Viral Infections Activate Types I and III Interferon Genes through a Common Mechanism

Viral infections trigger innate immune responses, including the production of type I interferons (IFN-α and -β) and other proinflammatory cytokines. Novel antiviral cytokines IFN-λ1, IFN-λ2, and IFN-λ3 are classified as type III IFNs and have evolved independently of type I IFNs. Type III IFN genes are regulated at the level of transcription and induced by viral infection. Although the regulatory mechanism of type I IFNs is well elucidated, the expression mechanism of IFN-λs is not well understood. Here, we analyzed the mechanism by which IFN-λ gene expression is induced by viral infections. Loss- and gain-of-function experiments revealed the involvement of RIG-I (retinoic acid-inducible gene I), IPS-1, TBK1, and interferon regulatory factor-3, key regulators of the virus-induced activation of type I IFN genes. Consistent with this, a search for the cis-regulatory element of the human ifnλ1 revealed a cluster of interferon regulatory factor-binding sites and a NF-κB-binding site. Functional analysis demonstrated that all of these sites are essential for gene activation by the virus. These results strongly suggest that types I and III IFN genes are regulated by a common mechanism. Viral infections trigger innate immune responses, including the production of type I interferons (IFN-α and -β) and other proinflammatory cytokines. Novel antiviral cytokines IFN-λ1, IFN-λ2, and IFN-λ3 are classified as type III IFNs and have evolved independently of type I IFNs. Type III IFN genes are regulated at the level of transcription and induced by viral infection. Although the regulatory mechanism of type I IFNs is well elucidated, the expression mechanism of IFN-λs is not well understood. Here, we analyzed the mechanism by which IFN-λ gene expression is induced by viral infections. Loss- and gain-of-function experiments revealed the involvement of RIG-I (retinoic acid-inducible gene I), IPS-1, TBK1, and interferon regulatory factor-3, key regulators of the virus-induced activation of type I IFN genes. Consistent with this, a search for the cis-regulatory element of the human ifnλ1 revealed a cluster of interferon regulatory factor-binding sites and a NF-κB-binding site. Functional analysis demonstrated that all of these sites are essential for gene activation by the virus. These results strongly suggest that types I and III IFN genes are regulated by a common mechanism. Interferon (IFN) 2The abbreviations used are: IFN, interferon; IRF, interferon regulatory factor; IRAK, interleukin-1 receptor-associated kinase; MEF, mouse embryonic fibroblast; RIG-I, retinoic acid-inducible gene I; IPS-1, IFN-β promoter stimulator 1; CARD, caspase recruitment domain; RACE, rapid amplification of cDNA ends; EMSA, electrophoresis mobility shift assay; NDV, Newcastle disease virus; TRIF, Toll/IL-1 receptor domain-containing adapter inducing IFN-β; TICAM-1, Toll/IL-1 receptor-containing adapter molecule 1; TANK, Traf family member-associated NF-κB activator. plays a critical role in innate as well as adaptive immune responses against viral infections (1De Maeyer E. De Maeyer-Guignard J. Int. Rev. Immunol. 1998; 17: 53-73Crossref PubMed Scopus (94) Google Scholar, 2Samuel C.E. Clin. Microbiol. Rev. 2001; 14: 778-809Crossref PubMed Scopus (2168) Google Scholar). Viral infections trigger the activation of type I IFNs (IFN-α and IFN-β). The mechanism behind the virus-induced expression of type I IFNs is well documented (3Akira S. Takeda K. Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6751) Google Scholar, 4Honda K. Yanai H. Takaoka A. Taniguchi T. Int. Immunol. 2005; 17: 1367-1378Crossref PubMed Scopus (285) Google Scholar). Plasmacytoid-dendritic cells, which are responsible for a high level of IFN-α in serum, detect virus-associated molecular patterns via Toll-like receptor-7 or -9 receptors. The signal is transduced to IRAK1/IRAK4/IKK-α kinases in a MyD88-dependent manner resulting in the activation of interferon regulatory factor 7 (IRF-7) through its specific phosphorylation. In other cells, extracellular double-stranded RNA is detected by Toll-like receptor-3, transducing a signal through the TRIF/TICAM-1 adaptor in a MyD88-independent manner and then activating TANK-binding kinase 1 (TBK1) or IκB kinase I (IKK-i) kinase and subsequently IRF-3 through its phosphorylation. The third class of receptors for virus-associated molecular patterns resides in the cytoplasm and detects replicating viral RNA. The retinoic acid inducible-gene I (RIG-I) family of RNA helicases are shown to recognize viral double-stranded RNA by their helicase domain and transmit signals to downstream molecules via their caspase recruitment domain (CARD) (5Yoneyama M. Kikuchi M. Matsumoto K. Imaizumi T. Miyagishi M. Taira K. Foy E. Loo Y.M. Gale Jr., M. Akira S. Yonehara S. Kato A. Fujita T. J. Immunol. 