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The ubiquitin E3 ligase TRIM31 promotes aggregation and activation of the signaling adaptor MAVS through Lys63-linked polyubiquitination

Nature Immunology volume 18, pages 214–224 (2017)Cite this article

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Abstract

The signaling adaptor MAVS forms prion-like aggregates to activate an innate antiviral immune response after viral infection. However, the molecular mechanisms that regulate MAVS aggregation are poorly understood. Here we identified TRIM31, an E3 ubiquitin ligase of the TRIM family of proteins, as a regulator of MAVS aggregation. TRIM31 was recruited to mitochondria after viral infection and specifically regulated antiviral signaling mediated by RLR pattern-recognition receptors. TRIM31-deficient mice were more susceptible to infection with RNA virus than were wild-type mice. TRIM31 interacted with MAVS and catalyzed the Lys63 (K63)-linked polyubiquitination of Lys10, Lys311 and Lys461 on MAVS. This modification promoted the formation of prion-like aggregates of MAVS after viral infection. Our findings reveal new insights in the molecular regulation of MAVS aggregation and the cellular antiviral response through TRIM31-mediated K63-linked polyubiquitination of MAVS.

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Figure 1: TRIM31 regulates RLR-induced IFN-β signaling.
Figure 2: TRIM31 deficiency impairs cellular antiviral response.
Figure 3: TRIM31 regulates infection with an RNA virus in vivo.
Figure 4: TRIM31 promotes RLR-mediated innate immune signaling.
Figure 5: TRIM31 targets MAVS.
Figure 6: TRIM31 catalyzes the K63-linked polyubiquitination of MAVS.
Figure 7: TRIM31 promotes the formation of MAVS aggregates.

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References

  1. Kawai, T. & Akira, S. The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors. Nat. Immunol. 11, 373–384 (2010).

    Article  CAS  PubMed  Google Scholar 

  2. Cao, X. Self-regulation and cross-regulation of pattern-recognition receptor signaling in health and disease. Nat. Rev. Immunol. 16, 35–50 (2016).

    Article  CAS  PubMed  Google Scholar 

  3. Wu, J. & Chen, Z.J. Innate immune sensing and signaling of cytosolic nucleic acids. Annu. Rev. Immunol. 32, 461–488 (2014).

    Article  CAS  PubMed  Google Scholar 

  4. Kato, H., Takahasi, K. & Fujita, T. RIG-I-like receptors: cytoplasmic sensors for non-self RNA. Immunol. Rev. 243, 91–98 (2011).

    Article  PubMed  Google Scholar 

  5. Unterholzner, L. et al. IFI16 is an innate immune sensor for intracellular DNA. Nat. Immunol. 11, 997–1004 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Sun, L., Wu, J., Du, F., Chen, X. & Chen, Z.J. Cyclic GMP–AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786–791 (2013).

    CAS  PubMed  Google Scholar 

  7. Takeuchi, O. & Akira, S. Pattern-recognition receptors and inflammation. Cell 140, 805–820 (2010).

    Article  CAS  PubMed  Google Scholar 

  8. Yoneyama, M. & Fujita, T. Structural mechanism of RNA recognition by the RIG-I-like receptors. Immunity 29, 178–181 (2008).

    Article  CAS  PubMed  Google Scholar 

  9. Meylan, E. et al. Cardif is an adaptor protein in the RIG-I antiviral pathway and is targeted by hepatitis C virus. Nature 437, 1167–1172 (2005).

    Article  CAS  PubMed  Google Scholar 

  10. Seth, R.B., Sun, L., Ea, C.K. & Chen, Z.J. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-κB and IRF3. Cell 122, 669–682 (2005).

    Article  CAS  PubMed  Google Scholar 

  11. Xu, L.G. et al. VISA is an adapter protein required for virus-triggered IFN-β signaling. Mol. Cell 19, 727–740 (2005).

    Article  CAS  PubMed  Google Scholar 

  12. Kawai, T. et al. IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I interferon induction. Nat. Immunol. 6, 981–988 (2005).

    Article  CAS  PubMed  Google Scholar 

  13. Hou, F. et al. MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response. Cell 146, 448–461 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Peisley, A., Wu, B., Xu, H., Chen, Z.J. & Hur, S. Structural basis for ubiquitin-mediated antiviral signal activation by RIG-I. Nature 509, 110–114 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Moore, C.B. et al. NLRX1 is a regulator of mitochondrial antiviral immunity. Nature 451, 573–577 (2008).

