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The NEMO adaptor bridges the nuclear factor-κB and interferon regulatory factor signaling pathways

Nature Immunology volume 8, pages 592–600 (2007)Cite this article

Abstract

Intracellular detection of RNA virus infection is mediated by the RNA helicase RIG-I, which is recruited to mitochondria by the adaptor protein MAVS and triggers activation of the transcription factors NF-κB, IRF3 and IRF7. Here we demonstrate that virus-induced activation of IRF3 and IRF7 depended on the NF-κB modulator NEMO, which acted 'upstream' of the kinases TBK1 and IKKε. IRF3 phosphorylation, formation of IRF3 dimers and DNA binding, as well as IRF3-dependent gene expression, were abrogated in NEMO-deficient cells. IRF3 phosphorylation and interferon production were restored by ectopic expression of NEMO. Thus, NEMO, like MAVS, acts as an adaptor protein that allows RIG-I to activate both the NF-κB and IRF signaling pathways.

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Figure 1: Responses to RNA virus infection in NEMO-deficient MEFs.
Figure 2: NEMO is required for the activation of ISRE and IFNA promoters.
Figure 3: Impaired IRF3 and IRF7 activation in Ikbkg−/− MEFs.
Figure 4: Defective antiviral response in Ikbkg−/− MEFs.
Figure 5: TANK links IKKε and TBK1 to NEMO.
Figure 6: NEMO domains required for ISRE activation.
Figure 7: NEMO point substitutions affecting activation of the ISRE promoter.

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References

  1. Honda, K., Takaoka, A. & Taniguchi, T. Type I inteferon gene induction by the interferon regulatory factor family of transcription factors. Immunity 25, 349–360 (2006).

    Article  CAS  Google Scholar 

  2. Kawai, T. & Akira, S. Innate immune recognition of viral infection. Nat. Immunol. 7, 131–137 (2006).

    Article  CAS  Google Scholar 

  3. Meylan, E. & Tschopp, J. Toll-like receptors and RNA helicases: two parallel ways to trigger antiviral responses. Mol. Cell 22, 561–569 (2006).

    Article  CAS  Google Scholar 

  4. Stetson, D.B. & Medzhitov, R. Type I interferons in host defense. Immunity 25, 373–381 (2006).

    Article  CAS  Google Scholar 

  5. Pichlmair, A. et al. RIG-I-mediated antiviral responses to single-stranded RNA bearing 5′-phosphates. Science 314, 997–1001 (2006).

    Article  CAS  Google Scholar 

  6. Hornung, V. et al. 5′-Triphosphate RNA is the ligand for RIG-I. Science 314, 994–997 (2006).

    Article  Google Scholar 

  7. Yoneyama, M. 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).

    Article  CAS  Google Scholar 

  8. Maniatis, T. et al. Structure and function of the interferon-β enhanceosome. Cold Spring Harb. Symp. Quant. Biol. 63, 609–620 (1998).

    Article  CAS  Google Scholar 

  9. Taniguchi, T., Ogasawara, K., Takaoka, A. & Tanaka, N. IRF family of transcription factors as regulators of host defense. Annu. Rev. Immunol. 19, 623–655 (2001).

    Article  CAS  Google Scholar 

  10. Hiscott, J., Nguyen, T.L., Arguello, M., Nakhaei, P. & Paz, S. Manipulation of the nuclear factor-κB pathway and the innate immune response by viruses. Oncogene 25, 6844–6867 (2006).

    Article  CAS  Google Scholar 

  11. van Boxel-Dezaire, A.H., Rani, M.R. & Stark, G.R. Complex modulation of cell type-specific signaling in response to type I interferons. Immunity 25, 361–372 (2006).

    Article  CAS  Google Scholar 

  12. Chen, Z.J. Ubiquitin signalling in the NF-κB pathway. Nat. Cell Biol. 7, 758–765 (2005).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Seth, R.B., Sun, L., Ea, C.K. & Chen, Z.J. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-κB and IRF3. Cell 122, 669–682 (2005).

