FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2063125566> ?p ?o ?g. }

• W2063125566 endingPage "878" @default.
• W2063125566 startingPage "866" @default.
• W2063125566 abstract "The innate immune system detects viral infection predominantly by sensing viral nucleic acids. We report the identification of a viral sensor, consisting of RNA helicases DDX1, DDX21, and DHX36, and the adaptor molecule TRIF, by isolation and sequencing of poly I:C-binding proteins in myeloid dendritic cells (mDCs). Knockdown of each helicase or TRIF by shRNA blocked the ability of mDCs to mount type I interferon (IFN) and cytokine responses to poly I:C, influenza A virus, and reovirus. Although DDX1 bound poly I:C via its Helicase A domain, DHX36 and DDX21 bound the TIR domain of TRIF via their HA2-DUF and PRK domains, respectively. This sensor was localized within the cytosol, independent of the endosomes. Thus, the DDX1-DDX21-DHX36 complex represents a dsRNA sensor that uses the TRIF pathway to activate type I IFN responses in the cytosol of mDCs. The innate immune system detects viral infection predominantly by sensing viral nucleic acids. We report the identification of a viral sensor, consisting of RNA helicases DDX1, DDX21, and DHX36, and the adaptor molecule TRIF, by isolation and sequencing of poly I:C-binding proteins in myeloid dendritic cells (mDCs). Knockdown of each helicase or TRIF by shRNA blocked the ability of mDCs to mount type I interferon (IFN) and cytokine responses to poly I:C, influenza A virus, and reovirus. Although DDX1 bound poly I:C via its Helicase A domain, DHX36 and DDX21 bound the TIR domain of TRIF via their HA2-DUF and PRK domains, respectively. This sensor was localized within the cytosol, independent of the endosomes. Thus, the DDX1-DDX21-DHX36 complex represents a dsRNA sensor that uses the TRIF pathway to activate type I IFN responses in the cytosol of mDCs. DExD/H-box helicases DDX1-DDX21-DHX36 complex senses dsRNA TIR in TRIF domains mediate signaling in response to dsRNA DDX1-DDX21-DHX36 complex sense cytosolic poly I:C in dendritic cells DDX1-DDX21-DHX36 complex signaling is independent on RIG-I and MDA5 signaling Type I interferon (IFN) response represents the most important and powerful innate immune response against viral infection (García-Sastre and Biron, 2006García-Sastre A. Biron C.A. Type 1 interferons and the virus-host relationship: A lesson in détente.Science. 2006; 312: 879-882Crossref PubMed Scopus (676) Google Scholar). The innate immune system detects viral infection predominantly by sensing viral nucleic acids to produce type I IFN (Theofilopoulos et al., 2005Theofilopoulos A.N. Baccala R. Beutler B. Kono D.H. Type I interferons (alpha/beta) in immunity and autoimmunity.Annu. Rev. Immunol. 2005; 23: 307-336Crossref PubMed Scopus (974) Google Scholar, Gilliet et al., 2008Gilliet M. Cao W. Liu Y.J. Plasmacytoid dendritic cells: Sensing nucleic acids in viral infection and autoimmune diseases.Nat. Rev. Immunol. 2008; 8: 594-606Crossref PubMed Scopus (893) Google Scholar.). During the past decade, major efforts with genomic and genetic approaches have identified three major classes of innate immune receptors for sensing microbial nucleic acids including Toll-like receptors (TLR; TLR3, 7, 8, 9) (Iwasaki and Medzhitov, 2004Iwasaki A. Medzhitov R. Toll-like receptor control of the adaptive immune responses.Nat. Immunol. 2004; 5: 987-995Crossref PubMed Scopus (3186) Google Scholar, Takeuchi and Akira, 2007Takeuchi O. Akira S. Recognition of viruses by innate immunity.Immunol. Rev. 2007; 220: 214-224Crossref PubMed Scopus (274) Google Scholar), retinoic acid-inducible gene I-like helicases (RLH: RIG-I, LGP2, MDA-5) (Kato et al., 2006Kato H. Takeuchi O. Sato S. Yoneyama M. Yamamoto M. Matsui K. Uematsu S. Jung A. Kawai T. Ishii K.J. et al.Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses.Nature. 2006; 441: 101-105Crossref PubMed Scopus (2707) Google Scholar, Myong et al., 2009Myong S. Cui S. Cornish P.V. Kirchhofer A. Gack M.U. Jung J.U. Hopfner K.P. Ha T. Cytosolic viral sensor RIG-I is a 5′-triphosphate-dependent translocase on double-stranded RNA.Science. 