IFN-Mediated Antiviral Response STAT3 Negatively Regulates Type I

STAT3 Negatively Regulates Type I IFN-Mediated Antiviral Response Wei-Bei Wang, David E. Levy and Chien-Kuo Lee This information is current as J Immunol published online 1 August 2011 of October 5, 2021. http://www.jimmunol.org/content/early/2011/07/31/jimmun ol.1004128

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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 © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published August 1, 2011, doi:10.4049/jimmunol.1004128 The Journal of Immunology

STAT3 Negatively Regulates Type I IFN-Mediated Antiviral Response

Wei-Bei Wang,* David E. Levy,† and Chien-Kuo Lee*

Type I IFNs are crucial cytokines of innate immunity for combating viral infections. Signaling through type I IFN receptors triggers the activation of STAT proteins, including STAT1, STAT2, and STAT3. Although an essential role of STAT1 and STAT2 for type I IFN-induced antiviral response has been well established by studies of gene-targeted mice and human mutations, the role of STAT3 for this response remains unclear. Using gain-of-function and loss-of-function approaches, we demonstrated that STAT3 negatively regulates type I IFN-mediated response. STAT3 knockdown or knockout cells displayed enhanced gene expression and antiviral activity in response to IFN-a/b. Restoration of STAT3 to STAT3KO cells resulted in attenuation of the response. Upon viral infection, increased type I IFN production in STAT3KO cells resulted in enhanced STAT activation and ISG expression. One mechanism for the enhanced IFN production and response in the absence of STAT3 might operate through an MDA5-dependent

manner. STAT3 also appeared to suppress IFN response directly in a manner dependent on its N-terminal domain and indepen- Downloaded from dent of its function as a transcriptional factor. Taken together, these results deﬁne STAT3 as a negative regulator of type I IFN response and provide a therapeutic target for viral infections. The Journal of Immunology, 2011, 187: 000–000.

ype I IFNs, composed of an IFN-b and several IFN-a STAT3 is a signaling mediator of IL-6 and IL-10 family mem- species, are critical cytokines of innate immunity for trig- bers and other cytokines such as leptin and G-CSF (6, 8). Struc- T gering antiviral response. Antiviral functions of type I turally, STAT3, similar to other STAT family members, consists of http://www.jimmunol.org/ IFNs are mediated by induction of IFN-stimulated genes (ISGs), an N-terminal domain (NTD) for dimerization and tetrameriza- including protein kinase R (PKR), 29,59-oligoadenylate synthetase tion, a coiled-coil domain (CCD) for protein–protein interaction, a (OAS), RNase L, inducible NO synthase (iNOS), and IFN regu- DNA-binding domain (DBD) for specific binding to IFN-g–acti- latory factors (IRFs), which interfere with virus replication or vated site element, a Src homology 2 domain for receptor re- trigger cell apoptosis to avoid viral spread (1). The signaling cruitment and STAT dimerization, and a transactivation domain pathways resulting in induction of the ISGs involve activation of at the C terminus (6). Conventional knockout of STAT3 results STAT family members, including STAT1, STAT2, and STAT3, by in embryonic lethality, underscoring a critical role of STAT3 in tyrosine and serine phosphorylation (2). Activated STAT1, STAT2, embryonic development (9). Conditional targeting of STAT3 in and IRF9 form the ISGF3 complex, binding to the IFN-stimulated distinct tissues or organs reveals versatile roles of STAT3 in vivo, by guest on October 5, 2021 response element (ISRE) in the promoters of ISGs (3). Activated including cell survival and/or apoptosis, migration, development, STAT1 and STAT3 form a homodimer, or heterodimer, and bind and differentiation (6). STAT3 also plays a key role in promoting to IFN-g–activated site. Whereas the homodimer of STAT1 or tumor formation (10). Constitutive activation of STAT3 is found STAT3 and the heterodimer of STAT1-STAT3 can bind to similar in tumors and immune cells in the tumor environment, serving as cognate sites in vitro, the in vivo binding sites for these dimers are a mediator for crosstalk between tumors and immunological en- most likely to be different because the downstream targets of vironment, leading to tumor-induced immunosuppression (11, 12). STAT1 and STAT3 and the phenotypes of STAT1- and STAT3- Targeted deletion of STAT1 or STAT2 gene in mice (5, 7, 13) deficient mice are quite distinct (4–7). or mutation-associated STAT1 deficiency in humans (14) reveals a pivotal role of either molecule in antiviral response. Although a limited number of groups have studied the role of STAT3 in type I IFN-mediated biological response, its function has remained *Graduate Institute of Immunology, National Taiwan University College of Medi- cine, Taipei 100, Taiwan; and †Department of Pathology, New York University controversial (15–18). Using gain-of-function and loss-of-function School of Medicine, New York, NY 10016 approaches, we demonstrated that STAT3 inhibits antiviral acti- Received for publication December 21, 2010. Accepted for publication June 21, vity of type I IFNs. Whereas knockdown or knockout of STAT3 2011. resulted in enhanced antiviral response, restoration of STAT3 This work was supported by Grant NSC 99-2320-B-002-010-MY3 from the National in STAT3KO mouse embryonic fibroblasts (MEFs) or hyperacti- Science Council, Taiwan. vation of STAT3 in wild-type (WT) MEFs attenuated the response. Address correspondence and reprint requests to Prof. Chien-Kuo Lee, No. 1, Section Overexpression of WT or mutant STAT3 lacking DNA-binding or 1, Jen-Ai Road, Room 513, Taipei 100, Taiwan. E-mail address: [email protected] transactivation ability suppressed IFN-driven reporter activity. The online version of this article contains supplemental material. Interestingly, STAT3 NTD is sufficient to confer the suppressive Abbreviations used in this article: BMM, bone marrow-derived macrophage; CCD, coiled-coil domain; CHX, cycloheximide; DBD, DNA-binding domain; EMCV, en- effect. Therefore, these results support a negative role of STAT3 in cephalomyocarditis virus; HA, hemagglutinin; iNOS, inducible NO synthase; IRF, type I IFN-mediated response. IFN regulatory factor; ISG, IFN-stimulated gene; ISRE, IFN-stimulated response element; MEF, mouse embryonic fibroblast; MOI, multiplicity of infection; NTD, N-terminal domain; OAS, 29,59-oligoadenylate synthetase; PKR, protein kinase R; Materials and Methods QPCR, quantitative PCR; shMDA5, short hairpin MDA5; shSTAT3, short hairpin Animals and cells STAT3; shRNA, short hairpin RNA; WT, wild-type. Generation and mating of MxCre-STAT3f/f mice and induction of STAT3 Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 deletion have been described previously (19, 20). These animals were