2005; 175: 2851-2858Crossref PubMed Scopus (1302) Google Scholar, 6Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (3136) Google Scholar). Although the precise mechanism involved has not been elucidated, IFN-β promoter stimulator 1 (IPS-1), a mitochondrial protein containing CARD, is thought to further transmit the signal to IRF-3 kinases, TBK1, and IKK-i (7Kawai T. Takahashi K. Sato S. Coban C. Kumar H. Kato H. Ishii K.J. Takeuchi O. Akira S. Nat. Immunol. 2005; 6: 981-988Crossref PubMed Scopus (2032) Google Scholar, 8Meylan E. Curran J. Hofmann K. Moradpour D. Binder M. Barten-schlager R. Tschopp J. Nature. 2005; 437: 1167-1172Crossref PubMed Scopus (1973) Google Scholar, 9Seth R.B. Sun L. Ea C.K. Chen Z.J. Cell. 2005; 122: 669-682Abstract Full Text Full Text PDF PubMed Scopus (2518) Google Scholar, 10Xu L.G. Wang Y.Y. Han K.J. Li L.Y. Zhai Z. Shu H.B. Mol. Cell. 2005; 19: 727-740Abstract Full Text Full Text PDF PubMed Scopus (1518) Google Scholar). Recently, a study using mice with disrupted genes for key molecules of the three different pathways of type I IFN gene activation was reported (11Gitlin L. Barchet W. Gilfillan S. Cella M. Beutler B. Flavell R.A. Diamond M.S. Colonna M. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8459-8464Crossref PubMed Scopus (919) Google Scholar, 12Kato H. Takeuchi O. Sato S. Yoneyama M. Yamamoto M. Matsui K. Uematsu S. Jung A. Kawai T. Ishii K.J. Yamaguchi O. Otsu K. Tsujimura T. Koh C.S. Reis e Sousa C. Matsuura Y. Fujita T. Akira S. Nature. 2006; 441: 101-105Crossref PubMed Scopus (2933) Google Scholar). Interestingly, RIG-I family helicases RIG-I and melanoma differentiation-associated gene 5 play key protective roles in infections of Japanese encephalitis virus and encephalomyelitis virus, respectively. It appeared that disruption of either of the Toll-like receptor pathways did not have a strong effect on protection against these viruses, suggesting a central role for the RIG-I family in host protection in vivo. In recent years, several novel interleukin-10-related cytokines have been identified (13Kotenko S.V. Gallagher G. Baurin V.V. Lewis-Antes A. Shen M. Shah N.K. Langer J.A. Sheikh F. Dickensheets H. Donnelly R.P. Nat. Immunol. 2003; 4: 69-77Crossref PubMed Scopus (1548) Google Scholar, 14Pestka S. Krause C.D. Sarkar D. Walter M.R. Shi Y. Fisher P.B. Annu. Rev. Immunol. 2004; 22: 929-979Crossref PubMed Scopus (950) Google Scholar, 15Sheppard P. Kindsvogel W. Xu W. Henderson K. Schlutsmeyer S. Whitmore T.E. Kuestner R. Garrigues U. Birks C. Roraback J. Ostrander C. Dong D. Shin J. Presnell S. Fox B. Haldeman B. Cooper E. Taft D. Gilbert T. Grant F.J. Tackett M. Krivan W. McKnight G. Clegg C. Foster D. Klucher K.M. Nat. Immunol. 2003; 4: 63-68Crossref PubMed Scopus (1325) Google Scholar). Although IFN-λs, a family of interleukin-10-related cytokines, are evolutionarily distantly related to type I IFNs, they exhibit antiviral activity similar to type I IFNs. Thus, they are collectively known as type III IFNs. Type III IFNs interact with cell surface receptors composed of interleukin-10 receptor β and interleukin-28 receptor α, distinct from those for types I or II IFNs. The binding of a type III IFN to its receptor results in the intracellular activation of Janus kinase 1 and signal transducers and activators of transcription STAT1 and STAT2 and then the subsequent formation of the IFN-stimulated gene factor 3 complex. Similar to type I IFNs, type III IFNs are induced to express by viral infection or treatment with poly(I·C) or lipopolysaccharide (16Coccia E.M. Severa M. Giacomini E. Monneron D. Remoli M.E. Julkunen I. Cella M. Lande R. Uze G. Eur. J. Immunol. 