2009; 323: 1070-1074Crossref PubMed Scopus (272) Google Scholar, Pippig et al., 2009Pippig D.A. Hellmuth J.C. Cui S. Kirchhofer A. Lammens K. Lammens A. Schmidt A. Rothenfusser S. Hopfner K.P. The regulatory domain of the RIG-I family ATPase LGP2 senses double-stranded RNA.Nucleic Acids Res. 2009; 37: 2014-2025Crossref PubMed Scopus (110) Google Scholar, Takeuchi and Akira, 2009Takeuchi O. Akira S. Innate immunity to virus infection.Immunol. Rev. 2009; 227: 75-86Crossref PubMed Scopus (842) Google Scholar), and nucleotide-binding domain and leucine-rich repeat containing (NLR) proteins (Martinon and Tschopp, 2005Martinon F. Tschopp J. NLRs join TLRs as innate sensors of pathogens.Trends Immunol. 2005; 26: 447-454Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, Kufer et al., 2005Kufer T.A. Fritz J.H. Philpott D.J. NACHT-LRR proteins (NLRs) in bacterial infection and immunity.Trends Microbiol. 2005; 13: 381-388Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Poly I:C is a synthetic form of RNA that mimics double-stranded viral RNA. Many cell types, such as epithelial cells, fibroblasts, and myeloid cells, sense poly I:C to produce type I IFN via at least three different sensor systems, including: (1) the TLR3-TRIF endosomal pathway (Akira and Takeda, 2004Akira S. Takeda K. Toll-like receptor signalling.Nat. Rev. Immunol. 2004; 4: 499-511Crossref PubMed Scopus (6339) Google Scholar, Alexopoulou et al., 2001Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3.Nature. 2001; 413: 732-738Crossref PubMed Scopus (4693) Google Scholar, Yamamoto et al., 2002Yamamoto M. Sato S. Mori K. Hoshino K. Takeuchi O. Takeda K. Akira S. Cutting edge: A novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-beta promoter in the Toll-like receptor signaling.J. Immunol. 2002; 169: 6668-6672PubMed Google Scholar, Yamamoto et al., 2003Yamamoto M. Sato S. Hemmi H. Hoshino K. Kaisho T. Sanjo H. Takeuchi O. Sugiyama M. Okabe M. Takeda K. Akira S. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway.Science. 2003; 301: 640-643Crossref PubMed Scopus (2388) Google Scholar); (2) the RIG-I or MDA5-IPS-1 mitochondria pathway (Kato et al., 2005Kato H. Sato S. Yoneyama M. Yamamoto M. Uematsu S. Matsui K. Tsujimura T. Takeda K. Fujita T. Takeuchi O. Akira S. Cell type-specific involvement of RIG-I in antiviral response.Immunity. 2005; 23: 19-28Abstract Full Text Full Text PDF PubMed Scopus (1059) Google Scholar, Yoneyama et al., 2005Yoneyama M. Kikuchi M. Matsumoto K. Imaizumi T. Miyagishi M. Taira K. Foy E. Loo Y.M. Gale Jr., M. Akira S. et al.Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity.J. Immunol. 2005; 175: 2851-2858PubMed Google Scholar, Meylan et al., 2005Meylan E. Curran J. Hofmann K. Moradpour D. Binder M. Bartenschlager R. Tschopp J. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus.Nature. 2005; 437: 1167-1172Crossref PubMed Scopus (1871) Google Scholar, Seth et al., 2005Seth R.B. Sun L. Ea C.K. Chen Z.J. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3.Cell. 2005; 122: 669-682Abstract Full Text Full Text PDF PubMed Scopus (2278) Google Scholar, Matsui et al., 2006Matsui K. Kumagai Y. Kato H. Sato S. Kawagoe T. Uematsu S. Takeuchi O. Akira S. Cutting edge: Role of TANK-binding kinase 1 and inducible IkappaB kinase in IFN responses against viruses in innate immune cells.J. Immunol. 2006; 177: 5785-5789PubMed Google Scholar, Yoneyama and Fujita, 2007Yoneyama M. Fujita T. Function of RIG-I-like receptors in antiviral innate immunity.J. Biol. Chem. 2007; 282: 15315-15318Crossref PubMed Scopus (243) Google Scholar); and (3) the PKR cytosolic pathway (Williams, 2001Williams B.R. Signal integration via PKR.Sci. STKE. 2001; 2001: re2Crossref PubMed Scopus (356) Google Scholar, García et al., 2007García M.A. Meurs E.F. Esteban M. The dsRNA protein kinase PKR: Virus and cell control.Biochimie. 