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1004128 2 STAT3 SUPPRESSES TYPE I IFN RESPONSE maintained and housed in specific germ-free conditions in the Animal Core In vitro antiviral assay and plaque formation assay Facility at the National Taiwan University College of Medicine. Proce- dures and use of these animals were reviewed and approved by the In- MEFs were pretreated with or without 2-fold serial dilution of IFN-a stitutional Animal Care and Use Committee at National Taiwan University starting from 1000 to 1.8 U/ml for 24 h. EMCV at a multiplicity of in- College of Medicine. WT, STAT1KO, and STAT3KO MEFs were gener- fection (MOI) of 0.1 was added in the cells using serum-free DMEM for ated, as described previously (21, 22). 45 min at 37˚C. Viral supernatant was then removed, and the cells were refreshed with complete medium. Sixteen to 18 h postinfection, the me- Abs, cytokines, and DNA construct dium was removed and cells were fixed with 10% formaldehyde solution for 20 min at room temperature. After fixation, cells were visualized with The sources of the cytokines are as follows: recombinant human IFNa-2a crystal violet. The excessive dye was then removed by immersing the plate (Roche), recombinant murine IFN-a (Merck), recombinant murine IFN-b in water. Each treatment was performed in duplicate. For plaque formation (Prospec). The sources of Abs are as follows: anti–b-actin (Chemicon), assay, different dilutions of supernatant from virus-infected cells (typically anti–a-tubulin (Sigma-Aldrich), anti–phospho-STAT1 (Y701) (Invitrogen), MEFs 16–18 h postinfection and BMMs 48 h postinfection) were used to anti-STAT1 (homemade), anti–phospho-STAT2 (Y689) (Millipore), anti- infect Vero cells in serum-free DMEM for 1 h, followed by overlaying 2% STAT2 (homemade), anti–phospho-STAT3 (Y705), anti-STAT3 (Cell FBS in DMEM containing 1% SeaPlaque agarose (Lonza) to immobilize Signaling Technology), anti-MDA5 (Axxora), anti-IFNAR1 Ab (eBio- the virus. After 24 h, cells were fixed and visualized with crystal violet, science), and anti-hemagglutinin (HA; 12CA5, homemade). Mouse IFN-a and the plaques were enumerated. ELISA kit was purchased from PBL. WT STAT3 and DBD mutant carrying 5 aa substitutions in the DBD were reported previously (23). Transfection and retroviral transduction of MEFs IRES-STAT3b was a gift of H. Yu (Department of Cancer Immunother- apeutics & Tumor Immunology, Beckman Research Institute of City of Transfection of vector encoding different STAT3 into MEFs was done using Hope) (24). HA-tagged full-length STAT3 1–770 or truncated mutants of Turbofect (Fermentas), according to manufacturer’s instruction. Retroviral STAT3, including 1–134, 1–317, and 318–770 aa, were gifts of J.-Y. Chen transduction of MEFs was conducted, as described (28). Briefly, a retro-

(Institute of Biomedical Sciences, Academia Sinica, Taiwan) (25). Flag- viral vector Pallino encoding WT STAT3 and GFP, respectively, was Downloaded from MDA5 was a gift of T. Fujita (Institute for Virus Research, Kyoto cotransfected with a helper plasmid into HEK293T cells for 2 d before University) (26). collecting the culture supernatant containing pseudotyped virus. MEFs were incubated with the viral supernatant in the presence of 8 mg/ml Quantitative RT-PCR polybrene and spun at 1100 3 g for 90 min at 30˚C. Two days after the infection, GFP-positive cells were further puriﬁed up to 90–95% using the Total RNA was prepared from MEFs or primary bone marrow-derived FACSAria cell sorting system (BD Biosciences). macrophages (BMMs) using a TRIzol reagent (Invitrogen). A total of 1–3

