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Systems-based analysis of RIG-I-dependent signalling identifies KHSRP as an inhibitor of RIG-I receptor activation

Nature Microbiology volume 2, Article number: 17022 (2017) Cite this article

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Abstract

Retinoic acid-inducible gene I (RIG-I) receptor recognizes 5′-triphosphorylated RNA and triggers a signalling cascade that results in the induction of type-I interferon (IFN)-dependent responses. Its precise regulation represents a pivotal balance between antiviral defences and autoimmunity. To elucidate the cellular cofactors that regulate RIG-I signalling, we performed two global RNA interference analyses to identify both positive and negative regulatory nodes operating on the signalling pathway during virus infection. These factors were integrated with experimentally and computationally derived interactome data to build a RIG-I protein interaction network. Our analysis revealed diverse cellular processes, including the unfolded protein response, Wnt signalling and RNA metabolism, as critical cellular components governing innate responses to non-self RNA species. Importantly, we identified K-Homology Splicing Regulatory Protein (KHSRP) as a negative regulator of this pathway. We find that KHSRP associates with the regulatory domain of RIG-I to maintain the receptor in an inactive state and attenuate its sensing of viral RNA (vRNA). Consistent with increased RIG-I antiviral signalling in the absence of KHSRP, viral replication is reduced when KHSRP expression is knocked down both in vitro and in vivo. Taken together, these data indicate that KHSRP functions as a checkpoint regulator of the innate immune response to pathogen challenge.

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Figure 1: Genome-wide RNAi screens to identify regulators of the RIG-I signalling pathway.
Figure 2: Confirmation studies of the putative negative regulators on the RIG-I pathway.
Figure 3: KHSRP negatively regulates RIG-I signalling.
Figure 4: KHSRP associates with the regulatory domain of RIG-I.
Figure 5: KHSRP maintains RIG-I in an inactive state and attenuates its sensing of vRNA.
Figure 6: KHSRP inhibits replication of RNA viruses.

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Acknowledgements

The authors acknowledge the Krogan laboratory for running and analysis of the AP-MS experiments, M. Schneider for help with editing the manuscript and the Chanda laboratory for discussions and advice. The authors thank R. Albrecht for providing the H1N1 PR8/34 IAV stocks used in the in vivo mouse experiments and C. Lässig and K.-P. Hopfner for providing recombinant RIG-I protein for in vitro assays. The authors acknowledge support from the National Institutes of Health (U19 AI106754 and P50 GM085764). This work was also supported by a grant from the James B. Pendleton Charitable Trust and by CRIP (Center for Research on Influenza Pathogenesis), an NIAID funded Center of Excellence for Influenza Research and Surveillance (CEIRS, contract no. HHSN272201400008C).

Author information

Authors and Affiliations

  1. Immunity and Pathogenesis Program, Infectious and Inflammatory Disease Center, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, 92037, California, USA

    Stephen Soonthornvacharin, Ariel Rodriguez-Frandsen, Felipe Galvez, Nicholas J. Huffmaster, Atsushi Inoue, Paul D. De Jesus, Quy Nguyen, Renate König, Sunnie M. Yoh & Sumit K. Chanda

  2. The San Diego Center for Systems Biology (SDCSB), La Jolla, 92093, California, USA

    Stephen Soonthornvacharin, Ariel Rodriguez-Frandsen, Felipe Galvez, Nicholas J. Huffmaster, Paul D. De Jesus, Quy Nguyen, Sunnie M. Yoh & Sumit K. Chanda

  3. Graduate School of Biomedical Sciences, Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, 92037, California, USA

    Stephen Soonthornvacharin

  4. Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, 92121, California, USA

    Yingyao Zhou

  5. Department of Microbiology, Icahn School of Medicine at Mount Sinai, 1468 Madison Avenue, New York, 10029, New York, USA

    Shashank Tripathi, Vinod R. M. T. Balasubramaniam, Elisa de Castro, María Teresa Sánchez-Aparicio & Adolfo García-Sastre

  6. Global Health and Emerging Pathogen Institute, Icahn School of Medicine at Mount Sinai, 1468 Madison Avenue, New York, 10029, New York, USA

    Shashank Tripathi, Vinod R. M. T. Balasubramaniam, Elisa de Castro, María Teresa Sánchez-Aparicio & Adolfo García-Sastre

  7. Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University Corvallis, 450 SW 30th Street, 97331, Oregon, USA

    Hong Moulton & David A. Stein

  8. Host-Pathogen-Interactions, Paul-Ehrlich-Institute, German Center for Infection Research (DZIF), Paul-Ehrlich-Straße 51–59, 63225 Langen, Germany

    Renate König

  9. Department of Cellular and Molecular Pharmacology, University of California San Francisco, 1700 4th Street, Byers Hall 308D, Box 2530, San Francisco, 94158, California, USA

    Nevan J. Krogan

  10. Department of Medicine, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, 1468 Madison Avenue, New York, 10029, New York, USA

    Adolfo García-Sastre

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  1. Stephen Soonthornvacharin
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Contributions

S.S., S.M.Y. and S.K.C. wrote the manuscript. S.S., R.K., S.M.Y. and S.K.C. designed experiments and interpreted data. Y.Z. and A.G.-S. interpreted data. S.S., A.R.-F., S.M.Y., F.G., N.J.H., S.T., V.R.M.T.B., A.I., M.T.S.-A., E.d.C., P.D.D.J., Q.N. and N.J.K. performed experiments. H.M. and D.A.S. provided reagents.

Corresponding authors

Correspondence to Sunnie M. Yoh or Sumit K. Chanda.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1–12, Supplementary Discussion, Supplementary Methods, Supplementary References. (PDF 2297 kb)

Supplementary Table 1

RSA analysis of RIG-I positive regulator screen. (XLSX 3747 kb)

Supplementary Table 2

RSA analysis of RIG-I negative regulator screen. (XLSX 2425 kb)

Supplementary Table 3

Summary of RIG-I negative regulator selection strategy. (XLSX 102 kb)

Supplementary Table 4

AP-MS analysis of RIG-I signalling factors. (XLSX 373 kb)

Supplementary Table 5

Gene ontology and functional enrichment of the RIG-I protein network. (XLSX 53 kb)

Supplementary Table 6

Binary interactions of the RIG-I pathway interaction map (Fig. 1d). (XLSX 51 kb)

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Soonthornvacharin, S., Rodriguez-Frandsen, A., Zhou, Y. et al. Systems-based analysis of RIG-I-dependent signalling identifies KHSRP as an inhibitor of RIG-I receptor activation. Nat Microbiol 2, 17022 (2017). https://doi.org/10.1038/nmicrobiol.2017.22

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