The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses

Article metrics


Intracellular double-stranded RNA (dsRNA) is a chief sign of replication for many viruses. Host mechanisms detect the dsRNA and initiate antiviral responses. In this report, we identify retinoic acid inducible gene I (RIG-I), which encodes a DExD/H box RNA helicase that contains a caspase recruitment domain, as an essential regulator for dsRNA-induced signaling, as assessed by functional screening and assays. A helicase domain with intact ATPase activity was responsible for the dsRNA-mediated signaling. The caspase recruitment domain transmitted 'downstream' signals, resulting in the activation of transcription factors NF-κB and IRF-3. Subsequent gene activation by these factors induced antiviral functions, including type I interferon production. Thus, RIG-I is key in the detection and subsequent eradication of the replicating viral genomes.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Identification of the CARD of RIG-I as a positive regulator for type I interferon.
Figure 2: The RIG-I CARD constitutively activates both IRF-3 and NF-κB.
Figure 3: Helicase activity is indispensable for signal transduction by RIG-I.
Figure 4: RIG-I is required for activation of IRF-3 and virus-induced gene expression.
Figure 5: Reduction in viral yield by RIG-I.
Figure 6: Dominant negative RIG-I inhibits NDV-induced but not TLR3-mediated activation of IRF-3.
Figure 7: Dominant negative RIG-I inhibits NDV-induced activation of IRF-3 in primary mouse fibroblasts.


  1. 1

    De Maeyer, E. & De Maeyer-Guignard, J. Type I interferons. Int. Rev. Immunol. 17, 53–73 (1998).

  2. 2

    Farrar, J.D. & Murphy, K.M. Type I interferons and T helper development. Immunol. Today 21, 484–489 (2000).

  3. 3

    Samuel, C.E. Antiviral actions of interferons. Clin. Microbiol. Rev. 14, 778–809 (2001).

  4. 4

    Lin, R., Heylbroeck, C., Pitha, P.M. & Hiscott, J. Virus-dependent phosphorylation of the IRF-3 transcription factor regulates nuclear translocation, transactivation potential, and proteasome-mediated degradation. Mol. Cell. Biol. 18, 2986–2996 (1998).

  5. 5

    Sato, M., Tanaka, N., Hata, N., Oda, E. & Taniguchi, T. Involvement of the IRF family transcription factor IRF-3 in virus-induced activation of the IFN-β gene. FEBS Lett. 425, 112–116 (1998).

  6. 6

    Wathelet, M.G. et al. Virus infection induces the assembly of coordinately activated transcription factors on the IFN-β enhancer in vivo. Mol. Cell 1, 507–518 (1998).

  7. 7

    Weaver, B.K., Kumar, K.P. & Reich, N.C. Interferon regulatory factor 3 and CREB-binding protein/p300 are subunits of double-stranded RNA-activated transcription factor DRAF1. Mol. Cell. Biol. 18, 1359–1368 (1998).

  8. 8

    Yoneyama, M. et al. Direct triggering of the type I interferon system by virus infection: activation of a transcription factor complex containing IRF-3 and CBP/p300. EMBO J. 17, 1087–1095 (1998).

  9. 9

    Fujita, T., Miyamoto, M., Kimura, Y., Hammer, J. & Taniguchi, T. Involvement of a cis-element that binds an H2TF-1/NF-κB like factor(s) in the virus-induced interferon-β gene expression. Nucl. Acids Res. 17, 3335–3346 (1989).

  10. 10

    Lenardo, M.J., Fan, C.M., Maniatis, T. & Baltimore, D. The involvement of NF-κB in β-interferon gene regulation reveals its role as widely inducible mediator of signal transduction. Cell 57, 287–294 (1989).

  11. 11

    Visvanathan, K.V. & Goodbourn, S. Double-stranded RNA activates binding of NF-κB to an inducible element in the human β-interferon promoter. EMBO J. 8, 1129–1138 (1989).

  12. 12

    Du, W. & Maniatis, T. An ATF/CREB binding site is required for virus induction of the human interferon β gene. Proc. Natl. Acad. Sci. USA 89, 2150–2154 (1992).

  13. 13

    Sato, M. et al. Distinct and essential roles of transcription factors IRF-3 and IRF-7 in response to viruses for IFN-α/β gene induction. Immunity 13, 539–548 (2000).

  14. 14

    Suhara, W., Yoneyama, M., Kitabayashi, I. & Fujita, T. Direct involvement of CREB-binding protein/p300 in sequence-specific DNA binding of virus-activated interferon regulatory factor-3 holocomplex. J. Biol. Chem. 277, 22304–22313 (2002).

  15. 15

    Fitzgerald, K.A. et al. IKKε and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4, 491–496 (2003).

  16. 16

    Sharma, S. et al. Triggering the interferon antiviral response through an IKK-related pathway. Science 300, 1148–1151 (2003).