mg RNA was subjected to reverse-transcriptase reaction, and cDNA was http://www.jimmunol.org/ then subjected to quantitative PCR (QPCR) by iCycler IQ (Bio-Rad) using Reporter assay the following primer sets. Each sample was performed in duplicate. Luciferase assays for pISRE-luc (Stratagene) containing 53 ISRE from OAS, forward 59-GCCATTGCACGCTCGCCTACTAC-39, reverse 59-CT- Ifi54 gene were performed in duplicate by transfection into HEK293-TLR3 CCTGCCATCCGGGTTTTTCA-39; PKR, forward 59-TGCGCAGACA- cells (a gift of K. Fitzgerald, University of Massachusetts) and STAT3KO ATGTATGGTAC-39, reverse 59-ATGTGACAACGCTAGAGGATG-39; MEFs in conjunction with different STAT3-expressing constructs using iNOS, forward 59-AGCGCTACAACATCCTGGAG-39, reverse 59-GAC- jetPEI (Polyplus-transfection) and Turbofect (Fermantas), respectively, CAGCCAAATCCAGTCTG-39; IRF1, forward 59-ATAACTCCAGCACT- according to manufacturer’s instruction. After transfection for 24–48 h, the GTCACCGTG-39, reverse 59-ATCCTCGTCTGTTGCGGCTT-39; IRF7, cells were treated with or without various doses of human IFNa-2a forward 59-AGCAAGACCGTGTTTACGAC-39, reverse 59-AGTGCTGA- (Roche) or mouse IFN-a (Merck) for 8 and 3 h, respectively, followed by AGTCGAAGATGG-39; IFN-a, forward 59-CTTCCACAGGAT ACT- measuring luciferase activities. Luciferase activities were measured ac- GTGTACCT-39, reverse 59-TTCTGCTCTGACCACCCTCCC-39; IFN-b, cording to manufacturer’s instructions (Promega) using Orion II micro- by guest on October 5, 2021 forward 59-CCTGTGTGATGCAGGAACC-39, reverse 59-TCACCTCC- plate luminometer (Berthold). CAGGCACAGA-39; MDA5, forward 59-GAGCCAGAGCTGATGARA- GC-39, reverse 59-TCTTATGWGCATACTCCTCTGG-39; RIG-I, forward Lentivirus-mediated knockdown of STAT3 and MDA5 59-GCATATTGACTGGACGTGGCA-39, reverse 59-CAGTCATGGCTG- CAGTTCTGTC-39; TLR3, forward 59-TAACCACGCACTCTGTTTGC- The lentiviruses carrying short hairpin RNA (shRNA) targeting STAT3, 39, reverse 59-ACAGTATTGCGGATCCAAG-39 ; and b-actin, forward 59- MDA5, and luciferase were obtained from the National RNAi Core Fa- GTGGGGCGCCCCAGGCA CCA-39, reverse 59-CTCCTTATTGTCAC- cility located at the Institute of Molecular Biology/Genomic Research GCACGATTTC-39. To detect encephalomyocarditis virus (EMCV) RNA Center, Academia Sinica, Taiwan. The core facility is part of The RNAi in infected cells, total RNA was first subjected to reverse transcription Consortium. The sequence targeting mouse STAT3 is 59-CCTGAGTTG- using a random hexamer (Invitrogen) as a primer. The cDNA was then AATTATCAGCTT-39 (TRCN0000071456), MDA5 is 59-CCACAGAATC- subjected to QPCR using the following primers to amplify the 2A-2B AGACACAAGTT-39 (TRCN0000103646), and a control construct targeting region of EMCV: forward 59-AATGCCCACTACGCTGGT-39, reverse luciferase is 59-CAAATCACAGAATCGTCGTAT-39 (TRCN0000072246). 59-GTCGTTCGGCAGTAGGGT-39 (27). Relative mRNA was calculated Viral supernatant was used to infect 1 3 105 MEF cells at a MOI of 2. by normalizing the values of indicated genes to that of b-actin. Lentivirus production and infection procedures were as described in the Web site (http://rnai.genmed.sinica.edu.tw/Protocols.asp). Following Microarray analysis infection, the cells were cultured in a selection medium containing 5 mg/ml puromycin for 5 d to enrich infected cells before experiments Total RNA from WT or STAT3KO MEFs stimulated with or without 1000 were performed. U/ml IFN-a for 2 h was obtained using the TRIzol reagent (Life Tech- nologies), followed by cleanup and DNase I treatment with QIAGEN RNeasy mini kit, according to the protocol provided by the kit. Quality Statistical analysis control was performed by Agilent Bioanalyser. The RNA was then sub- Unless otherwise indicated, all experiments were performed in triplicates. A jected to expressional microarray analysis using Illumina MouseWG-6 Student t test (two tailed) was performed for statistical analysis. *p , 0.05, v1.1 Expression BeadChip. The complete dataset was available at Na- **p , 0.01. tional Center for Biotechnology Information GEO accession GSE25044 (http://www.ncbi.nlm.nih.gov/gds/?term=GSE25044). Western blot Results STAT3 knockdown in WT MEFs results in enhanced type I IFN Total cell lysates were prepared by lysing cells in lysis buffer (300 mM response NaCl, 50 mM HEPES [pH 7.6], 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100, 10 mM NaPyrPO4, 20 mM NaF, 1 mM EGTA, 0.1 mM EDTA, 1 To study the role of STAT3 in type I IFN response, STAT3 was mM DTT, 1 mM PMSF, and 1 mM Na4VO3) at 4˚C for 15 min. Lysates knocked down in WT MEF using lentivirus carrying shRNA to were first clarified by centrifugation at 12,000 3 g for 20 min. Equal amounts of samples were resolved in 7–10% SDS-PAGE, followed by STAT3. shRNA to firefly luciferase was used as a control. As transferring to nitrocellulose or polyvinylidene difluoride membranes shown in Fig. 1A, following short hairpin STAT3 (shSTAT3) (Millipore) and blotting with indicated Abs. treatment, STAT3 protein levels were reduced to ∼40% to that of The Journal of Immunology 3

FIGURE 1. STAT3 knockdown enhances type I IFN response. A, WT MEFs infected with lentivirus carrying shLuc or shSTAT3 were stimulated with or without 1000 U/ml IFN-a for 15 min. Total cell lysates were subjected to immunoblotting using Abs to pSTAT1, pSTAT2, pSTAT3, STAT3, and tubulin. Total RNA from the cells received the same treatments, as described in A, for different times was subjected to RT- QPCR using primers for PKR (B), OAS (C), IRF1 (D), and b-actin. Relative mRNA was calculated by normalizing the values of the indicated genes to that of b-actin. E, WT MEFs infected with lentivirus-shLuc (upper) or lentivirus-shSTAT3 (lower) were pretreated with or without 2-fold serial dilution of IFN-a starting from 125 U/ml. Following the treatment, the cells were infected with or without EMCV at a MOI of 0.1 for 16 h, followed by visualizing the viable cells with crystal violet. *p , 0.05, **p , 0.01. Downloaded from shLuc control. Moreover, basal and IFN-induced STAT3 phos- STAT3KO MEFs and macrophages display enhanced type I phorylation at Y705 was also greatly reduced as compared with IFN response that of control, whereas the levels of pSTAT1 (pY701) and We next conﬁrmed the suppressive effect of STAT3 using MEFs pSTAT2 (pY689) remained comparable. We next examined the lacking STAT3. As shown in Fig. 2A and 2B, following IFN-a http://www.jimmunol.org/ effect of STAT3 knockdown on expression of ISGs. As shown in treatment, STAT3KO MEFs expressed higher basal and induced Fig. 1B–D, following stimulation, the expression of PKR, OAS, levels of PKR and OAS than did WT MEFs. A similar phenom- and IRF1 was increased in cells treated with shSTAT3 as com- enon was also observed in other IFN-a downstream genes, such pared with that of shLuc control. We next investigated whether as RNase L, IRF7, IRF1, and IP-10 (data not shown). We next IFN-mediated antiviral activity was affected or not by the treat- employed DNA microarray to conduct a genome-wide expression ment. As shown in Fig. 1E, antiviral response in STAT3 knock- proﬁling for WT and STAT3KO MEFs in response to IFN-a. down cells was greater than that of control cells, revealed by the Expression of a variety of ISGs was also increased in STAT3KO increased resistance of lytic activity of EMCV following infection. MEFs as compared with WT MEFs (Supplemental Table 1; GEO Together, these results suggest that STAT3 may have a negative GSE25044, http://www.ncbi.nlm.nih.gov/gds/?term=GSE25044), by guest on October 5, 2021 effect on IFN response. suggesting that the hyperresponsiveness of IFN-a in STAT3KO

