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Regulation of Signal Transduction by Enzymatically Inactive Antiviral RNA Helicase Proteins MDA5, RIG-I, and LGP2

2009; Elsevier BV; Volume: 284; Issue: 15 Linguagem: Inglês

10.1074/jbc.m807365200

ISSN

1083-351X

Autores

Darja Bamming, Curt M. Horvath,

Tópico(s)

Cytokine Signaling Pathways and Interactions

Resumo

Intracellular pattern recognition receptors MDA5, RIG-I, and LGP2 are essential components of the cellular response to virus infection and are homologous to the DEXH box subfamily of RNA helicases. However, the relevance of helicase activity in the regulation of interferon production remains elusive. To examine the importance of the helicase domain function for these signaling proteins, a series of mutations targeting conserved helicase sequence motifs were analyzed for enzymatic activity, RNA binding, interferon induction, and antiviral signaling. Results indicate that all targeted motifs are required for ATP hydrolysis, but a subset is involved in RNA binding. The enzymatically inactive mutants differed in their signaling ability. Notably, mutations to MDA5 motifs I, III, and VI and RIG-I motif III produced helicase proteins with constitutive antiviral activity, whereas mutations in RIG-I motif V retained ATP hydrolysis but failed to mediate signal transduction. These findings demonstrate that type I interferon production mediated by full-length MDA5 and RIG-I is independent of the helicase domain catalytic activity. In addition, neither enzymatic activity nor RNA binding was required for negative regulation of antiviral signaling by LGP2, supporting an RNA-independent interference mechanism. Intracellular pattern recognition receptors MDA5, RIG-I, and LGP2 are essential components of the cellular response to virus infection and are homologous to the DEXH box subfamily of RNA helicases. However, the relevance of helicase activity in the regulation of interferon production remains elusive. To examine the importance of the helicase domain function for these signaling proteins, a series of mutations targeting conserved helicase sequence motifs were analyzed for enzymatic activity, RNA binding, interferon induction, and antiviral signaling. Results indicate that all targeted motifs are required for ATP hydrolysis, but a subset is involved in RNA binding. The enzymatically inactive mutants differed in their signaling ability. Notably, mutations to MDA5 motifs I, III, and VI and RIG-I motif III produced helicase proteins with constitutive antiviral activity, whereas mutations in RIG-I motif V retained ATP hydrolysis but failed to mediate signal transduction. These findings demonstrate that type I interferon production mediated by full-length MDA5 and RIG-I is independent of the helicase domain catalytic activity. In addition, neither enzymatic activity nor RNA binding was required for negative regulation of antiviral signaling by LGP2, supporting an RNA-independent interference mechanism. The first line of defense against virus infection is provided by the cellular antiviral response and innate immune system. The immediate response includes the induction of type I interferon (IFNα and IFNβ, referred to herein as IFN 2The abbreviations used are: IFN, type I interferon; IPS-1, interferon-β promoter stimulator-1; CARD caspase activation and recruitment domain; dsRNA, double-stranded RNA; ssRNA, single-stranded RNA; nt, nucleotides; VSV, vesicular stomatitis virus; PFU, plaque-forming unit; MOPS, 4-morpholinepropanesulfonic acid; RT, reverse transcription; CTD, C-terminal domain.) and other cytokines. Virus infections are sensed by cellular receptors that can recognize pathogen-associated molecular patterns such as viral nucleic acids (1Akira S. Uematsu S. Takeuchi O. Cell. 2006; 124: 783-801Abstract Full Text Full Text PDF PubMed Scopus (8790) Google Scholar). In addition to the transmembrane Toll-like receptors (2Akira S. Takeda K. Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6702) Google Scholar), a family of cytoplasmic RNA helicases has been identified that can detect cytosolic non-self RNA. Two members of this group are MDA5 (melanoma differentiation-associated gene 5) and RIG-I (retinoic acid-inducible gene I) (3Yoneyama 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 (3122) Google Scholar). These proteins are characterized by the combination of two caspase activation and recruitment domain (CARD) motifs linked to an RNA helicase domain. MDA5 and RIG-I can detect foreign RNAs and transmit a signal through the CARD-containing mitochondrial adaptor molecule IPS-1 (also named Cardif, MAVS, and VISA) (4Kawai 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 (2018) Google Scholar, 5Meylan E. Curran J. Hofmann K. Moradpour D. 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Nature. 2006; 439: 208-211Crossref PubMed Scopus (707) Google Scholar, 9Saha S.K. Pietras E.M. He J.Q. Kang J.R. Liu S.Y. Oganesyan G. Shahangian A. Zarnegar B. Shiba T.L. Wang Y. Cheng G. EMBO J. 2006; 25: 3257-3263Crossref PubMed Scopus (347) Google Scholar) which are responsible for transcriptional activation of a variety of antiviral effectors, including the IFNβ gene that is fundamental to the antiviral response. Despite their similarities in domain structure and amino acid sequence, MDA5 and RIG-I are nonredundant and are involved in recognition of different types of non-self RNAs and therefore different viruses (10Gitlin 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 (911) Google Scholar, 11Kato 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 (2906) Google Scholar, 12Loo Y.M. Fornek J. Crochet N. Bajwa G. Perwitasari O. Martinez-Sobrido L. Akira S. Gill M.A. Garcia-Sastre A. Katze M.G. Gale Jr., M. J. Virol. 2008; 82: 335-345Crossref PubMed Scopus (821) Google Scholar). RIG-I exhibits ligand specificity for short ( 2 kbp) dsRNA (17Kato H. Takeuchi O. Mikamo-Satoh E. Hirai R. Kawai T. Matsushita K. Hiiragi A. Dermody T.S. Fujita T. Akira S. J. Exp. Med. 2008; 205: 1601-1610Crossref PubMed Scopus (1160) Google Scholar) and is a cytosolic receptor for the synthetic RNA, poly(I-C) (10Gitlin 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 (911) Google Scholar, 11Kato 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 (2906) Google Scholar). Recent data indicate that the two helicases also differ in regulation and signaling mechanisms. In RIG-I the C-terminal domain (CTD) contains a basic RNA binding pocket suitable for accommodation of 5′-triphosphate RNA (15Cui S. Eisenacher K. Kirchhofer A. Brzozka K. Lammens A. Lammens K. Fujita T. Conzelmann K.K. Krug A. Hopfner K.P. Mol. Cell. 2008; 29: 169-179Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 16Takahasi K. Yoneyama M. Nishihori T. Hirai R. Kumeta H. Narita R. Gale Jr., M. Inagaki F. Fujita T. Mol. Cell. 2008; 29: 428-440Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 19Saito T. Hirai R. Loo Y.M. Owen D. Johnson C.L. Sinha S.C. Akira S. Fujita T. Gale Jr., M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 582-587Crossref PubMed Scopus (583) Google Scholar). Sequence comparisons suggest the overall structure of the MDA5 CTD may be conserved but that MDA5 lacks key residues in the CTD RNA binding pocket (15Cui S. Eisenacher K. Kirchhofer A. Brzozka K. Lammens A. Lammens K. Fujita T. Conzelmann K.K. Krug A. Hopfner K.P. Mol. Cell. 2008; 29: 169-179Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 16Takahasi K. Yoneyama M. Nishihori T. Hirai R. Kumeta H. Narita R. Gale Jr., M. Inagaki F. Fujita T. Mol. Cell. 2008; 29: 428-440Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). In addition, the RIG-I CTD autoregulatory domain function was not observed for the MDA5 CTD (19Saito T. Hirai R. Loo Y.M. Owen D. Johnson C.L. Sinha S.C. Akira S. Fujita T. Gale Jr., M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 582-587Crossref PubMed Scopus (583) Google Scholar). A third related protein, LGP2, has high sequence similarity to MDA5 and RIG-I helicase domains but lacks the N-terminal CARD homology. Expression of LGP2 from a plasmid vector acts as a negative regulator of IFN production and antiviral signaling (19Saito T. Hirai R. Loo Y.M. Owen D. Johnson C.L. Sinha S.C. Akira S. Fujita T. Gale Jr., M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 582-587Crossref PubMed Scopus (583) Google Scholar, 20Komuro A. Horvath C.M. J. Virol. 2006; 80: 12332-12342Crossref PubMed Scopus (243) Google Scholar, 21Yoneyama 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 (1290) Google Scholar, 22Rothenfusser S. Goutagny N. DiPerna G. Gong M. Monks B.G. Schoenemeyer A. Yamamoto M. Akira S. Fitzgerald K.A. J. Immunol. 