  17. 17

    McWhirter, S.M. et al. IFN-regulatory factor 3-dependent gene expression is defective in Tbk1-deficient mouse embryonic fibroblasts. Proc. Natl. Acad. Sci. USA 101, 233–238 (2004).

  18. 18

    Krieg, A.M. CpG motifs in bacterial DNA and their immune effects. Annu. Rev. Immunol. 20, 709–760 (2002).

  19. 19

    Sing, A. et al. Bacterial induction of β interferon in mice is a function of the lipopolysaccharide component. Infect. Immun. 68, 1600–1607 (2000).

  20. 20

    Alexopoulou, L., Holt, A.C., Medzhitov, R. & Flavell, R.A. Recognition of double-stranded RNA and activation of NF-κB by Toll-like receptor 3. Nature 413, 732–738 (2001).

  21. 21

    Poltorak, A. et al. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science 282, 2085–2088 (1998).

  22. 22

    Hoshino, K. et al. Cutting edge: Toll-like receptor 4 (TLR4)-deficient mice are hyporesponsive to lipopolysaccharide: evidence for TLR4 as the Lps gene product. J. Immunol. 162, 3749–3752 (1999).

  23. 23

    Heil, F. et al. Species-specific recognition of single-stranded RNA via Toll-like receptor 7 and 8. Science 303, 1526–1529 (2004).

  24. 24

    Diebold, S.S., Kaisho, T., Hemmi, H., Akira, S. & Reis, E.S.C. Innate antiviral responses by means of TLR7-mediated recognition of single-stranded RNA. Science 303, 1529–1531 (2004).

  25. 25

    Lund, J.M. et al. Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc. Natl. Acad. Sci. USA 101, 5598–5603 (2004).

  26. 26

    Hemmi, H. et al. A Toll-like receptor recognizes bacterial DNA. Nature 408, 740–745 (2000).

  27. 27

    Akira, S., Takeda, K. & Kaisho, T. Toll-like receptors: critical proteins linking innate and acquired immunity. Nat. Immunol. 2, 675–680 (2001).

  28. 28

    Hoshino, K., Kaisho, T., Iwabe, T., Takeuchi, O. & Akira, S. Differential involvement of IFN-β in Toll-like receptor-stimulated dendritic cell activation. Int. Immunol. 14, 1225–1231 (2002).

  29. 29

    Hoebe, K. et al. Identification of Lps2 as a key transducer of MyD88-independent TIR signalling. Nature 424, 743–748 (2003).

  30. 30

    Oshiumi, H., Matsumoto, M., Funami, K., Akazawa, T. & Seya, T. TICAM-1, an adaptor molecule that participates in Toll-like receptor 3–mediated interferon-β induction. Nat. Immunol. 4, 161–167 (2003).

  31. 31

    Yamamoto, M. et al. Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-β promoter in the Toll-like receptor signaling. J. Immunol. 169, 6668–6672 (2002).

  32. 32

    Yamamoto, M. et al. Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway. Science 301, 640–643 (2003).

  33. 33

    Diebold, S.S. et al. Viral infection switches non-plasmacytoid dendritic cells into high interferon producers. Nature 424, 324–328 (2003).

  34. 34

    Smith, E.J., Marie, I., Prakash, A., Garcia-Sastre, A. & Levy, D.E. IRF3 and IRF7 phosphorylation in virus-infected cells does not require double-stranded RNA-dependent protein kinase R or IκB kinase but is blocked by vaccinia virus E3L protein. J. Biol. Chem. 276, 8951–8957 (2001).

  35. 35

    Iwamura, T. et al. PACT, a double-stranded RNA binding protein acts as a positive regulator for type I interferon gene induced by Newcastle disease virus. Biochem. Biophys. Res. Commun. 282, 515–523 (2001).

  36. 36

    Tanner, N.K. & Linder, P. DExD/H box RNA helicases: from generic motors to specific dissociation functions. Mol. Cell 8, 251–262 (2001).

  37. 37

    Tijsterman, M., Ketting, R.F. & Plasterk, R.H. The genetics of RNA silencing. Annu. Rev. Genet. 36, 489–519 (2002).

  38. 38

    Sun, Y.W. RIG-I, a Human Homolog Gene of RNA Helicase, Is Induced by Retinoic Acid During the Differentiation of Acute Promyelocytic Leukemia Cell. Thesis, Shanghai Second Medical Univ. (1997).

  39. 39

    Zhang, X., Wang, C., Schook, L.B., Hawken, R.J. & Rutherford, M.S. An RNA helicase, RHIV-1, induced by porcine reproductive and respiratory syndrome virus (PRRSV) is mapped on porcine chromosome 10q13. Microb. Pathog. 28, 267–278 (2000).