FIGURE 2. STAT3 knockout enhances IFN-a response. Total RNA prepared from WT (filled) or STAT3KO (open) MEFs that were stimulated with or without 1000 U/ml IFN-a for 2 h was subjected to RT-QPCR using primers for PKR (A), OAS (B), and b-actin. Total RNA prepared from BMMs of WT (filled) or STAT3KO (open) mice that were stimulated with or without 100 U/ml IFN-a for 4 h was subjected to RT-QPCR using primers for PKR (C) and iNOS (D). Relative mRNA was calculated by normalizing the values of the indicated genes to that of b-actin. E, WT (upper), STAT1KO (middle), and STAT3KO (lower) MEFs pretreated overnight with 2-fold serial dilution of IFN-a from 1000 U/ml were infected with EMCV at a MOI of 0.1 for 18 h, followed by visualizing live cells with crystal violet. WT (filled) and STAT3KO (open) MEFs pretreated with the indicated doses of IFN-a were infected with EMCV (F)or vesicular stomatitis virus (G) at a MOI of 0.1 for 12 and 18 h, respectively, followed by measuring viral titers in the culture supernatant using plaque formation assay. *p , 0.05, **p , 0.01. 4 STAT3 SUPPRESSES TYPE I IFN RESPONSE cells was a general phenomenon. We next examined IFN response in primary BMMs. As shown in Fig. 2C and 2D, STAT3KO BMMs also showed enhanced expression of PKR and iNOS following IFN stimulation. We next examined STAT activation in MEFs following IFN treatment. As shown in Supplemental Fig. 1, whereas activation of STAT1 and STAT2 was marginally increased in STAT3KO MEFs as opposed to WT MEFs, STAT1 activation was, however, comparable between STAT3KO and WT BMMs. Due to STAT3KO MEFs showing a greater IFN response, we next investigated whether IFN-mediated antiviral activity was also enhanced. WT and STAT3KO MEFs were pretreated with or without 2-fold serial dilution of IFN-a, followed by infection with EMCV. Additionally, we also included STAT1KO MEFs as a control. As shown in Fig. 2E, increased doses of IFN-a reduced the lytic activity of EMCV in WT MEFs, indicating that IFN-a conferred antiviral response in a dose-dependent manner. How- ever, the activity was dramatically abolished in STAT1KO MEFs, confirming a pivotal role of STAT1 for IFN-mediated innate im- FIGURE 3. Restoration of STAT3 to STAT3KO MEFs reduces type I munity to viral infection (5, 7). By contrast, STAT3KO MEFs dis- IFN response. A, Empty vector (EV)- or STAT3-restored STAT3KO MEFs Downloaded from played an enhanced antiviral response, as revealed by increased were treated with 20 ng/ml IFN-b for 30 min. Total cell lysates were then resistance to EMCV-induced lytic activity. We next investigated subjected to immunoblotting using Abs to pSTAT1, STAT1, pSTAT2, whether the increased survival of STAT3KO MEFs was accom- STAT2, pSTAT3, STAT3, and tubulin. Total RNA prepared from EV-re- plished by reduced viral titers. Indeed, STAT3KO MEFs reduced stored (open) or STAT3-restored (filled) STAT3KO MEFs that were treated EMCV viral titers before and after IFN-a treatment (Fig. 2F). with or without 1000 U/ml IFN-a for 2 h was subjected to RT-QPCR using This result was consistent with the enhanced antiviral state in primers for PKR (B), OAS (C), and b-actin. Relative mRNA was calcu-

lated by normalizing the values of the indicated genes to that of b-actin. D, http://www.jimmunol.org/ STAT3KO MEFs. In addition to EMCV, STAT3KO MEFs also EV- or STAT3-restored STAT3KO MEFs pretreated overnight with 2-fold exerted a greater antiviral activity against vesicular stomatitis vi- serial dilution of IFN-a from 1000 U/ml were infected with EMCV at rus infection than did their WT counterparts (Fig. 2G) in response a MOI of 1 for 24 h, followed by visualizing live cells with crystal violet. to IFN-a. Together, these results suggest that STAT3 suppresses E, Supernatants of EV (open)- or STAT3-restored (filled) STAT3KO MEFs type I IFN-mediated antiviral response. that were pretreated with the indicated doses of IFN-a, followed by EMCV infection for 24 h, were subjected to plaque formation assay to determine Restoration of STAT3 reverses the otherwise enhanced type I the viral titers. **p , 0.01. IFN response in STAT3KO MEFs To examine whether the enhanced IFN response in STAT3KO by guest on October 5, 2021 cells was intrinsic to the loss of STAT3, we restored STAT3a compared with WT MEFs (Fig. 4B). We next investigated whether into STAT3KO MEFs by retroviral transduction. The levels of the enhanced STAT activation in STAT3KO MEFs was due to restored STAT3 were slightly lower than that of WT cells (data not increased production of type I IFNs during viral infection. As shown). However, whereas re-expression of STAT3 did not shown in Fig. 4C, STAT3KO MEFs expressed higher levels of change the levels of IFN-b–activated pSTAT1 or pSTAT2 (Fig. IFN-b mRNA than did WT MEFs after the infection. A similar 3A), it attenuated IFN-a–stimulated expression of PKR and OAS phenotype was also observed in primary BMMs. The levels of (Fig. 3B,3C). pSTAT1 (Fig. 4D) and the expression of ISGs, including PKR We next determined the effect of gain of function of STAT3 in (Fig. 4E), OAS, iNOS, IRF1, IRF7, and TLR3 (Supplemental Fig. antiviral response. Compared with that restored with the empty 2A–E), were also enhanced in STAT3KO BMMs after the in- vector, STAT3-restored cells showed increased lytic activity and fection. Additionally, STAT3KO BMMs also expressed signifi- viral titers (Fig. 3D,3E). Similar results were also observed in WT cantly higher levels of IFN-b mRNA (Fig. 4F) and IFN-a protein MEFs when STAT3 was hyperactivated by cotreatment of IFN-a (Fig. 4G) than did WT BMMs upon infection. and IL-6 (data not shown). Taken together, these results suggest We next investigated whether the increased production of type that enhanced gene expression and antiviral activity of type I IFN I IFNs contributed to the enhanced activation of STAT1 and in STAT3KO MEFs are intrinsic to the loss of STAT3. STAT2 in STAT3KO MEFs after the infection. STAT3KO MEFs were first pretreated with a neutralizing Ab to IFN-a/b receptor Increased expression of type I IFNs in STAT3KO cells during 1 (IFNAR1), followed by infection with EMCV. As shown in viral infection Supplemental Fig. 2H, the anti-IFNAR1 Ab attenuated pSTAT1 During viral infection, the binding of viral components to pattern and pSTAT2 levels in STAT3KO MEFs, suggesting that virus- recognition receptors triggers the production of type I IFNs, which induced type I IFNs facilitate the enhancement of IFN-a signaling in turn activates STAT proteins. Therefore, we next examined the in STAT3KO cells. role of STAT3 in viral infection in the absence of exogenous IFN-a. We first assessed STAT activation as an indicator of IFN-a MDA5 knockdown impedes enhanced antiviral response in signaling. As shown in Fig. 4A, at MOI of 0.1, EMCV infection STAT3KO cells failed to induce detectable tyrosine phosphorylation of STAT1 RIG-I and MDA5, two RLR family members of cytosolic sensors and STAT2 in WT MEFs, which might be due to an insufficient of viral RNA, are themselves IFN inducible (29, 30). We reasoned amount of type I IFN production. By contrast, the levels of that STAT3 might indirectly regulate antiviral response through pSTAT1 and pSTAT2 were increased in STAT3KO MEFs fol- modulating the expression of these two molecules. As shown in lowing the infection for 6 h. Concomitantly, infection-induced Fig. 5, STAT3KO MEFs (Fig. 5A,5B) and BMMs (Supplemental PKR expression was also augmented in STAT3KO MEFs as Fig. 2F,2G) expressed higher levels of MDA5 and RIG-I than did The Journal of Immunology 5