2004; 34: 796-805Crossref PubMed Scopus (421) Google Scholar). However, the regulatory mechanisms involved are not well understood. Here we analyzed how type III IFN genes are regulated by viral infections. We took advantage of a comparative approach using the well established molecular mechanism regulating type I IFN genes. We looked at the effect of loss and gain of function of the signaling molecules critical for type I IFN genes. We mapped and analyzed cis-regulatory elements that function as virus-inducible enhancers. Our results demonstrate a central role for a pathway involving RIG-I, IRF-3, and NF-κB. Cell Culture, Transfection, Preparation of Cell Extracts, and Luciferase Assay—Mouse embryonic fibroblasts (MEFs) and 293T cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and penicillin-streptomycin. L929 cells were maintained in minimum essential medium with 5% fetal bovine serum and penicillin-streptomycin. 293T and L929 cells were transiently transfected with the calcium-phosphate method and FuGENE 6 (Roche Applied Science), respectively. For the preparation of cell extracts, cells were lysed with lysis buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1mm EDTA, 1% Nonidet P-40, 0.1 mg/ml leupeptin, 1 mm phenylmethylsulfonyl fluoride, and 1 mm sodium orthovanadate) and centrifuged at 245,000 × g for 10 min. The supernatant was used for immunoblotting. The Dual-Luciferase reporter assay system (Promega) was used for luciferase assays. As an internal control for the Dual-Luciferase assay, the Renilla luciferase construct pRL-TK (Promega) was used. Plasmid Constructs—We used pEF-FLAG-IRF-3 5D and pEF-FLAG-RIG-I CARD as described previously (6Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (3136) Google Scholar, 17Mori M. Yoneyama M. Ito T. Takahashi K. Inagaki F. Fujita T. J. Biol. Chem. 2004; 279: 9698-9702Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). IPS-1 cDNA was purchased from the Biological Resource Center of the National Institute of Technology and Evaluation of Japan. We amplified this cDNA with an oligonucleotide for the N-terminal FLAG tag by PCR and inserted it into the XbaI-SpeI site of pEF-BOS(+) (pEF-FLAG-IPS-1). The promoter region of human ifnλ1 was amplified by genomic PCR (upper primer, 5′-CTAGGTCGACGGGCAACAAGAGCAAAACTA-3′, lower primer, 5′-GTGGATCCGCTAAATCGCAACTGCTTCC-3′), and the PCR fragment was inserted into the SalI-BamHI site of luciferase vector (pλ1-(-554/+14)Luc). The nucleotide sequence was confirmed with the BigDye DNA sequencing kit (Applied Biosystems). We chemically synthesized fragments encompassing the promoter sequence of the ifnλ1 between -225 and -36 and inserted them into the SalI-BamHI site of p-55Luc (p-55λ1-(-225/-36)Luc). Synthesized fragments of p-55λ1mut.IRF-Luc, p-55λ1mut. NF-κBLuc, and p-55λ1mut.IRF/mut. NF-κBLuc were designed as shown in Fig. 4A and inserted into the SalI-BamHI site of p-55Luc. Quantitative Real Time-PCR—Total RNA was prepared with TRIzol reagent (Invitrogen), treated with DNase I (Roche Diagnostics), and amplified by real time-PCR with the ABI PRISM 7500 sequence detection system (Applied Biosystems). Taq-Man reverse transcription reagents and the TaqMan Universal PCR mix (Applied Biosystems) were used for cDNA synthesis and PCR, respectively. We used commercial TaqMan primerprobe sets (Applied Biosystems) for mouse ifnλ2, human ifnλ1, and human ifnλ2. As an internal control for the comparative threshold cycle method, a primer-probe set for eukaryotic 18 S rRNA (Applied Biosystems) was used. The results were normalized to the abundance of internal 18 S rRNA and were reproducibly obtained in two independent transfection experiments. Rapid Amplification of cDNA Ends (RACE)—RACE analysis was performed using the FirstChoice RNA ligase-mediated RACE (Ambion) according to the manufacturer's instructions. Total RNA was prepared as for quantitative real time-PCR. Nested PCR reaction was performed using recombinant TaqDNA polymerase (TaKaRa), ifnλ1-specific outer primer (5′-GGCCACATATTTGAGGTCTC-3′), and ifnλ1-specific inner primer (5′-CGCGGATCCAGAAGCCTCAGGTCCCAATT-3′) according to the manufacturer. The RACE product was cloned into pBluescript II vector, and 10 random clones were sequenced. Electrophoresis Mobility Shift Assay (EMSA)—The