    Article  CAS  Google Scholar 

  16. Xu, L.G. et al. VISA is an adapter protein required for virus-triggered IFN-β signaling. Mol. Cell 19, 727–740 (2005).

    Article  CAS  Google Scholar 

  17. Kawai, T. et al. IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nat. Immunol. 6, 981–988 (2005).

    Article  CAS  Google Scholar 

  18. Meylan, E. et al. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 437, 1167–1172 (2005).

    Article  CAS  Google Scholar 

  19. Pomerantz, J.L. & Baltimore, D. NF-κB activation by a signaling complex containing TRAF2, TANK and TBK1, a novel IKK-related kinase. EMBO J. 18, 6694–6704 (1999).

    Article  CAS  Google Scholar 

  20. Marie, I., Durbin, J.E. & Levy, D.E. Differential viral induction of distinct interferon-α genes by positive feedback through interferon regulatory factor-7. EMBO J. 17, 6660–6669 (1998).

    Article  CAS  Google Scholar 

  21. Zhang, S.Q., Kovalenko, A., Cantarella, G. & Wallach, D. Recruitment of the IKK signalosome to the p55 TNF receptor: RIP and A20 bind to NEMO (IKKγ) upon receptor stimulation. Immunity 12, 301–311 (2000).

    Article  CAS  Google Scholar 

  22. Servant, M.J. et al. Identification of the minimal phosphoacceptor site required for in vivo activation of interferon regulatory factor 3 in response to virus and double-stranded RNA. J. Biol. Chem. 278, 9441–9447 (2003).

    Article  CAS  Google Scholar 

  23. 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).

    Article  CAS  Google Scholar 

  24. Chariot, A. et al. Association of the adaptor TANK with the IκB kinase (IKK) regulator NEMO connects IKK complexes with IKKε and TBK1 kinases. J. Biol. Chem. 277, 37029–37036 (2002).

    Article  CAS  Google Scholar 

  25. Guo, B. & Chang, G. Modulation of the interferon antiviral response by the TBK1/IKKI adaptor protein tank. J. Biol. Chem. 282, 11817–11826 (2007).

    Article  CAS  Google Scholar 

  26. Agou, F. et al. The trimerization domain of NEMO is composed of the interacting C-terminal CC2 and LZ coiled-coil subdomains. J. Biol. Chem. 279, 27861–27869 (2004).

    Article  CAS  Google Scholar 

  27. Uzel, G. The range of defects associated with nuclear factor κB essential modulator. Curr. Opin. Allergy Clin. Immunol. 5, 513–518 (2005).

    Article  CAS  Google Scholar 

  28. Ku, C.L. et al. NEMO mutations in two unrelated boys with severe infections and conical teeth. Pediatrics 115, e615–e619 (2005).

    Article  Google Scholar 

  29. Wu, C.J., Conze, D.B., Li, T., Srinivasula, S.M. & Ashwell, J.D. NEMO is a sensor of Lys 63-linked polyubiquitination and functions in NF-κB activation. Nat. Cell Biol. 8, 398–406 (2006).

    Article  CAS  Google Scholar 

  30. Ea, C.K., Deng, L., Xia, Z.P., Pineda, G. & Chen, Z.J. Activation of IKK by TNFα requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Mol. Cell 22, 245–257 (2006).

    Article  CAS  Google Scholar 

  31. Rudolph, D. et al. Severe liver degeneration and lack of NF-κB activation in NEMO/IKKγ-deficient mice. Genes Dev. 14, 854–862 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Makris, C. et al. Female mice heterozygous for IKKγ/NEMO deficiencies develop a dermatopathy similar to the human X-linked disorder incontinentia pigmenti. Mol. Cell 5, 969–979 (2000).