FIGURE 4. Enhanced expression of type I IFNs in STAT3KO cells after viral infection. WT or STAT3KO MEFs (A) or BMMs (D) were infected with EMCV at a MOI of 0.1 and 10, respectively, for the indicated times. Total cell lysates were subjected to immunoblotting using Abs to pSTAT1, STAT1, pSTAT2, STAT2, pSTAT3, STAT3, and b-actin. Total RNA prepared from EMCV- infected MEFs (B, C) or BMMs (E, F) for the indicated times was subjected to RT-QPCR using primers for PKR (B, E), IFN-b (C, F), and b-actin. Relative mRNA was calculated by normalizing the values of the indicated genes to that of b-actin. G, Production of IFN-a by WT (ﬁlled) or STAT3KO (open) BMMs after viral infection for 48 h was measured by ELISA. *p , 0.05, **p , 0.01. Downloaded from

WT controls following EMCV infection. Although MDA5 has EMCV (Fig. 5I) in STAT3KO MEFs. These results suggest that been shown to play a critical role in clearance of EMCV infection increased MDA5 expression contributes to enhanced type I IFN (31), its effector function in the absence of STAT3 remained to be response in STAT3KO MEFs. verified. Therefore, we first overexpressed MDA5 in STAT3KO STAT3 suppresses type I IFN response independent of its MEFs. As shown in Fig. 5C–E, increased MDA5 accentuated DNA-binding and transactivation ability http://www.jimmunol.org/ infection-induced IFN-a expression and antiviral activity. We next To investigate whether the suppressive effect of STAT3 on IFN-a confirmed the role of MDA5 in STAT3KO MEFs by a knockdown response requires de novo protein synthesis, we pretreated cells approach using lentivirus carrying shRNA to MDA5. To rule out with cycloheximide (CHX). As shown in Fig. 6, CHX pre- off-target effects, shRNA to luciferase was used as a control. As treatment could not block the enhanced expression of PKR and shown in Fig. 5F, whereas the expression of MDA5 was further OAS in STAT3KO MEFs (Fig. 6A,6B) and elevated expression of induced by IFN-a treatment, short hairpin MDA5 (shMDA5), but PKR and iNOS in STAT3KO BMMs (Fig. 6C,6D) in response not shLuc, greatly reduced the protein levels of MDA5. In- to IFN-a. Although these results suggest that the suppressive ef- terestingly, shMDA5 treatment resulted in decreased IFN-a ex- fect of STAT3 is independent of its downstream effector mole- by guest on October 5, 2021 pression (Fig. 5G), increased EMCV infection, as revealed by cules, we still cannot rule out the possibility of involvement of elevated viral RNA (Fig. 5H), and increased lytic activity of CHX-sensitive inhibitors of IFN-a response. Therefore, we next

FIGURE 5. Gain or loss of function of MDA5 in STAT3KO MEFs during viral infection. Total RNA from WT (filled) or STAT3KO (open) MEFs that were infected with EMCV at a MOI of 0.1 for the indicated times was subjected to RT-QPCR using primers for MDA5 (A), RIG-I (B), and b-actin. Relative mRNA was calculated by normalizing the values of the indicated genes to that of b-actin. C, Empty vector (EV) or vector encoding flag-MDA5 was transfected into STAT3KO MEFs for 24 h, followed by immunoblotting using Abs to flag and tubulin. EV- or MDA5-transfected MEFs were subjected to EMCV infection at a MOI of 0.1 for 3 and 6 h, followed by RT-QPCR using primers for IFN-a (D) and EMCV 2A-2B (E), respectively. F, STAT3KO MEFs infected with lentivirus carrying shLuc or shMDA5 were stimulated with 1000 U/ml IFN-a for the indicated times, followed by immunoblotting using Abs to MDA5 and tubulin. shLuc- or shMDA5-treated STAT3KO MEFs were infected with EMCV at a MOI of 0.1 for 9 and 4 h, followed by RT-QPCR using primers for IFN-a (G) and EMCV 2A-2B (H), respectively. Relative mRNA was calculated by normalizing the values of IFN-a to that of b-actin. I, shLuc- or shMDA5-treated STAT3KO MEFs were infected with or without indicated doses of EMCV for 24 h, followed by visualizing live cells with crystal violet. *p , 0.05, **p , 0.01. 6 STAT3 SUPPRESSES TYPE I IFN RESPONSE Downloaded from