2007; 89: 799-811Crossref PubMed Scopus (418) Google Scholar). Several studies suggested that different cell types may use different receptors to sense poly I:C or viral dsRNA. Although TLR3 was reported to play a key role in sensing poly I:C by epithelial cells (Guillot et al., 2005Guillot L. Le Goffic R. Bloch S. Escriou N. Akira S. Chignard M. Si-Tahar M. Involvement of toll-like receptor 3 in the immune response of lung epithelial cells to double-stranded RNA and influenza A virus.J. Biol. Chem. 2005; 280: 5571-5580Crossref PubMed Scopus (547) Google Scholar, Rudd et al., 2006Rudd B.D. Smit J.J. Flavell R.A. Alexopoulou L. Schaller M.A. Gruber A. Berlin A.A. Lukacs N.W. Deletion of TLR3 alters the pulmonary immune environment and mucus production during respiratory syncytial virus infection.J. Immunol. 2006; 176: 1937-1942PubMed Google Scholar, Matsukura et al., 2007Matsukura S. Kokubu F. Kurokawa M. Kawaguchi M. Ieki K. Kuga H. Odaka M. Suzuki S. Watanabe S. Homma T. et al.Role of RIG-I, MDA-5, and PKR on the expression of inflammatory chemokines induced by synthetic dsRNA in airway epithelial cells.Int. Arch. Allergy Immunol. 2007; 143: 80-83Crossref PubMed Scopus (53) Google Scholar), it only played a moderate or minor role in sensing poly I:C in macrophages or conventional DCs (Alexopoulou et al., 2001Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3.Nature. 2001; 413: 732-738Crossref PubMed Scopus (4693) Google Scholar, Yamamoto et al., 2003Yamamoto M. Sato S. Hemmi H. Hoshino K. Kaisho T. Sanjo H. Takeuchi O. Sugiyama M. Okabe M. Takeda K. Akira S. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway.Science. 2003; 301: 640-643Crossref PubMed Scopus (2388) Google Scholar, López et al., 2004López C.B. Moltedo B. Alexopoulou L. Bonifaz L. Flavell R.A. Moran T.M. TLR-independent induction of dendritic cell maturation and adaptive immunity by negative-strand RNA viruses.J. Immunol. 2004; 173: 6882-6889PubMed Google Scholar). By contrast, RIG-I and MDA5 were found to play a more important role than TLR3 in sensing poly I:C in fibroblasts and DCs (Kato et al., 2005Kato H. Sato S. Yoneyama M. Yamamoto M. Uematsu S. Matsui K. Tsujimura T. Takeda K. Fujita T. Takeuchi O. Akira S. Cell type-specific involvement of RIG-I in antiviral response.Immunity. 2005; 23: 19-28Abstract Full Text Full Text PDF PubMed Scopus (1059) Google Scholar, Kato et al., 2006Kato H. Takeuchi O. Sato S. Yoneyama M. Yamamoto M. Matsui K. Uematsu S. Jung A. Kawai T. Ishii K.J. et al.Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses.Nature. 2006; 441: 101-105Crossref PubMed Scopus (2707) Google Scholar, Kato et al., 2008Kato H. Takeuchi O. Mikamo-Satoh E. Hirai R. Kawai T. Matsushita K. Hiiragi A. Dermody T.S. Fujita T. Akira S. Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5.J. Exp. Med. 2008; 205: 1601-1610Crossref PubMed Scopus (1057) Google Scholar). Although MDA5-IPS-1 was found to preferentially sense long-form poly I:C (over 1000 bp), RIG-I-IPS-1 was found to preferentially sense short form poly I:C (>300 bp and < 1000 bp) or dsRNA with 5′ triphosphate (Kato et al., 2006Kato H. Takeuchi O. Sato S. Yoneyama M. Yamamoto M. Matsui K. Uematsu S. Jung A. Kawai T. Ishii K.J. et al.Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses.Nature. 2006; 441: 101-105Crossref PubMed Scopus (2707) Google Scholar, Kato et al., 2008Kato H. Takeuchi O. Mikamo-Satoh E. Hirai R. Kawai T. Matsushita K. Hiiragi A. Dermody T.S. Fujita T. Akira S. Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5.J. Exp. Med. 2008; 205: 1601-1610Crossref PubMed Scopus (1057) Google Scholar, Schlee et al., 2009aSchlee M. Roth A. Hornung V. Hagmann C.A. Wimmenauer V. Barchet W. Coch C. Janke M. Mihailovic A. Wardle G. et al.Recognition of 5′ triphosphate by RIG-I helicase requires short blunt double-stranded RNA as contained in panhandle of negative-strand virus.Immunity. 2009; 31: 25-34Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar, Schlee et al., 2009bSchlee M. Hartmann E. Coch C. Wimmenauer V. Janke M. Barchet W. Hartmann G. Approaching the RNA ligand for RIG-I?.Immunol. Rev. 2009; 227: 66-74Crossref PubMed Scopus (64) Google Scholar). The genomic and genetic approaches have left a major gap in our understanding of how these receptors bind nucleic acids and whether additional receptors or coreceptors exist. For example, although it is known that TLR3 uses TRIF as an adaptor molecule for signal transduction when sensing poly I:C, cells derived from TRIF-deficient mice displayed more reduced IFN-β and NF-κB responses than those of cells derived from TLR3-deficient mice in response to poly I:C (Yamamoto et al., 2003Yamamoto M. Sato S. Hemmi H. Hoshino K. Kaisho T. Sanjo H. Takeuchi O. Sugiyama M. Okabe M. Takeda K. Akira S. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway.Science. 2003; 301: 640-643Crossref PubMed Scopus (2388) Google Scholar), suggesting the presence of a TLR3-independent, TRIF-dependent poly I:C sensor. We investigated this issue by isolating and characterizing poly I:C-binding proteins in mDCs by using biotinylated poly I:C and protein pull-down experiments, followed by protein sequencing with liquid chromatography (LC)-mass spectrometry. We found that poly I:C pulled down two known dsRNA sensors, PKR and LGP2, as well as three new members of the DExD/H-box helicase family, DDX1, DDX21, and DHX36 (Fuller-Pace, 2006Fuller-Pace F.V. DExD/H box RNA helicases: Multifunctional proteins with important roles in transcriptional regulation.Nucleic Acids Res. 2006; 34: 4206-4215Crossref PubMed Scopus (295) Google Scholar, Linder, 2006Linder P. Dead-box proteins: A family affair—active and passive players in RNP-remodeling.Nucleic Acids Res. 2006; 34: 4168-4180Crossref PubMed Scopus (335) Google Scholar). We demonstrated here that DDX1, DDX21, DHX36 represent a dsRNA sensor that uses the TRIF pathway to activate type I IFN responses. We generated biotinylated poly I:C that was 0.2–1 kb in length; poly I:C is frequently used to induce type I IFN responses. Biotinylated poly A:U (bio-poly A:U) was also generated to use as a control (Table S1 and Figure S1 available online). To purify poly I:C-bound protein complexes, we initially incubated D2SC cells with culture medium, poly I:C, bio-poly I:C, biotin, or bio-poly A:U for 8 hr. Whole-cell lysates from the treated D2SC cells were prepared and subjected to purification with NeutrAvidin beads (NA beads). The proteins bound to bio-poly I:C were separated by gel electrophoresis. As shown in Figure 1A , we identified several protein bands that were unique to the bio-poly I:C, yet absent from the controls. The protein bands within 250 kD to 25 kD from bio-poly I:C and poly I:C pull-downs were excised from the gel and analyzed by LC-mass spectrometry. We obtained ∼50 unique sequences with five or more hits. We found two known dsRNA sensors, PKR and LGP2, which validated our method for isolating dsRNA-binding proteins in mDCs. However, we did not find the two other known dsRNA sensors, RIG-I and MDA5. We also identified three members of the DExD/H helicase family, DDX1, DDX21, and DHX36. Because members of this family, including RIG-I (DDX58) (Yoneyama et al., 2004Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses.Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (2934) Google Scholar), LGP2 (DHX58) (Rothenfusser et al., 2005Rothenfusser S. Goutagny N. DiPerna G. Gong M. Monks B.G. Schoenemeyer A. Yamamoto M. Akira S. Fitzgerald K.A. The RNA helicase Lgp2 inhibits TLR-independent sensing of viral replication by retinoic acid-inducible gene-I.J. Immunol. 2005; 175: 5260-5268PubMed Google Scholar), MDA5 (IFIH1) (Kato et al., 2006Kato H. Takeuchi O. Sato S. Yoneyama M. Yamamoto M. Matsui K. Uematsu S. Jung A. Kawai T. Ishii K.J. et al.Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses.Nature. 2006; 441: 101-105Crossref PubMed Scopus (2707) Google Scholar), and Dicer (Deddouche et al., 2008Deddouche S. Matt N. Budd A. Mueller S. Kemp C. Galiana-Arnoux D. Dostert C. Antoniewski C. Hoffmann J.A. Imler J.L. The DExD/H-box helicase Dicer-2 mediates the induction of antiviral activity in drosophila.Nat. Immunol. 