vallejo-Ruiz, J. Reyes-Leyva, Chapter 6: Unit 6.3. L. Flores-Mendoza, I. Herrera-Camacho, N. H. Rosas-Murrieta, and G. Santos- 41. Paquin, A., O. O. Onabajo, W. Tang, and L. Prokunina-Olsson. 2016. Compar- Lo´pez. 2015. Interferon lambda inhibits dengue virus replication in epithelial ative functional analysis of 12 mammalian IFN-lambda4 orthologs. J. Interferon cells. Virol. J. 12: 150. Cytokine Res.36: 30–36. 65. Bierne, H., L. Travier, T. Mahlako˜iv, L. Tailleux, A. Subtil, A. Lebreton, 42. Roisman, L. C., D. A. Jaitin, D. P. Baker, and G. Schreiber. 2005. Mutational A. Paliwal, B. Gicquel, P. Staeheli, M. Lecuit, and P. Cossart. 2012. Activation of analysis of the IFNAR1 binding site on IFNalpha2 reveals the architecture of a type III interferon genes by pathogenic bacteria in infected epithelial cells and weak ligand-receptor binding-site. J. Mol. Biol. 353: 271–281. mouse placenta. PLoS One 7: e39080.