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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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).
The authors declare no competing financial interests.
Supplementary Figures 1–12, Supplementary Discussion, Supplementary Methods, Supplementary References. (PDF 2297 kb)
RSA analysis of RIG-I positive regulator screen. (XLSX 3747 kb)
RSA analysis of RIG-I negative regulator screen. (XLSX 2425 kb)
Summary of RIG-I negative regulator selection strategy. (XLSX 102 kb)
AP-MS analysis of RIG-I signalling factors. (XLSX 373 kb)
Gene ontology and functional enrichment of the RIG-I protein network. (XLSX 53 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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