2005; 175: 5260-5268Crossref PubMed Scopus (486) Google Scholar), but analysis of mice deficient for LGP2 suggests disparate functions for LGP2 in response to different viruses (23Venkataraman T. Valdes M. Elsby R. Kakuta S. Caceres G. Saijo S. Iwakura Y. Barber G.N. J. Immunol. 2007; 178: 6444-6455Crossref PubMed Scopus (333) Google Scholar), acting as either a positive or negative regulator of antiviral signaling. Great progress has been made in understanding the roles of MDA5, RIG-I, and LGP2 in RNA sensing and antiviral immune responses. Recent reports demonstrated in vitro unwinding activity of RIG-I for double-stranded oligonucleotides with 3′ overhangs. Interestingly, a reverse correlation between suitable unwinding substrates and immunogenic oligonucleotides was found (16Takahasi K. Yoneyama M. Nishihori T. Hirai R. Kumeta H. Narita R. Gale Jr., M. Inagaki F. Fujita T. Mol. Cell. 2008; 29: 428-440Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). However, the significance of the DEXH box helicase domain for the role of proteins in innate immune responses still remains elusive. MDA5, RIG-I, and LGP2 can be categorized as DEXH box RNA helicases belonging to the helicase superfamily 2 (24Gorbalenya A.E. Koonin E.V. Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (1034) Google Scholar). This family is characterized by six highly conserved sequence motifs (I–VI) within the helicase domain (25Cordin O. Banroques J. Tanner N.K. Linder P. Gene (Amst.). 2006; 367: 17-37Crossref PubMed Scopus (734) Google Scholar). Structural studies of a variety of helicase family members reveal a general folding into two RecA-like globular domains, with domain 1 encompassing motifs I–III and domain 2 containing motifs IV–VI (26Caruthers J.M. McKay D.B. Curr. Opin. Struct. Biol. 2002; 12: 123-133Crossref PubMed Scopus (454) Google Scholar). Low resolution structural data of LGP2 (27Murali A. Li X. Ranjith-Kumar C.T. Bhardwaj K. Holzenburg A. Li P. Kao C.C. J. Biol. Chem. 2008; 283: 15825-15833Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) as well as the crystal structure of MDA5 domain 1 (PDB code 3b6e) suggest the general helicase fold to be conserved in these family members. Available evidence from structural and biochemical studies in other helicases has implicated specific motifs in different aspects of helicase functions. Motif I and motif II, also referred to as Walker A and B motif, respectively (28Walker J.E. Saraste M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4257) Google Scholar), are indispensable for ATP binding and hydrolysis. In most DEXH box helicases motif V and VI are also implicated in nucleotide binding (25Cordin O. Banroques J. Tanner N.K. Linder P. Gene (Amst.). 2006; 367: 17-37Crossref PubMed Scopus (734) Google Scholar, 29Tanner N.K. Linder P. Mol. Cell. 2001; 8: 251-262Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar). In some studies, RNA binding has been assigned to motif IV and V (25Cordin O. Banroques J. Tanner N.K. Linder P. Gene (Amst.). 2006; 367: 17-37Crossref PubMed Scopus (734) Google Scholar, 29Tanner N.K. Linder P. Mol. Cell. 2001; 8: 251-262Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar), whereas motif III has been implicated in forming intramolecular interactions between the domains. Indeed, interdomain interactions are also likely to occur between other motifs, to form a stabilized helicase catalytic core (30Sengoku T. Nureki O. Nakamura A. Kobayashi S. Yokoyama S. Cell. 2006; 125: 287-300Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, 31Kim J.L. Morgenstern K.A. Griffith J.P. Dwyer M.D. Thomson J.A. Murcko M.A. Lin C. Caron P.R. Structure (Lond.). 1998; 6: 89-100Abstract Full Text Full Text PDF PubMed Scopus (581) Google Scholar). To better understand the importance of helicase activity in cytoplasmic RNA sensing and antiviral signal transduction, we have systematically analyzed the catalytic core by site-directed mutagenesis. Results indicate that signaling by full-length RIG-I and MDA5 can occur independent of enzymatic activity. Mutations in the conserved helicase motifs produced a subset of RIG-I and MDA5 mutants that dissociate enzymatic activity from signal transduction activity, either producing enzymatically defective constitutive activators of IFNβ transcription or enzymatically active proteins defective in antiviral signaling. Furthermore, LGP2 mutants revealed that its ability to negatively regulate antiviral signaling is independent of both enzymatic activity and RNA binding activity. Plasmids and Mutagenesis-FLAG-tagged MDA5 and RIG-I cDNA in expression vector pEFBos were provided by M. Gale, Jr. (University of Washington). LGP2 cDNA was obtained by PCR amplification of Human Universal Quick Clone II (Clontech) and cloned into p3xFLAG-CMV10 (Sigma) as described previously (20Komuro A. Horvath C.M. J. Virol. 