2008; 9: 1425-1432Crossref PubMed Scopus (239) Google Scholar), have been shown to play key roles in sensing dsRNA and viral infection, we investigated the function of DDX1, DDX21, and DHX36. We established stable D2SC cell lines, which are derived from BALB/c mice spleen primary cells and have functional attributes of immature DCs, expressing small heteroduplex RNA (shRNA) to knockdown expression of DDX1, DDX21, DHX36, RIG-I, MDA5, IPS-1, or TLR3. Two different clones of shRNA for DDX1, DDX21, and DHX36 were selected after screening the shRNA sets from Open Biosystems. A scrambled shRNA served as the control. Efficient knockdown of protein expression was confirmed, as shown in Figure 1B. The cells were then stimulated by a short poly I:C (0.2-1 kb) or long poly I:C (1.5–8 kb) delivered with or without Lipofectamine 2000. The production of type I IFN (IFN-α and IFN-β), TNF-α, and IL-6 by the cultured cells was measured by ELISA. D2SC mDCs treated with scrambled shRNA produced high amounts of type I IFN, TNF-α, and IL-6 after stimulation with a short poly I:C delivered with Lipofectamine 2000. This cytokine response was strongly attenuated (∼80%) in D2SC mDCS expressing shRNA targeting DDX1, DDX21, DHX36, TRIF, or IPS-1, partially attenuated (∼50%) in D2SC mDCs expressing shRNA targeting RIG-I and was not affected in D2SC mDCs expressing shRNA targeting TLR3 or MDA5 (Figure 1C and Figure S2). Because Lipofectamine 2000 may deliver the majority of poly I:C into the cytosol, we determined the cytokines produced by D2SC cells in response to poly I:C without Lipofectamine 2000, in order to measure the endosomal poly I:C sensing by TLR3 (Figure 1C). DDX1, DDX21, and DHX36 knockdown led to a 60% reduction in type I IFN production, whereas RIG-I, IPS-1, and TRIF knockdown led to 80% reduction in type I IFN. Again, MDA5 knockdown had no effect on type I IFN production by D2SC mDCs in response to short poly I:C with or without Lipofectamine 2000, confirming a previous report showing that MDA5 only plays an important role in sensing long poly I:C (Kato et al., 2006Kato H. Takeuchi O. Sato S. Yoneyama M. Yamamoto M. Matsui K. Uematsu S. Jung A. Kawai T. Ishii K.J. et al.Differential roles of MDA5 and RIG-I helicases in the recognition of RNA viruses.Nature. 2006; 441: 101-105Crossref PubMed Scopus (2707) Google Scholar). Although TLR3 knockdown had no marked effect on the cytokine responses of D2SC cells to poly I:C with Lipofectamine 2000, it led to a 60% and 70% reduction in IFN-α and IFN-β production, respectively, by D2SC cells in response to poly I:C without Lipofectamine 2000, confirming a previous study showing that TLR3 only senses poly I:C in the endosomes (Diebold et al., 2003Diebold S.S. Montoya M. Unger H. Alexopoulou L. Roy P. Haswell L.E. Al-Shamkhani A. Flavell R. Borrow P. Reis e Sousa C. Viral infection switches non-plasmacytoid dendritic cells into high interferon producers.Nature. 2003; 424: 324-328Crossref PubMed Scopus (498) Google Scholar). D2SC cells treated with TRIF shRNA displayed more reduced cytokine responses than the D2SC cells treated with TLR3 shRNA in response to poly I:C, suggesting the presence of TLR3-independent, TRIF-dependent poly I:C sensors. A similar effect of the above molecules on TNF-α and IL-6 production by D2SC in response to poly I:C without Lipofectamine 2000 was observed (Figure S2). These data indicate that DDX1, DDX21, DHX36, TRIF, RIG-I, and IPS-1 all play important roles in sensing short poly I:C, MDA-5 plays no role in sensing short poly I:C, and TLR3 only senses short poly I:C in the endosomes. We next investigated whether DDX1, DDX21, and DHX36 also play a role in sensing long poly I:C (1.5–8 kb) in D2SC cells. DDX1, DDX21, DHX36, and TRIF knockdown resulted in about a 40%–50% reduction in IFN-β production by D2SC cells in response to long poly I:C with or without Lipofectamine delivery. Whereas MDA5 and IPS-1 knockdown led to a 90% of reduction in type I IFN, RIG-I and TLR3 knockdown had little effect on type I IFN production by D2SC cells in response to long poly I:C (Figure 1D). Our data suggest that DDX1, DDX21, DHX36, and TRIF all play important roles in sensing both short (0.2–1 kb) and long (1.5–8 kb) poly I:C in D2SC cells. Our study also confirms a previous study showing that MDA5-IPS-1 preferentially sense long poly I:Cs and RIG-I-IPS-1 preferentially sense short poly I:Cs in DCs (Kato et al., 2008Kato H. Takeuchi O. Mikamo-Satoh E. Hirai R. Kawai T. Matsushita K. Hiiragi A. Dermody T.S. Fujita T. Akira S. Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5.J. Exp. Med. 2008; 205: 1601-1610Crossref PubMed Scopus (1057) Google Scholar). In addition, TLR3 only plays an important role in sensing short poly I:C without Lipofectamine delivery. To further confirm the role of DDX1, DDX21, and DHX36 in sensing poly I:C, we overexpressed DDX1, DDX21, DHX36, TRIF, or RIG-I in L929 cells, a mouse fibroblast cell line that was used previously for RIG-I overexpression experiments (Yoneyama et al., 2004Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses.Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (2934) Google Scholar). We found that overexpression of DDX1, DDX21, DHX36, or TRIF led to increased IFN-β promoter activation in L929 cells after stimulation with short or long poly I:C (Figure 1E). Overexpression of RIG-I led to increased IFN-β promoter activation in L929 cells after stimulation with short poly I:C, but not with long poly I:C. To determine whether DDX1, DDX21, and DHX36 sense other nucleic acids, we stimulated D2SC cells treated with shRNA with 5′ triphosphate RNA (RIG-I ligand) (Myong et al., 2009Myong S. Cui S. Cornish P.V. Kirchhofer A. Gack M.U. Jung J.U. Hopfner K.P. Ha T. Cytosolic viral sensor RIG-I is a 5′-triphosphate-dependent translocase on double-stranded RNA.Science. 2009; 323: 1070-1074Crossref PubMed Scopus (272) Google Scholar) or poly dA-dT. As shown in Figure S2, DDX1, DDX21, and DHX36 knockdown, as well as MDA5 knockdown, had little effect on the production of type I IFN by the mDCs in response to poly dA-dT or 5′ triphosphate RNA, whereas IPS-1 and RIG-I knockdown led to a 90% reduction in type I IFN levels in response to RIG-I ligand 5′ triphosphate RNA. The size of RNA ligands used is shown in Figure S2. To determine the function of DDX1, DDX21, and DHX36 in sensing viral infection, we cultured D2SC cells treated with shRNA with influenza A virus. DDX1, DDX21, and DHX36 knockdown resulted in about a 60%–70% reduction in IFN-β production and a 40%–50% reduction in TNF-α production by D2SC cells in response to influenza A virus (Figure 1F). We confirmed a previous study showing that TRIF, RIG-I, and IPS-1 play critical roles, TLR3 plays a moderate role, and MDA5 plays no role in sensing influenza A viral infection (Guillot et al., 2005Guillot L. Le Goffic R. Bloch S. Escriou N. Akira S. Chignard M. Si-Tahar M. Involvement of toll-like receptor 3 in the immune response of lung epithelial cells to double-stranded RNA and influenza A virus.J. Biol. Chem. 2005; 280: 5571-5580Crossref PubMed Scopus (547) Google Scholar, Loo et al., 2008Loo Y.M. Fornek J. Crochet N. Bajwa G. Perwitasari O. Martinez-Sobrido L. Akira S. Gill M.A. García-Sastre A. Katze M.G. Gale Jr., M. Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity.J. Virol. 2008; 82: 335-345Crossref PubMed Scopus (784) Google Scholar, Kato et al., 2008Kato H. Takeuchi O. Mikamo-Satoh E. Hirai R. Kawai T. Matsushita K. Hiiragi A. Dermody T.S. Fujita T. Akira S. Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma differentiation-associated gene 5.J. Exp. Med. 2008; 205: 1601-1610Crossref PubMed Scopus (1057) Google Scholar). To further determine whether DDX1/DDX21/DHX36 sense other RNA viruses, we cultured D2SC cells treated with shRNA with reovirus. DDX1, DDX21, and DHX36 knockdown all resulted in about a 70%–80% reduction in IFN-β production and a 60%–70% reduction in TNF-α production by D2SC cells in response to reovirus (Figure 1G). To determine whether the three helicases directly bind poly I:C, we prepared recombinant HA-tagged helicases by transfecting HEK293T cells with plasmids encoding the recombinant proteins and then purifying them with anti-HA beads. Each purified helicase was then incubated with bio-poly I:C. Only DDX1, but not DDX21 or DHX36, was found to bind poly I:C (Figure 2A ). By performing competition experiments using increasing amounts of unlabeled poly I:C, poly A:U, CpG, and poly A, we found that only unlabeled poly I:C could block the binding of bio-poly I:C to DDX1 (Figure 2B). To map the poly I:C-binding site of DDX1, we prepared truncated versions of DDX1. Bio-poly I:C pull-down experiments indicated that the Helicase A domain of DDX1 binds poly I:C (Figure 2C). To determine whether recombinant DDX1 could rescue the DDX1 shRNA-induced defect, HA-DDX1a (full size DDX1) or HA-DDX1g (deletion of poly I:C binding site) was expressed in the DDX1 