FIGURE 6. STAT3 suppresses type I IFN response independent of its DNA-binding and transactivation activity. WT (filled) or STAT3KO (open) MEFs (A, B) or BMMs (C, D) were pretreated with or without 20 mg/ml CHX, followed by 1000 U/ml IFN-a stimulation for the indicated times. Total RNA of the treated cells was then subjected to RT-QPCR using primers for PKR (A, C), OAS (B), iNOS (D), and b-actin. Relative mRNA was calculated by normalizing the values of the indicated genes to that of b-actin. E, Different doses of vector encoding WT STAT3 was cotransfected with pISRE-luc and pCMV-RL into HEK293-TLR3, followed by treating the cells with 1000 IU/ml recombinant human IFNa-2a or without (medium) for 8 h before measuring luciferase activity. F, Empty vector (EV) or vector encoding WT STAT3, DBD STAT3, or STAT3b was cotransfected with pISRE-luc and pCMV-RL into http://www.jimmunol.org/ HEK293-TLR3, followed by treating the cells with or without IFN-a. G, EVor vector encoding STAT3 full-length 1–770 aa, 1–134 aa, 1–317 aa, and 318– 770 aa was cotransfected with pISRE-luc and pCMV-RL into HEK293-TLR3 cells for 24 h, followed by stimulation with or without IFN-a. Relative luciferase activity was calculated by normalizing firefly luciferase activity to that of Renilla luciferase. *p , 0.05, **p , 0.01. performed reporter assays to examine whether STAT3 was able to NTD is sufficient to confer the suppressive activity. These results suppress IFN-driven transactivation activity. Expression of WT are also consistent with the dispensable role of DNA-binding and STAT3 suppressed IFN-driven ISRE reporter activity in a dose- transactivation ability of STAT3. Taken together, these results dependent manner (Fig. 6E). Interestingly, both DBD mutant of suggest that STAT3 may directly suppress type I IFN response,

STAT3 and STAT3b were also capable of suppressing the reporter and the effect is independent of its DNA-binding and transcrip- by guest on October 5, 2021 activity (Fig. 6F), suggesting that DNA-binding and transac- tional function. tivation ability of STAT3 were not required for the suppressive effect. To dissect further the functional domains of STAT3 re- NTD of STAT3 is sufﬁcient to suppress type I IFN response quired for the effect, different truncation mutants of STAT3 were To further conﬁrm the antagonistic effect of STAT3 1–134 aa, we used. As shown in Fig. 6G, 1–134 aa (NTD only) or 1–317 aa of restored HA-tagged WT or STAT3 1–134 aa into STAT3KO STAT3 (NTD and CCD) remained the inhibitory activity, whereas MEFs. As shown in Fig. 7A, the expression of WT or mutant the activity was almost abolished in cells expressing STAT3 318– STAT3 was detected by anti-HA Ab. STAT3 1–134 aa exerted 770 aa lacking NTD and CCD. These results suggest that STAT3 almost the same inhibitory activity as did WT STAT3 in reporter

FIGURE 7. STAT3 N-terminal 1–134 aa is sufficient to suppress type I IFN response. A, Empty vector (EV), vector encoding HA-tagged WTor STAT31–134 aa was transfected into STAT3KO MEFs for 48 h. Total cell lysates were then subjected to immunoblotting using anti-HA and anti-tubulin Abs. B,EV,WT,orSTAT31– 134 aa was cotransfected with pISRE-luc and a vector carrying GFP into STAT3KO MEFs for 3 h, followed by treating the cells with IFN-a. Relative luciferase activity was calculated by normalizing firefly luciferase activity to that of GFP-positive percentage. STAT3KO MEFs transfected with EV, WT, or STAT3 1–134 aa were treated with or without IFN-a for the indicated times. Total RNA of the treated cells was subjected to RT-QPCR using primers for PKR (C), OAS (D), or b-actin. Relative mRNA was calculated by normalizing the values of the indicated genes to that of b-actin. E, STAT3KO MEFs transduced with EV or vector encoding STAT3 1–134 aa were treated with 2-fold serial dilution of IFN-a from 1000 U/ml for 24 h, followed by infection with EMCV at a MOI of 1 for 24 h. Live cells were visualized with crystal violet. *p , 0.05. The Journal of Immunology 7 assay (Fig. 7B) and IFN-stimulated expression of PKR (Fig. 7C) flammatory cytokines. This is due to the lack of suppressive effect and OAS (Fig. 7D). Moreover, restoration of STAT3 1–134 aa of IL-10 on cytokine production from macrophages and neu- also showed a compromised antiviral response as compared with trophils in the absence of STAT3 (38). The results of this study empty vector control (Fig. 7E). Together, these results suggest define a new negative role of STAT3 in IFN-mediated functions that NTD of STAT3 is sufficient to trigger negative effect on type I and provide another mechanism to fine-tune type I IFN response. IFN response. Additionally, they underscore a potential therapeutic target of STAT3 for viral infections.