oligonucleotides containing self-complementary sequences for NF-κB(5′-GGGAAATTCTCTTAGCTTGAGAATTTCC-3′) or mutated NF-κB(5′-GCCGAATTCTCTTAGCTTGAGAATTCGG-3′) of human ifnλ1 were used as a probe. The method for EMSA was described previously (18Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (691) Google Scholar). We used commercial anti-NF-κB antibodies for human p50, p65, and IRF-3 (IBL; catalog numbers 18661, 18667, and 18781, respectively). The Effect of Loss of Function of IRF-3, TBK1, and RIG-I on Virus-induced Activation of ifnλ2—It has been reported that the expression of IFN-λ is induced by stimuli such as viral infections and treatment with poly(I·C) or lipopolysaccharide in a parallel fashion to that of type I IFN genes (16Coccia E.M. Severa M. Giacomini E. Monneron D. Remoli M.E. Julkunen I. Cella M. Lande R. Uze G. Eur. J. Immunol. 2004; 34: 796-805Crossref PubMed Scopus (421) Google Scholar). We investigated the role of the transcription factor IRF-3 and its regulatory kinase, TBK1, using MEFs derived from respective knock-out mice (19Hemmi H. Takeuchi O. Sato S. Yamamoto M. Kaisho T. Sanjo H. Kawai T. Hoshino K. Takeda K. Akira S. J. Exp. Med. 2004; 199: 1641-1650Crossref PubMed Scopus (465) Google Scholar, 20Sato M. Suemori H. Hata N. Asagiri M. Ogasawara K. Nakao K. Nakaya T. Katsuki M. Noguchi S. Tanaka N. Taniguchi T. Immunity. 2000; 13: 539-548Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar). Because mice have no ortholog gene of the human ifnλ1, we examined the expression level of the mouse ifnλ2. MEFs were mock-treated or infected with Newcastle disease virus (NDV), and the endogenous mouse ifnλ2 mRNA level was determined by quantitative real time-PCR. Although a significant level of expression of the mouse ifnλ2 gene was observed in NDV-infected wild-type cells, IRF-3-/- cells were totally deficient in ifnλ2 (Fig. 1A). TBK1-/- cells exhibited a significantly suppressed expression of the ifnλ2 (Fig. 1B). The residual induction is likely due to IKK-i (IKK-ε). Viral infections are shown to activate type I IFN genes through the activation of the RNA helicase RIG-I, which acts as a sensor for replicating viral RNA in cells (6Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (3136) Google Scholar, 21Kato H. Sato S. Yoneyama M. Yamamoto M. Uematsu S. Matsui K. Tsujimura T. Takeda K. Fujita T. Takeuchi O. Akira S. Immunity. 2005; 23: 19-28Abstract Full Text Full Text PDF PubMed Scopus (1118) Google Scholar). We investigated the involvement of RIG-I in the NDV-induced activation of the ifnλ2 by using RIG-I knock-out As shown in Fig. cells to activate the ifnλ2, suggesting RIG-I is critical for this the was obtained in experiments using mouse L929 cells not We not the gene expression between Although the of the mice is the MEFs were prepared at different embryonic for IRF-3, TBK1, and RIG-I the effect of was prepared at the embryonic or from cell are different between different gene are between Thus, is between and of Function of IRF-3 and RIG-I in the Activation of IFN-λ an we of IRF-3 and RIG-I in 293T cells and endogenous human ifnλ1 and ifnλ2 mRNA by quantitative real time-PCR. IRF-3 5D is a with with IRF-3 5D is at by in human cells and is of activating including type I IFN genes M. Yoneyama M. Ito T. Takahashi K. Inagaki F. Fujita T. J. Biol. Chem. 2004; 279: 9698-9702Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). of a of RIG-I, which CARD CARD, Fig. results in the activation of type I IFN genes (6Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (3136) Google Scholar). expression of IRF-3 and RIG-I in 293T cells (Fig. in the activation of human ifnλ1 and ifnλ2 (Fig. and These results strongly suggest that IFN-λ genes are regulated by a mechanism common to type I IFN-λ are by IPS-1 known as or was identified as an adaptor molecule of RIG-I signaling (7Kawai T. Takahashi K. Sato S. Coban C. Kumar H. Kato H. Ishii K.J. Takeuchi O. Akira S. Nat. Immunol. 2005; 6: 981-988Crossref PubMed Scopus (2032) Google Scholar, 8Meylan E. Curran J. Hofmann K. Moradpour D. Binder M. Barten-schlager R. Tschopp J. Nature. 