shRNA cells (Figure 2D). This shRNA selectively targets the 3′ UTR of DDX1 mRNA so that only the expression of endogenous DDX1 was knocked down. As shown in Figure 2E, the full size DDX1 could rescue the IFN-β responses to short and long poly I:C, whereas The DDX1 with deletion of the poly I:C-binding domain failed to rescue. These data indicate that DDX1 dsRNA binding activity is necessary for eliciting the type I IFN response to poly I:C. Because DDX1, DDX21, DHX36, and TRIF knockdown displayed similar effects on the cytokine responses of D2SC cells to short and long poly I:C, we hypothesized that DDX1, DDX21, and DHX36 may use TRIF as an adaptor molecule for signal transduction. A Myc-tagged version of TRIF was incubated with HA-tagged versions of DDX1, DHX36, or DDX21. Anti-Myc pull-down experiments showed that TRIF binds DDX21 and DHX36, but not the poly I:C binding DDX1 (Figure 3A ). To map the binding site of TRIF with DDX21 and DHX36, we prepared truncated versions of TRIF and conducted pull-down assays with DDX21 or DHX36. As indicated in Figure 3B, we observed that the TIR domain of TRIF interacts with DDX21 and DHX36. To determine which domains of DDX21 and DHX36 mediate interaction with TRIF, we incubated Myc-TRIF with truncated versions of DDX21 or DHX36. As indicated in Figures 3D and 3E, DDX21 and DHX36 bind TRIF via their PRK and HA2-DUF domains, respectively. Because DDX1, DDX21, and DHX36 appear to play similar roles in sensing poly I:C in D2SC cells, we investigated whether the three helicases have the ability to form a complex. Myc-tagged versions of DDX1 or DDX21 were incubated with HA-tagged versions of DDX1, DHX36, or DDX21. Anti-Myc pull-down experiments showed that DDX1 binds DDX21, DDX21 binds DHX36, and DDX21 can bind itself, whereas there is no direct interaction between DDX1 and DHX36 (Figure 3A). The domains involved in DDX1, DDX21, and DHX36 interactions were further determined by mutagenesis experiments. HA-tagged versions of DDX1, DDX21, and DHX36 were prepared and incubated with Myc-tagged versions of DDX1, DDX21, or DHX36. Pull-down experiments indicated that the SPRY domain of DDX1 binds the PRK domain of DDX21 (Figures 3C and 3E). The PRK domain of DDX21 binds the Helicase C-HA2-DUF domains of DHX36 (Figures 3D and 3E). The N terminus of DDX21 can bind together (Figure 3E). To further determine whether endogenous DDX1, DDX21, and DHX36 exist as a complex in D2SC cells, we cultured the cells with medium or poly I:C for 16 hr and then immunoprecipitated them by using antibody to DDX1, DDX21, DHX36, TRIF, or RIG-I. DDX1 antibody precipitated DDX21 and DDX36, DDX21 antibody precipitated DDX1 and DDX36, and the DDX36 antibody precipitated DDX1 and DDX21 (Figure 4A ). Interestingly, the antibody against TRIF (Figure 4A), but not RIG-I or MDA5 (data not shown), precipitated all three helicases. These data showed that endogenous DDX1, DDX21, DHX36, and TRIF proteins exist as a complex in D2SC cells, with or without poly I:C stimulation. To determine whether poly I:C stimulation modified the expression of the endogenous DDX1, DDX21, DHX36, and TRIF protein complex, D2SC cells were incubated with bio-poly I:C for 10, 20, and 30 min. Whole-cell lysates from the treated D2SC cells were prepared and subjected to precipitation with avidin-conjugated beads. The proteins bound to bio-poly I:C were detected by antibodies against DDX1, DDX21, DHX36, and RIG-I. DDX1, DDX21, and DHX36, but not RIG-I, could be detected after stimulation for 10, 20, and 30 min (Figure 4B). These data indicate that the DDX1, DDX21, DHX36, and TRIF complex exists in resting cells and the complex formation does not require stimulation. Because anti" @default.
• W2063125566 created "2016-06-24" @default.
• W2063125566 creator A5002701262 @default.
• W2063125566 creator A5007234866 @default.
• W2063125566 creator A5018805094 @default.
• W2063125566 creator A5029777489 @default.
• W2063125566 creator A5044949766 @default.
• W2063125566 creator A5059452261 @default.
• W2063125566 creator A5064099072 @default.
• W2063125566 creator A5072050974 @default.
• W2063125566 creator A5082109212 @default.
• W2063125566 date "2011-06-01" @default.