2006; 80: 12332-12342Crossref PubMed Scopus (243) Google Scholar). Site-directed mutagenesis was carried out using QuickChangeXL mutagenesis kit (Stratagene). Introduced mutations were confirmed by DNA sequence analysis. For recombinant baculovirus production helicase cDNA sequences were amplified by PCR and cloned into pBac2cp (Novagen). Cell Culture, Transfections, and Cytokine Treatment-Sf9 insect cells were maintained in Grace’s insect cell medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 1% penicillin/streptomycin (Invitrogen). HEK293T, 2fTGH, U6A (a STAT2-deficient 2fTGH cell derivative), HeLa, Vero, and Huh 7.5 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% cosmic calf serum (HyClone) and 1% penicillin/streptomycin (Invitrogen). Transfections of HEK293T cells were carried out using the standard CaPO4 method (32Ausubel F.M.B.R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. John Wiley and Sons, Inc., New York1994: 9.1.4-9.1.11Google Scholar) or FuGENE transfection reagent (Roche Applied Science). 2fTGH cells were transfected with Superfect (Qiagen). Transfection of HeLa, Vero, and Huh 7.5 cells and all poly(I-C) (Amersham Biosciences) transfections were performed with Lipofectamine transfection reagent (Invitrogen). All transfections were carried out according to manufacturers’ protocol. Purified human IFNβ at 100,000 pg/ml was obtained from PBL InterferonSource and added to growth media at indicated final concentrations. Protein Purification-HEK293T cells were transfected for expression of recombinant wild type or mutant helicase proteins with N-terminal FLAG tags. Cell lysates were precleared with Sepharose 6B beads (Sigma) and subsequently incubated with anti-FLAG M2 affinity gel (Sigma). Elution was achieved by addition of 3× FLAG peptide (Sigma). Proteins expressed by recombinant baculovirus infection of Sf9 insect cells were tagged with a His6 epitope, purified using Ni2+-nitrilotriacetic acid beads (Novagen), and eluted in buffer containing 250 mm imidazole. ATP Hydrolysis Assay-500 ng of purified protein were incubated in 50 μl of ATPase reactions, containing 2 μg of poly(I-C), 0.5 mm ATP (Sigma), 0.66 nm [γ-32P]ATP (PerkinElmer Life Sciences) in 5 mm MOPS, pH 6.5, 0.3 mm MgCl2, 0.2 mm dithiothreitol, for 30 min at 37 °C. 10% of the reactions were loaded onto thin layer chromatography plates (Sigma) and analyzed by phosphorimaging with a Storm scanner (GE Healthcare) and subsequently quantified using ImageQuant™ software. The remaining reaction was analyzed by SDS-PAGE and subjected to silver staining. Luciferase Reporter Gene Assays-Cells were cotransfected with reporter gene and expression vectors for Renilla luciferase as well as empty vector or the helicase of interest. The reporter gene contains the firefly luciferase open reading frame under the control of the interferon β promoter (–110). The next day cells were stimulated by infection with Sendai virus (Cantell strain, 3 × 106 PFU/well) infection or poly(I-C) (5 μg/well) transfection for 6 h before harvest. Relative luciferase activity was measured using Dual-Luciferase™ reporter assay (Promega). Data are plotted as average values (n = 3) with error bars representing standard deviation. RT-PCR Analysis-HEK293T cells were transfected with helicase expression plasmids and 24 h later transfected with poly(I-C) or infected with Sendai virus (Cantell strain, 3 × 106 PFU/well). Total RNA was extracted using TRIzol reagent (Invitrogen). Samples were treated with DNase I (Invitrogen), and 1 μg of RNA was subjected to reverse transcriptase Superscript III (Invitrogen) for cDNA production. Quantitative real time PCR was carried out using the MX3000P real time PCR system (Stratagene) with SYBR green detection. Antiviral Activity Assay-Antiviral responses were measured by cytopathic effect protection assay using VSV (Indiana strain) as a reporter virus. HEK293T cells were transfected for expression of helicase proteins. 