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TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity

Abstract

Retinoic-acid-inducible gene-I (RIG-I; also called DDX58) is a cytosolic viral RNA receptor that interacts with MAVS (also called VISA, IPS-1 or Cardif) to induce type I interferon-mediated host protective innate immunity against viral infection1,2,3,4,5,6. Furthermore, members of the tripartite motif (TRIM) protein family, which contain a cluster of a RING-finger domain, a B box/coiled-coil domain and a SPRY domain, are involved in various cellular processes, including cell proliferation and antiviral activity7. Here we report that the amino-terminal caspase recruitment domains (CARDs) of RIG-I undergo robust ubiquitination induced by TRIM25 in mammalian cells. The carboxy-terminal SPRY domain of TRIM25 interacts with the N-terminal CARDs of RIG-I; this interaction effectively delivers the Lys 63-linked ubiquitin moiety to the N-terminal CARDs of RIG-I, resulting in a marked increase in RIG-I downstream signalling activity. The Lys 172 residue of RIG-I is critical for efficient TRIM25-mediated ubiquitination and for MAVS binding, as well as the ability of RIG-I to induce antiviral signal transduction. Furthermore, gene targeting demonstrates that TRIM25 is essential not only for RIG-I ubiquitination but also for RIG-I-mediated interferon-β production and antiviral activity in response to RNA virus infection. Thus, we demonstrate that TRIM25 E3 ubiquitin ligase induces the Lys 63-linked ubiquitination of RIG-I, which is crucial for the cytosolic RIG-I signalling pathway to elicit host antiviral innate immunity.

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Figure 1: The 2CARD of RIG-I undergoes robust ubiquitination.
Figure 2: Interaction between RIG-I and TRIM25.
Figure 3: TRIM25 is a primary E3 ubiquitin ligase of RIG-I.
Figure 4: Role of TRIM25-mediated ubiquitination in RIG-I antiviral activity.

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Acknowledgements

This work was supported by US Public Health Service grants (J.U.J.), the exchange programme between Harvard Medical School and the graduate training programme 1071 at the Friedrich-Alexander University Erlangen-Nuremberg, Germany (M.U.G.), and a Korea Research Foundation Grant (C.-H.J.). We thank A. Garcia-Sastre, D.-E. Zhang and S. Whelan for providing reagents, and R. Tomaino and J. Nagel for mass spectrometry. We also thank all members of the Tumor Virology Division, New England Primate Research Center, for discussions.

Author Contributions M.U.G. performed all aspects of this study. Y.C.S., C.-H.J. and C.L. assisted in experimental design and in collecting the data. T.U. and S.I. performed the in vitro ubiquitination assay and generated Trim25-/- MEFs. L.S. and Z.C. generated the MAVS construct and RIG-I antibody. T.O. and S.A. generated the RIG-I construct and RIG-I-/- MEFs. M.U.G. and J.U.J. organized this study and wrote the paper. All authors discussed the results and commented on the manuscript.

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Correspondence to Jae U. Jung.

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Supplementary Figures

This file contains Supplementary Figures 1-10 with Legends. The Supplementary Figures provide additional information on the Lys63-linked ubiquitination of RIG-I and the role of RIG-I ubiquitination in its signaling activity. They also show more data to support the essential role of TRIM25-mediated ubiquitination in RIG-I signal transduction and host anti-viral activity. (PDF 3300 kb)

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Gack, M., Shin, Y., Joo, CH. et al. TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature 446, 916–920 (2007). https://doi.org/10.1038/nature05732

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