Discussion Acknowledgments This work highlights a previously unappreciated role of STAT3 in We are grateful to Drs. Lih-Hwa Hwang, Yi-Ling Lin, John Kung, Jeou- type I IFN response. Using loss- and gain-of-function approaches, Yuan Chen, Kate Fitzgerald, Hua Yu, and Takashi Fujita for providing we demonstrated that STAT3 negatively regulates type I IFN- reagents, and Dr. Betty Wu-Hsieh for reading the manuscript. We also thank mediated responses. Whereas knockdown or knockout of STAT3 the sorting service provided by the Core Facility of Cell Sorting System at resulted in enhanced ISG induction and antiviral activity in re- the National Taiwan University College of Medicine. shRNA reagents were sponse to type I IFNs, restoration of STAT3 in STAT3KO MEFs obtained from the National RNAi Core Facility at the Institute of Molecular attenuated the response. Interestingly, viral infection of STAT3KO Biology/Genomic Research Center, Academia Sinica, supported by the National Research Program for Genomic Medicine. cells induced higher levels of type I IFN expression and activation of STAT1 and STAT2, leading to the expression of elevated levels of ISGs, including a viral sensor MDA5. The results of MDA5 Disclosures overexpression and knockdown experiments suggest a positive role The authors have no financial conflicts of interest. Downloaded from of MDA5 in regulating type I IFN production and antiviral response in the absence of STAT3. In addition, our truncation experiment References showed that STAT3 1–134 aa is sufficient to confer antagonism of 1. Pitha, P. M., and M. S. Kunzi. 2007. Type I interferon: the ever unfolding story. IFN response, demonstrating that the inhibitory effect of STAT3 Curr. Top. Microbiol. Immunol. 316: 41–70. on type I IFN response is independent of its DNA-binding and 2. Schindler, C., D. E. Levy, and T. Decker. 2007. JAK-STAT signaling: from interferons to cytokines. J. Biol. Chem. 282: 20059–20063. http://www.jimmunol.org/ transcriptional activity. Taken together, we demonstrated that 3. Qureshi, S. A., M. Salditt-Georgieff, and J. E. Darnell Jr. 1995. Tyrosine- STAT3 suppresses antiviral response through directly regulating phosphorylated Stat1 and Stat2 plus a 48-kDa protein all contact DNA in forming interferon-stimulated-gene factor 3. Proc. Natl. Acad. Sci. USA 92: IFN response and inducing viral sensors and IRFs during viral 3829–3833. infection. 4. Baker, S. J., S. G. Rane, and E. P. Reddy. 2007. Hematopoietic cytokine receptor A negative role of STAT3 in type I IFN response is reported signaling. Oncogene 26: 6724–6737. 5. Durbin, J. E., R. Hackenmiller, M. C. Simon, and D. E. Levy. 1996. Targeted recently. Ho and Ivashkiv (15) showed that overexpression of disruption of the mouse Stat1 gene results in compromised innate immunity to STAT3 in THP-1 cells downregulated IFN-a–activated, STAT1- viral disease. Cell 84: 443–450. dependent genes such as IRF-1, CXCL9, and CXCL10, and 6. Levy, D. E., and C. K. Lee. 2002. What does Stat3 do? J. Clin. Invest. 109: 1143– 1148. knocking down STAT3 led to elevated expression of the same set 7. Meraz, M. A., J. M. White, K. C. Sheehan, E. A. Bach, S. J. Rodig, A. S. Dighe, by guest on October 5, 2021 of genes. Conversely, IFN-a–activated STAT3 supported ISRE- D. H. Kaplan, J. K. Riley, A. C. Greenlund, D. Campbell, et al. 1996. Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in driven genes such as OAS and Mx2. Enhanced STAT3 expres- the JAK-STAT signaling pathway. Cell 84: 431–442. sion did not affect tyrosine phosphorylation or nuclear trans- 8. Aggarwal, B. B., A. B. Kunnumakkara, K. B. Harikumar, S. R. Gupta, location of STAT1; instead, it sequestered STAT1 and suppressed S. T. Tharakan, C. Koca, S. Dey, and B. Sung. 2009. Signal transducer and activator of transcription-3, inflammation, and cancer: how intimate is the re- the formation of DNA-binding STAT1 homodimers. Contrary to lationship? Ann. N. Y. Acad. Sci. 1171: 59–76. their findings, we showed that STAT3 negatively regulated ISRE- 9. Takeda, K., K. Noguchi, W. Shi, T. Tanaka, M. Matsumoto, N. Yoshida, driven genes, including OAS, PKR, and IRF7. The discrepancy T. Kishimoto, and S. Akira. 1997. Targeted disruption of the mouse Stat3 gene leads to early embryonic lethality. Proc. Natl. Acad. Sci. USA 94: 3801–3804. between our system and theirs remains unclear. It could be that 10. Bromberg, J. F., M. H. Wrzeszczynska, G. Devgan, Y. Zhao, R. G. Pestell, we addressed the role of STAT3 in mouse MEFs and BMMs, C. Albanese, and J. E. Darnell Jr. 1999. Stat3 as an oncogene. Cell 98: 295–303. 11. Yu, H., M. Kortylewski, and D. Pardoll. 2007. Crosstalk between cancer and whereas Ho and Ivashkiv studied STAT3 in human monocytic cell immune cells: role of STAT3 in the tumour microenvironment. Nat. Rev. line. The advantage of studying STAT3 in mouse cells is that we Immunol. 7: 41–51. could apply both knockdown and knockout technologies to 12. Yu, H., D. Pardoll, and R. Jove. 2009. STATs in cancer inflammation and immunity: a leading role for STAT3. Nat. Rev. Cancer 9: 798–809. ensure consistent results. Concerning the suppressive mechanism 13. Park, C., S. Li, E. Cha, and C. Schindler. 2000. Immune response in Stat2 of STAT3, we showed that STAT3 1–134 aa is sufficient to knockout mice. Immunity 13: 795–804. antagonize IFN response, suggesting that competition for dimer 14. Dupuis, S., E. Jouanguy, S. Al-Hajjar, C. Fieschi, I. Z. Al-Mohsen, S. Al- Jumaah, K. Yang, A. Chapgier, C. Eidenschenk, P. Eid, et al. 2003. Impaired formation with STAT1, requiring Src homology 2 domain, is not response to interferon-alpha/beta and lethal viral disease in human STAT1 de- involved. Therefore, the sequestration mechanism does not seem ficiency. Nat. Genet. 33: 388–391. 15. Ho, H. H., and L. B. Ivashkiv. 2006. Role of STAT3 in type I interferon to operate in our system. responses: negative regulation of STAT1-dependent inflammatory gene activa- In most biological responses, STAT3 acts as a positive regulator tion. J. Biol. Chem. 281: 14111–14118. (6). However, we and others have shown that STAT3 also acts as 16. Waris, G., J. Turkson, T. Hassanein, and A. Siddiqui. 2005. Hepatitis C virus (HCV) constitutively activates STAT-3 via oxidative stress: role of STAT-3 in a negative regulator for G-CSF–mediated granulopoiesis (19, 32). HCV replication. J. Virol. 79: 1569–1580. As such, excessive granulocytes are produced in the bone marrow 17. Yang, C. H., A. Murti, and L. M. Pfeffer. 1998. STAT3 complements defects in and periphery in mice lacking STAT3 in the hematopoietic system. an interferon-resistant cell line: evidence for an essential role for STAT3 in interferon signaling and biological activities. Proc. Natl. Acad. Sci. USA 95: 5568– Reduced expression of suppressor of cytokine signaling 3, a potent 5572. negative regulator, in STAT3KO bone marrow in response to G- 18. Zhu, H., X. Shang, N. Terada, and C. Liu. 2004. STAT3 induces anti-hepatitis C viral activity in liver cells. Biochem. Biophys. Res. Commun. 324: 518–528. CSF is shown to account for the hyperproliferative activity of the 19. Lee, C. K., R. Raz, R. Gimeno, R. Gertner, B. Wistinghausen, K. Takeshita, cells (33, 34). STAT3 also functions as a negative regulator of R. A. DePinho, and D. E. Levy. 2002. STAT3 is a negative regulator of gran- TLR-mediated inflammatory response (35–37). Mice devoid of ulopoiesis but is not required for G-CSF-dependent differentiation. Immunity 17: 63–72. STAT3 in macrophages and neutrophils are highly susceptible to 20. Chou, W. C., D. E. Levy, and C. K. Lee. 2006. STAT3 positively regulates an LPS-induced endotoxic shock with increased production of in- early step in B-cell development. Blood 108: 3005–3011. 8 STAT3 SUPPRESSES TYPE I IFN RESPONSE