24 h later supernatant was harvested, diluted, and added to 2fTGH cells. Following 8 h of incubation with the supernatant, 2fTGH cells were infected with VSV (Indiana strain) at 6 × 103 PFU/well in serum-free media for 1 h. Media were changed and cells incubated for 18 h before staining with 1% methylene blue in 50% ethanol. RNA Binding Assay-For poly(I-C) interaction studies HEK293T cells were transfected for expression of helicase wild type and mutant proteins. Cell lysates were subjected to incubation with poly(I-C)-agarose beads (Sigma), washed in RNA binding buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl), and eluted by addition of SDS loading buffer. Bound protein was analyzed by SDS-PAGE and immunoblotting. Solution RNA binding assays were performed with immunopurified helicases proteins immobilized on anti-FLAG M2 affinity gel (Sigma). RNA substrates were prepared by in vitro transcription using Ambion Maxiscript T7 in the presence of [α-32P]UTP (PerkinElmer Life Sciences). dsRNA constructs were prepared by cotranscription and subsequent annealing of complementary strands. Approximately 500 ng of immobilized proteins and 1 μg of labeled RNA (at approximately 4 × 105 cpm/μg) were incubated for 2 h at 4 °C before washing in RNA binding buffer. Bound RNA was quantified by scintillation counting. Reactions were separated by SDS-PAGE and subjected to immunoblot analysis, and amounts of proteins were estimated using AutoChemi™ BioImaging Systems (UVP). Bound RNA was normalized to the relative amount of protein and presented as percent of wild type. Limited Proteolysis-Proteins were expressed by infection of Sf9 insect cells growing in suspension culture with respective recombinant baculovirus and purified by Ni2+-nitrilotriacetic acid-agarose (Novagen). 10 μg of protein in 120-μl reactions containing 50 mm Tris-HCl, pH 7, 50 mm KCl, 4 mm dithiothreitol, 4 mm MgCl2, 6.5% glycerol were incubated with 10 ng of chymotrypsin. The samples were incubated at 37 °C, and aliquots were removed and stopped by addition of SDS-PAGE loading buffer at indicated time points. Samples were analyzed by SDS-PAGE and immunoblot using antiserum against MDA5 (antigen amino acids 587–600). Identification and Mutation of Conserved Helicase Motifs-Comparison of the amino acid sequences of the MDA5, RIG-I, and LGP2 helicase domains with established consensus sequences of helicase families (33Abdelhaleem M. Maltais L. Wain H. Genomics. 2003; 81: 618-622Crossref PubMed Scopus (132) Google Scholar) clearly identifies characteristic helicase motifs clustered in two subdomains. Domain 1 contains motif I–III and domain 2 contains motif IV–VI (Fig. 1A). The sequences in individual motifs are highly conserved within MDA5, RIG-I, and LGP2 and despite slight variations from the consensus confidently characterize them as a related subset of DEXH box RNA helicases (Fig. 1B). Notably, the highest sequence identity is found between MDA5 and LGP2, which are 62% identical within helicase domain 2, encompassing motif IV–VI, whereas RIG-I and LGP2 or RIG-I and MDA5 are 43 and 44% identical in this region, respectively. The intervening sequences between motifs are also very similar among the three proteins but are poorly conserved with other DEXH box helicases. For example, the closest related human homologue is Dicer-1, sharing about 30% identity with LGP2, MDA5, and RIG-I within the helicase domain 1 and domain 2, but it lacks significant homology in surrounding regions. To examine the role of individual conserved helicase motifs, a series of mutations were engineered in all three helicase proteins (Fig. 1B). Motif I, also known as the Walker A motif, is an NTP-binding motif with a critical lysine residue required for coordination of the NTP. This lysine was targeted by an alanine substitution. Motifs II and III were substituted entirely by alanine residues. Motifs IV, V, and VI are larger motifs and were deleted. An exception is motif IV of MDA5, which was subjected to two alanine substitutions to Phe-724 and Thr-727 instead of a motif deletion as in LGP2 and RIG-I. Expression of the mutant proteins in HEK293T cells resulted in similar levels of protein accumulation with the exception of LGP2 motif II. Independent preparations confirmed this