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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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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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