Abstract
Lysine 63 (K63)-linked ubiquitination of RIG-I plays a critical role in the activation of type I interferon pathway, yet the molecular mechanism responsible for its deubiquitination is still poorly understood. Here we report that the deubiquitination enzyme ubiquitin-specific protease 3 (USP3) negatively regulates the activation of type I interferon signaling by targeting RIG-I. Knockdown of USP3 specifically enhanced K63-linked ubiquitination of RIG-I, upregulated the phosphorylation of IRF3 and augmented the production of type I interferon cytokines and antiviral immunity. We further show that there is no interaction between USP3 and RIG-I-like receptors (RLRs) in unstimulated or uninfected cells, but upon viral infection or ligand stimulation, USP3 binds to the caspase activation recruitment domain of RLRs and then cleaves polyubiquitin chains through cooperation of its zinc-finger Ub-binding domain and USP catalytic domains. Mutation analysis reveals that binding of USP3 to polyubiquitin chains on RIG-I is a prerequisite step for its cleavage of polyubiquitin chains. Our findings identify a previously unrecognized role of USP3 in RIG-I activation and provide insights into the mechanisms by which USP3 inhibits RIG-I signaling and antiviral immunity.
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Acknowledgements
We would like to thank Dr Jianhua Yang (Baylor College of Medicine) for USP3 and other USP (without tag) plasmids. This work was in part supported by grants (CA090327, CA101795, CA121191, CA116408, CA094327 and DA030338) from NCI, NIH and Cancer Prevention and Research Institute of Texas (CPRIT RP121048) (to RFW), and the National Natural Science Foundation of China (31000394 and 31370869), the National Key Basic Research Program of China (2014CB910800), Guangdong Innovative Research Team Program (2011Y035 and 201001Y0104687244) and Guangdong Natural Science Funds for Distinguished Young Scholar (S2013050014772).
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Supplementary information
Supplementary information, Figure S1
USP3 negatively regulates dsRNA and dsDNA induced type I IFN signaling. (PDF 198 kb)
Supplementary information, Figure S2
Knockdown of Usp3 enhances type I IFN signaling and IFN-β production. (PDF 328 kb)
Supplementary information, Figure S3
USP3 inhibits type I IFN signaling through RIG-I or MDA5. (PDF 340 kb)
Supplementary information, Figure S4
USP3 specifically interacts with RIG-I and MDA5 in a ligand-dependent manner. (PDF 113 kb)
Supplementary information, Figure S5
USP3 inhibits IRF3 activation by deubiquitinating K63-linked polyubiquitin chains. (PDF 274 kb)
Supplementary information, Figure S6
Effects of USP3 deletions and mutations on RIG-I ubiquitination and RIG-I (N)-induced ISRE-luc activity. (PDF 169 kb)
Supplementary information, Figure S7
USP3 specifically binds to the ubiquitinated RIG-I and MDA5 with a K63 linage. (PDF 153 kb)
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Cui, J., Song, Y., Li, Y. et al. USP3 inhibits type I interferon signaling by deubiquitinating RIG-I-like receptors. Cell Res 24, 400–416 (2014). https://doi.org/10.1038/cr.2013.170
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DOI: https://doi.org/10.1038/cr.2013.170
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