• W2063125566 modified "2023-10-15" @default.
• W2063125566 title "DDX1, DDX21, and DHX36 Helicases Form a Complex with the Adaptor Molecule TRIF to Sense dsRNA in Dendritic Cells" @default.
• W2063125566 cites W1569857352 @default.
• W2063125566 cites W1592851115 @default.
• W2063125566 cites W1965892231 @default.
• W2063125566 cites W1967686012 @default.
• W2063125566 cites W1974623065 @default.
• W2063125566 cites W1975296966 @default.
• W2063125566 cites W1993728991 @default.
• W2063125566 cites W1995323565 @default.
• W2063125566 cites W2000952771 @default.
• W2063125566 cites W2004465286 @default.
• W2063125566 cites W2010091770 @default.
• W2063125566 cites W2013601771 @default.
• W2063125566 cites W2015686762 @default.
• W2063125566 cites W2018828712 @default.
• W2063125566 cites W2028012273 @default.
• W2063125566 cites W2028575270 @default.
• W2063125566 cites W2032948492 @default.
• W2063125566 cites W2033431215 @default.
• W2063125566 cites W2042433752 @default.
• W2063125566 cites W2043379214 @default.
• W2063125566 cites W2052798287 @default.
• W2063125566 cites W2057704272 @default.
• W2063125566 cites W2066425910 @default.
• W2063125566 cites W2066728093 @default.
• W2063125566 cites W2070306789 @default.
• W2063125566 cites W2071727569 @default.
• W2063125566 cites W2076361525 @default.
• W2063125566 cites W2079778407 @default.
• W2063125566 cites W2085487477 @default.
• W2063125566 cites W2088534886 @default.
• W2063125566 cites W2090603241 @default.
• W2063125566 cites W2090640877 @default.
• W2063125566 cites W2093579074 @default.
• W2063125566 cites W2097556922 @default.
• W2063125566 cites W2099039895 @default.
• W2063125566 cites W2100665873 @default.
• W2063125566 cites W2107513655 @default.
• W2063125566 cites W2114084606 @default.
• W2063125566 cites W2115185894 @default.
• W2063125566 cites W2125566625 @default.
• W2063125566 cites W2130421043 @default.
• W2063125566 cites W2137303998 @default.
• W2063125566 cites W2138677670 @default.
• W2063125566 cites W2140430757 @default.
• W2063125566 cites W2140692786 @default.
• W2063125566 cites W2141137217 @default.
• W2063125566 cites W2148387062 @default.
• W2063125566 cites W2148985310 @default.
• W2063125566 cites W2156354476 @default.
• W2063125566 cites W2156602474 @default.
• W2063125566 cites W2159685069 @default.
• W2063125566 cites W2167457380 @default.
• W2063125566 cites W4361866506 @default.
• W2063125566 cites W2098751820 @default.
• W2063125566 doi "https://doi.org/10.1016/j.immuni.2011.03.027" @default.
• W2063125566 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/3652560" @default.
• W2063125566 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/21703541" @default.
• W2063125566 hasPublicationYear "2011" @default.
• W2063125566 type Work @default.
• W2063125566 sameAs 2063125566 @default.
• W2063125566 citedByCount "311" @default.
• W2063125566 countsByYear W20631255662012 @default.
• W2063125566 countsByYear W20631255662013 @default.
• W2063125566 countsByYear W20631255662014 @default.
• W2063125566 countsByYear W20631255662015 @default.
• W2063125566 countsByYear W20631255662016 @default.
• W2063125566 countsByYear W20631255662017 @default.
• W2063125566 countsByYear W20631255662018 @default.
• W2063125566 countsByYear W20631255662019 @default.
• W2063125566 countsByYear W20631255662020 @default.
• W2063125566 countsByYear W20631255662021 @default.
• W2063125566 countsByYear W20631255662022 @default.
• W2063125566 countsByYear W20631255662023 @default.
• W2063125566 crossrefType "journal-article" @default.
• W2063125566 hasAuthorship W2063125566A5002701262 @default.
• W2063125566 hasAuthorship W2063125566A5007234866 @default.
• W2063125566 hasAuthorship W2063125566A5018805094 @default.
• W2063125566 hasAuthorship W2063125566A5029777489 @default.
• W2063125566 hasAuthorship W2063125566A5044949766 @default.
• W2063125566 hasAuthorship W2063125566A5059452261 @default.
• W2063125566 hasAuthorship W2063125566A5064099072 @default.
• W2063125566 hasAuthorship W2063125566A5072050974 @default.
• W2063125566 hasAuthorship W2063125566A5082109212 @default.
• W2063125566 hasBestOaLocation W20631255661 @default.

• next