mutant protein to be unstable, possibly the result of mutation-induced misfolding and subsequent proteasomal degradation. To test the effect of these mutations on enzymatic activity, ATP hydrolysis was tested (Fig. 2). Proteins were expressed in HEK293T cells, purified by immunoaffinity chromatography, and incubated with [γ-32P]ATP in the presence or absence of the synthetic dsRNA analogue poly(I-C). The ATP hydrolysis activity of wild type MDA5 and RIG-I proteins was strongly stimulated by poly(I-C) (Fig. 2, A and B), consistent with prior reports (3Yoneyama 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 (3122) Google Scholar, 34Gee P. Chua P.K. Gevorkyan J. Klumpp K. Najera I. Swinney D.C. Deval J. J. Biol. Chem. 2008; 283: 9488-9496Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 35Kang D.C. Gopalkrishnan R.V. Wu Q. Jankowsky E. Pyle A.M. Fisher P.B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 637-642Crossref PubMed Scopus (516) Google Scholar, 36Kovacsovics M. Martinon F. Micheau O. Bodmer J.L. Hofmann K. Tschopp J. Curr. Biol. 2002; 12: 838-843Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Wild type LGP2 exhibited significant ATP hydrolysis activity in the absence of poly(I-C), and the activity was enhanced by addition of RNA (Fig. 2C). For all three proteins, every mutation eliminated ATP hydrolysis activity. These results authenticate the identified helicase motifs as crucial for enzymatic activity. Signal Transduction by Mutant RIG-I and MDA5 Proteins-RNA sensing by MDA5 and RIG-I results in IFNβ gene expression. Targeted gene disruption in mice has indicated that poly(I-C) is primarily detected by MDA5, whereas RIG-I is required for detection of diverse viruses, including Sendai virus (11Kato 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 (2906) Google Scholar). It has been demonstrated previously that Sendai virus Cantell strain preparations are prone to defective interfering particles that can strongly activate the RIG-I system (37Strahle L. Garcin D. Kolakofsky D. Virology. 2006; 351: 101-111Crossref PubMed Scopus (161) Google Scholar, 38Yount J.S. Kraus T.A. Horvath C.M. Moran T.M. Lopez C.B. J. Immunol. 2006; 177: 4503-4513Crossref PubMed Scopus (83) Google Scholar). Irrespective of their in vivo specificity, however, it has been observed that in cell culture models, both helicases can be activated by poly(I-C) and Sendai virus (3Yoneyama 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 (3122) Google Scholar, 21Yoneyama 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 (1290) Google Scholar). Both of these stimuli were used to test the ability of the helicase wild type and mutants to induce the expression of an IFNβ promoter luciferase reporter gene (Fig. 3). We and others (15Cui S. Eisenacher K. Kirchhofer A. Brzozka K. Lammens A. Lammens K. Fujita T. Conzelmann K.K. Krug A. Hopfner K.P. Mol. Cell. 2008; 29: 169-179Abstract Full Text Full Text PDF PubMed Scopus (430) Google Scholar, 39Joo C.H. Shin Y.C. Gack M. Wu L. Levy D. Jung J.U. J. Virol. 2007; 81: 8282-8292Crossref PubMed Scopus (108) Google Scholar) observe that HEK293T cells have a very low endogenous response to both poly(I-C) and Sendai virus in reporter gene assays. This response can be greatly elevated by ectopic expression of MDA5 (Fig. 3A) or RIG-I (Fig. 3C). As observed previously (19Saito T. Hirai R. Loo Y.M. Owen D. Johnson C.L. Sinha S.C. Akira S. Fujita T. Gale Jr., M. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 582-587Crossref PubMed Scopus (583) Google Scholar), expression of wild type MDA5 results in reporter gene activity, irrespective of stimulation, but the auto-regulated RIG-I does not activate the reporter gene in the absence of ligand stimulation. Our analysis of mutant proteins revealed that four MDA5 mutants, with mutations in motifs II or deletions in motifs IV, V and VI, were inactive for reporter gene transcription, resulting in similar profiles as the empty vector control. Two other mutants revealed an unexpected phenotype. Mutations to MDA5 motif I and motif III resulted in strongly induced reporter gene expression in the absence of stimulation, despite their inability to hydrolyze ATP. This strong constitutive activity was more than 3-fold compared with MDA5 wild type, even following poly(

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