    Article  CAS  Google Scholar 

  33. Nomura, F., Kawai, T., Nakanishi, K. & Akira, S. NF-κB activation through IKK-i-dependent I-TRAF/TANK phosphorylation. Genes Cells 5, 191–202 (2000).

    Article  CAS  Google Scholar 

  34. Sasai, M. et al. Cutting Edge: NF-κB-activating kinase-associated protein 1 participates in TLR3/Toll-IL-1 homology domain-containing adapter molecule-1-mediated IFN regulatory factor 3 activation. J. Immunol. 174, 27–30 (2005).

    Article  CAS  Google Scholar 

  35. Hiscott, J., Lin, R., Nakhaei, P. & Paz, S. MasterCARD: a priceless link to innate immunity. Trends Mol. Med. 12, 53–56 (2006).

    Article  CAS  Google Scholar 

  36. Saha, S.K. et al. Regulation of antiviral responses by a direct and specific interaction between TRAF3 and Cardif. EMBO J. 25, 3257–3263 (2006).

    Article  CAS  Google Scholar 

  37. Lin, R. et al. Dissociation of a MAVS/IPS-1/VISA/Cardif-IKKε molecular complex from the mitochondrial outer membrane by hepatitis C virus NS3–4A proteolytic cleavage. J. Virol. 80, 6072–6083 (2006).

    Article  CAS  Google Scholar 

  38. Nelson, D.L. NEMO, NFκB signaling and incontinentia pigmenti. Curr. Opin. Genet. Dev. 16, 282–288 (2006).

    Article  CAS  Google Scholar 

  39. Orange, J.S. et al. The presentation and natural history of immunodeficiency caused by nuclear factor κB essential modulator mutation. J. Allergy Clin. Immunol. 113, 725–733 (2004).

    Article  CAS  Google Scholar 

  40. Lin, R. et al. Negative regulation of the retinoic acid-inducible gene I-induced antiviral state by the ubiquitin-editing protein A20. J. Biol. Chem. 281, 2095–2103 (2006).

    Article  CAS  Google Scholar 

  41. Harris, J. et al. Nuclear accumulation of cRel following C-terminal phosphorylation by TBK1/IKKε. J. Immunol. 177, 2527–2535 (2006).

    Article  CAS  Google Scholar 

  42. Carter, R.S., Pennington, K.N., Ungurait, B.J. & Ballard, D.W. In vivo identification of inducible phosphoacceptors in the IKKγ/NEMO subunit of human IκB kinase. J. Biol. Chem. 278, 19642–19648 (2003).

    Article  CAS  Google Scholar 

  43. Morgenstern, J.P. & Land, H. Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18, 3587–3596 (1990).

    Article  CAS  Google Scholar 

  44. 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).

    Article  CAS  Google Scholar 

  45. Li, Z.W. et al. The IKKβ subunit of IκB kinase (IKK) is essential for nuclear factor κB activation and prevention of apoptosis. J. Exp. Med. 189, 1839–1845 (1999).

    Article  CAS  Google Scholar 

  46. Li, Q., Estepa, G., Memet, S., Israel, A. & Verma, I.M. Complete lack of NF-κB activity in IKK1 and IKK2 double-deficient mice: additional defect in neurulation. Genes Dev. 14, 1729–1733 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Zhou, J. & Aiken, C. Nef enhances human immunodeficiency virus type 1 infectivity resulting from intervirion fusion: evidence supporting a role for Nef at the virion envelope. J. Virol. 75, 5851–5859 (2001).

    Article  CAS  Google Scholar 

  48. Paz, S. et al. Induction of IRF-3 and IRF-7 phosphorylation following activation of the RIG-I pathway. Cell Mol. Biol. (Noisy-le-grand) 52, 17–28 (2006).

    CAS  Google Scholar 

  49. Lin, R., Mamane, Y. & Hiscott, J. Structural and functional analysis of interferon regulatory factor 3: localization of the transactivation and autoinhibitory domains. Mol. Cell. Biol. 19, 2465–2474 (1999).