2005; 437: 1167-1172Crossref PubMed Scopus (1973) Google Scholar, 9Seth R.B. Sun L. Ea C.K. Chen Z.J. Cell. 2005; 122: 669-682Abstract Full Text Full Text PDF PubMed Scopus (2518) Google Scholar, 10Xu L.G. Wang Y.Y. Han K.J. Li L.Y. Zhai Z. Shu H.B. Mol. Cell. 2005; 19: 727-740Abstract Full Text Full Text PDF PubMed Scopus (1518) Google Scholar). It has been that RIG-I with IPS-1 between respective CARD resulting in the activation of IRF-3, and type I IFN genes. Interestingly, of IPS-1 the expression of type I IFN genes a viral infection. We IPS-1 in 293T cells (Fig. and human ifnλ1 and ifnλ2 of the vector not vector induced the expression of endogenous human ifnλ1 and ifnλ2 (Fig. and suggesting that IFN-λ genes are regulated by the pathway by viral of the of the results suggest the of or virus-inducible elements the IFN-λ genes. these the transcription of human ifnλ1 was investigated by The RACE product was detected with NDV-infected RNA (Fig. The product was and sequenced. random 10 clones as shown in Fig. was no sequence at the we that ifnλ1 is a of the of the we analyzed virus-inducible elements the We the cell genomic fragment encompassing the human ifnλ1 to to the transcription site (Fig. The fragment was cloned into a luciferase reporter gene (Fig. For a reporter containing of the (Fig. was used. Although was significantly by IFN-β was not significantly by IFN-β with the that IFN-λ is of viral by type I IFN (Fig. N. H. C. K. J. 2006; PubMed Scopus Google Scholar). In infection strongly induced the activation of and that the genomic fragment a virus-inducible (Fig. Function of and NF-κB-binding the ifnλ1 the we binding sites for to and to (Fig. It is that sites and a NF-κB similar to the were in the human ifnλ2 (Fig. the function of these we chemically synthesized the region to of human ifnλ1 and cloned it into the of the promoter of human to a luciferase reporter gene (18Yoneyama M. Suhara W. Fukuhara Y. Fukuda M. Nishida E. Fujita T. EMBO J. 1998; 17: 1087-1095Crossref PubMed Scopus (691) Google Fig. that the fragment as a virus-inducible suggesting that the fragment the regulatory of the binding sites for either or NF-κB the level of virus-induced gene and disruption of these binding sites in further viral induction (Fig. and These results that these binding sites function as of a virus-inducible NF-κB to the NF-κB of reporter assay that the NF-κB is critical for IFN-λ gene We NF-κB binding to the by EMSA (Fig. infection induced binding activity to the wild-type not to the mutated suggesting that the is a NF-κB site (Fig. In or the specific (Fig. suggesting that to the ifnλ1 NF-κB site. The type I and III IFNs exhibit and are thought to have different types I and III IFNs through with distinct cell surface receptors. We of their IFN-λ genes are regulated by a mechanism common to type I IFNs. Our revealed the involvement of IRF-3, TBK1, and activation of IRF-3, RIG-I, and IPS-1 all type III IFN genes. These signaling molecules play a critical role in the virus-induced activation of type I IFN genes. Our search for a in the promoter of the human ifnλ1 revealed a cluster of sites and a NF-κB-binding site as of the virus-inducible is of type I IFN sites virus-inducible of IFN-α and and a site plays a critical role in the activation of Although sites are to by IFN through IFN-stimulated gene factor the expression of types I or III genes is induced by IFN treatment Thus, the common expression patterns of types I and III IFN genes are determined by the of these transcription factor-binding which independently through the of the respective genes. Consistent with P. J. Klucher K.M. J. S. Julkunen I. J. 2005; PubMed Scopus Google that IRF-3 to the site by a DNA the binding of promoter region nucleotide to from the transcription of the binding site did not the and T. disruption of the other NF-κB site to significantly the of and NF-κB sites significantly the the promoter exhibited suggesting that the DNA In they reported activation of ifnλ1 and ifnλ2. virus the expression of ifnλ1 not virus induced production of high of ifnλ1 and ifnλ2. It is binding sites for and NF-κB are in the promoter region of the ifnλ2 (Fig. DNA sequence to that of ifnλ1 is are to the mechanism behind the activation of type III IFN genes. Because of the of distinct types I and III IFNs likely not signal in and Type I IFNs have been used to viral infections and The study to a using IFN that or types I and III IFN genes to the IFN system for