21. Lee, C. K., E. Smith, R. Gimeno, R. Gertner, and D. E. Levy. 2000. STAT1 30. Takeuchi, O., and S. Akira. 2008. MDA5/RIG-I and virus recognition. Curr. affects lymphocyte survival and proliferation partially independent of its role Opin. Immunol. 20: 17–22. downstream of IFN-gamma. J. Immunol. 164: 1286–1292. 31. Kato, H., O. Takeuchi, S. Sato, M. Yoneyama, M. Yamamoto, K. Matsui, 22. Schlessinger, K., and D. E. Levy. 2005. Malignant transformation but not normal S. Uematsu, A. Jung, T. Kawai, K. J. Ishii, et al. 2006. Differential roles of cell growth depends on signal transducer and activator of transcription 3. Cancer MDA5 and RIG-I helicases in the recognition of RNA viruses. Nature 441: Res. 65: 5828–5834. 101–105. 23. Gough, D. J., A. Corlett, K. Schlessinger, J. Wegrzyn, A. C. Larner, and 32. Kamezaki, K., K. Shimoda, A. Numata, T. Haro, H. Kakumitsu, M. Yoshie, D. E. Levy. 2009. Mitochondrial STAT3 supports Ras-dependent oncogenic M. Yamamoto, K. Takeda, T. Matsuda, S. Akira, et al. 2005. Roles of Stat3 and transformation. Science 324: 1713–1716. ERK in G-CSF signaling. Stem Cells 23: 252–263. 24. Niu, G., K. H. Shain, M. Huang, R. Ravi, A. Bedi, W. S. Dalton, R. Jove, and 33. Croker, B. A., D. Metcalf, L. Robb, W. Wei, S. Mifsud, L. DiRago, L. A. Cluse, H. Yu. 2001. Overexpression of a dominant-negative signal transducer and ac- K. D. Sutherland, L. Hartley, E. Williams, et al. 2004. SOCS3 is a critical tivator of transcription 3 variant in tumor cells leads to production of soluble physiological negative regulator of G-CSF signaling and emergency gran- factors that induce apoptosis and cell cycle arrest. Cancer Res. 61: 3276–3280. ulopoiesis. Immunity 20: 153–165. 25. Lee, J. L., M. J. Wang, and J. Y. Chen. 2009. Acetylation and activation of 34. Kimura, A., I. Kinjyo, Y. Matsumura, H. Mori, R. Mashima, M. Harada, STAT3 mediated by nuclear translocation of CD44. J. Cell Biol. 185: 949–957. K. R. Chien, H. Yasukawa, and A. Yoshimura. 2004. SOCS3 is a physiological 26. Yoneyama, M., M. Kikuchi, K. Matsumoto, T. Imaizumi, M. Miyagishi, K. Taira, negative regulator for granulopoiesis and granulocyte colony-stimulating factor E. Foy, Y. M. Loo, M. Gale, Jr., S. Akira, et al. 2005. Shared and unique receptor signaling. J. Biol. Chem. 279: 6905–6910. functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral 35. Hu, X., S. D. Chakravarty, and L. B. Ivashkiv. 2008. Regulation of interferon and innate immunity. J. Immunol. 175: 2851–2858. Toll-like receptor signaling during macrophage activation by opposing feedfor- 27. Vangrysperre, W., and K. De Clercq. 1996. Rapid and sensitive polymerase chain ward and feedback inhibition mechanisms. Immunol. Rev. 226: 41–56. reaction based detection and typing of foot-and-mouth disease virus in clinical 36. Murray, P. J. 2006. Understanding and exploiting the endogenous interleukin-10/ samples and cell culture isolates, combined with a simultaneous differentiation STAT3-mediated anti-inflammatory response. Curr. Opin. Pharmacol. 6: 379– with other genomically and/or symptomatically related viruses. Arch. Virol. 141: 386. 331–344. 37. Kortylewski, M., M. Kujawski, A. Herrmann, C. Yang, L. Wang, Y. Liu, 28. Chen, L. S., P. C. Wei, T. Liu, C. H. Kao, L. M. Pai, and C. K. Lee. 2009. STAT2 R. Salcedo, and H. Yu. 2009. Toll-like receptor 9 activation of signal transducer hypomorphic mutant mice display impaired dendritic cell development and and activator of transcription 3 constrains its agonist-based immunotherapy. antiviral response. J. Biomed. Sci. 16: 22. Cancer Res. 69: 2497–2505. Downloaded from 29. Bergha¨ll, H., J. Sireń, D. Sarkar, I. Julkunen, P. B. Fisher, R. Vainionpaä¨, and 38. Takeda, K., B. E. Clausen, T. Kaisho, T. Tsujimura, N. Terada, I. Fo¨rster, and S. Matikainen. 2006. The interferon-inducible RNA helicase, mda-5, is involved S. Akira. 1999. Enhanced Th1 activity and development of chronic entero- in measles virus-induced expression of antiviral cytokines. Microbes Infect. 8: colitis in mice devoid of Stat3 in macrophages and neutrophils. Immunity 10: 2138–2144. 39–49. http://www.jimmunol.org/ by guest on October 5, 2021 The Journal of Immunology

Corrections

Wang, W.-B., D. E. Levy, and C.-K. Lee. 2011. STAT3 negatively regulates type I IFN-mediated antiviral response. J. Immunol. 187: 2578– 2585.

In Materials and Methods, the primer sequences of IFN-b provided in the Quantitative RT-PCR section should read “forward 59-ATGAGTGGTGGTTGCAGGC-39, reverse 59-TGACCTTTCAAATGCAGTAGATTCA-39”. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1190075