  40. 40

    Kang, D.C. et al. mda-5: An interferon-inducible putative RNA helicase with double-stranded RNA-dependent ATPase activity and melanoma growth-suppressive properties. Proc. Natl. Acad. Sci. USA 99, 637–642 (2002).

  41. 41

    Inohara, N. & Nunez, G. NODs: intracellular proteins involved in inflammation and apoptosis. Nat. Rev. Immunol. 3, 371–382 (2003).

  42. 42

    Ogura, Y. et al. Nod2, a Nod1/Apaf-1 family member that is restricted to monocytes and activates NF-κB. J. Biol. Chem. 276, 4812–4818 (2001).

  43. 43

    Watanabe, N., Sakakibara, J., Hovanessian, A.G., Taniguchi, T. & Fujita, T. Activation of IFN-β element by IRF-1 requires a posttranslational event in addition to IRF-1 synthesis. Nucl. Acids Res. 19, 4421–4428 (1991).

  44. 44

    Walker, J.E., Saraste, M., Runswick, M.J. & Gay, N.J. Distantly related sequences in the α- and β-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J. 1, 945–951 (1982).

  45. 45

    Bouchier-Hayes, L. & Martin, S.J. CARD games in apoptosis and immunity. EMBO Rep. 3, 616–621 (2002).

  46. 46

    Kovacsovics, M. et al. Overexpression of Helicard, a CARD-containing helicase cleaved during apoptosis, accelerates DNA degradation. Curr. Biol. 12, 838–843 (2002).

  47. 47

    Hoebe, K. et al. Upregulation of costimulatory molecules induced by lipopolysaccharide and double-stranded RNA occurs by Trif-dependent and Trif-independent pathways. Nat. Immunol. 4, 1223–1229 (2003).

  48. 48

    Takahasi, K. et al. X-ray crystal structure of IRF-3 and its functional implications. Nat. Struct. Biol. 10, 922–927 (2003).

  49. 49

    Qin, B.Y. et al. Crystal structure of IRF-3 reveals mechanism of autoinhibition and virus-induced phosphoactivation. Nat. Struct. Biol. 10, 913–921 (2003).

  50. 50

    Tabara, H., Yigit, E., Siomi, H. & Mello, C.C. The dsRNA binding protein RDE-4 interacts with RDE-1, DCR-1, and a DExH-box helicase to direct RNAi in C. elegans. Cell 109, 861–871 (2002).

  51. 51

    Kunkel, T.A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82, 488–492 (1985).

  52. 52

    Imaizumi, T. et al. Retinoic acid-inducible gene-I is induced in endothelial cells by LPS and regulates expression of COX-2. Biochem. Biophys. Res. Commun. 292, 274–279 (2002).

  53. 53

    Yoneyama, M. et al. Autocrine amplification of type I interferon gene expression mediated by interferon stimulated gene factor 3 (ISGF3). J. Biochem. (Tokyo) 120, 160–169 (1996).

  54. 54

    Mori, M. et al. Identification of Ser-386 of interferon regulatory factor 3 as critical target for inducible phosphorylation that determines activation. J. Biol. Chem. 279, 9698–9702 (2004).

  55. 55

    Miyagishi, M. & Taira, K. Strategies for generation of an siRNA expression library directed against the human genome. Oligonucleotides 13, 325–333 (2003).

  56. 56

    Karaghiosoff, M. et al. Central role for type I interferons and Tyk2 in lipopolysaccharide-induced endotoxin shock. Nat. Immunol. 4, 471–477 (2003).

Download references


We thank E.L. Barsoumian for critical reading of the manuscript; S. Saito and M. Kohase for plaque assay protocol and suggestions; and M. Kohara for suggestions on real-time PCR. Supported by the Research for the Future Program; Japan Society for the Promotion of Science; Ministry of Education, Culture, Sports, Science and Technology of Japan; Nippon Boehringer Ingelheim; and Toray Industries.

Author information

Correspondence to Takashi Fujita.

Ethics declarations

Competing interests

The authors of this manuscript have filed a patent regarding RIG-I in collaboration with Toray Industries.

Supplementary information

Supplementary Fig. 1

IFN-treatment is prerequisite to virus-induced activation of IRF-3 in human K562 cells. (PDF 58 kb)

Supplementary Fig. 2

RIG-IC inhibits dsRNA-induced activation of reported gene expression. (PDF 28 kb)

Supplementary Fig. 3

Effect of anti-type I IFN antiserum on viral replication. (PDF 28 kb)

Supplementary Fig. 4

Expression of TBK1 in MEFs. (PDF 36 kb)

Supplementary Fig. 5

IRF-3 is not activated by polyU in mouse primary fibroblasts. (PDF 64 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Yoneyama, M., Kikuchi, M., Natsukawa, T. et al. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol 5, 730–737 (2004) doi:10.1038/ni1087

Download citation

Further reading