    Article  CAS  Google Scholar 

  50. tenOever, B.R. et al. Activation of TBK1 and IKKepsilon kinases by vesicular stomatitis virus infection and the role of viral ribonucleoprotein in the development of interferon antiviral immunity. J. Virol. 78, 10636–10649 (2004).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank M. Schmidt-Supprian, S.C. Sun, S. Yamaoka, G. Sen and T. Dermody for reagents used in this study; D. Goubau and M. Solis for isolation of human primary macrophages; and members of the Molecular Oncology Group at the Lady Davis Institute for discussions. We thank J.D. Ashwell (National Institutes of Health) for plasmids encoding human full-length NEMO, NEMO constructs of amino acids 1–395 or 251–419, and the L329P NEMO substitution mutant; K. Mossman (McMaster University) for Irf3−/− or Irf3−/−Irf9−/− MEFs; M. Karin (University of California, San Diego) for Ikbkb−/−, Ikbkg−/− and wild-type MEFs; I. Verma (The Salk Institute) for MEFs derived from mice lacking IKKα and IKKβ; J. Bell (Ottawa Cancer Centre) for VSV whole virus antisera; and I. Julkunen (National Public Health Institute and University of Helsinki) for SV antisera. Supported by the Cancer Research Society (R.L.), Canadian Institutes of Health Research (R.L. and J.H.), the National Cancer Institute of Canada with support from the Canadian Cancer Society (J.H.), the National Institutes of Health (AI052379 and CA082556 to D.B.W.), Fonds de la Recherche en Santé Quebec (R.L.) and the Canadian Institutes of Health Research (J.H.).

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Authors and Affiliations

  1. Terry Fox Molecular Oncology Group, Lady Davis Institute for Medical Research, McGill University, Montreal, H3T 1E2, Canada

    Tiejun Zhao, Long Yang, Qiang Sun, Meztli Arguello, John Hiscott & Rongtuan Lin

  2. Department of Microbiology & Immunology, McGill University, Montreal, H3T 1E2, Canada

    Meztli Arguello & John Hiscott

  3. Department of Medicine, McGill University, Montreal, H3T 1E2, Canada

    Long Yang, John Hiscott & Rongtuan Lin

  4. Department of Microbiology and Immunology, Vanderbilt University Medical Center, Nashville, 37232, Tennessee, USA

    Dean W Ballard

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Contributions

R.L. designed the research, did experiments, analyzed data, supervised all experiments and wrote the paper; T.Z. did RT-PCR, ELISA, RNA interference and fluorescence microscopy assays; L.Y. did EMSAs, plaque assays and some of the immunoblots; Q.S. did the in vitro kinase assays; J.H. and M.A. wrote the paper together; and D.W.B. provided new reagents and contributed to discussions.

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Correspondence to Rongtuan Lin.

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Supplementary information

Supplementary Fig. 1

NEMO deletion mutants affecting activation of NF-κB promoter. (PDF 88 kb)

Supplementary Fig. 2

NEMO deletion mutants affecting cytocolic poly(dA:dT)-mediated activation of ISRE promoter. (PDF 74 kb)

Supplementary Fig. 3

NEMO point mutations affecting activation of NF-κB promoter. (PDF 88 kb)

Supplementary Fig. 4

Model of the role of NEMO in RNA virus–triggered activation of the RIG-I–MAVS signaling pathway. (PDF 57 kb)

Supplementary Table 1

List of the primer sequence for RT-PCR. (PDF 44 kb)

Supplementary Methods (PDF 21 kb)

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Zhao, T., Yang, L., Sun, Q. et al. The NEMO adaptor bridges the nuclear factor-κB and interferon regulatory factor signaling pathways. Nat Immunol 8, 592–600 (2007). https://doi.org/10.1038/ni1465

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