    Article  CAS  PubMed  Google Scholar 

  16. Yoo, Y.S. et al. The mitochondrial ubiquitin ligase MARCH5 resolves MAVS aggregates during antiviral signaling. Nat. Commun. 6, 7910 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Sugiura, T. & Miyamoto, K. Characterization of TRIM31, upregulated in gastric adenocarcinoma, as a novel RBCC protein. J. Cell. Biochem. 105, 1081–1091 (2008).

    Article  CAS  PubMed  Google Scholar 

  18. Shi, M. et al. TRIM30-α negatively regulates TLR-mediated NF-κB activation by targeting TAB2 and TAB3 for degradation. Nat. Immunol. 9, 369–377 (2008).

    Article  CAS  PubMed  Google Scholar 

  19. Wang, P., Zhao, W., Zhao, K., Zhang, L. & Gao, C. TRIM26 negatively regulates interferon-β production and antiviral response through polyubiquitination and degradation of nuclear IRF3. PLoS Pathog. 11, e1004726 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  20. Juang, Y.T. et al. Primary activation of interferon A and interferon B gene transcription by interferon regulatory factor 3. Proc. Natl. Acad. Sci. USA 95, 9837–9842 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Sugiura, T. The cellular level of TRIM31, an RBCC protein overexpressed in gastric cancer, is regulated by multiple mechanisms including the ubiquitin–proteasome system. Cell Biol. Int. 35, 657–661 (2011).

    Article  CAS  PubMed  Google Scholar 

  22. Gack, M.U. et al. TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity. Nature 446, 916–920 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Zeng, W. et al. Reconstitution of the RIG-I pathway reveals a signaling role of unanchored polyubiquitin chains in innate immunity. Cell 141, 315–330 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Nakhaei, P., Genin, P., Civas, A. & Hiscott, J. RIG-I-like receptors: sensing and responding to RNA virus infection. Semin. Immunol. 21, 215–222 (2009).

    Article  CAS  PubMed  Google Scholar 

  25. Kawai, T. & Akira, S. Toll-like receptor and RIG-I-like receptor signaling. Ann. NY Acad. Sci. 1143, 1–20 (2008).

    Article  CAS  PubMed  Google Scholar 

  26. Xia, Z.P. et al. Direct activation of protein kinases by unanchored polyubiquitin chains. Nature 461, 114–119 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Larabi, A. et al. Crystal structure and mechanism of activation of TANK-binding kinase 1. Cell Rep. 3, 734–746 (2013).

    Article  CAS  PubMed  Google Scholar 

  28. Heaton, S.M., Borg, N.A. & Dixit, V.M. Ubiquitin in the activation and attenuation of innate antiviral immunity. J. Exp. Med. 213, 1–13 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. You, F. et al. PCBP2 mediates degradation of the adaptor MAVS via the HECT ubiquitin ligase AIP4. Nat. Immunol. 10, 1300–1308 (2009).

    Article  CAS  PubMed  Google Scholar 

  30. Zhou, X., You, F., Chen, H. & Jiang, Z. Poly(C)-binding protein 1 (PCBP1) mediates housekeeping degradation of mitochondrial antiviral signaling (MAVS). Cell Res. 22, 717–727 (2012).

    Article  CAS  PubMed  Google Scholar 

  31. Zhong, B. et al. The E3 ubiquitin ligase RNF5 targets virus-induced signaling adaptor for ubiquitination and degradation. J. Immunol. 184, 6249–6255 (2010).

    Article  CAS  PubMed  Google Scholar 

  32. Arimoto, K. et al. Negative regulation of the RIG-I signaling by the ubiquitin ligase RNF125. Proc. Natl. Acad. Sci. USA 104, 7500–7505 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Wang, Y., Tong, X. & Ye, X. Ndfip1 negatively regulates RIG-I-dependent immune signaling by enhancing E3 ligase Smurf1-mediated MAVS degradation. J. Immunol. 189, 5304–5313 (2012).

    Article  CAS  PubMed  Google Scholar 

  34. Pan, Y. et al. Smurf2 negatively modulates RIG-I-dependent antiviral response by targeting VISA/MAVS for ubiquitination and degradation. J. Immunol. 192, 4758–4764 (2014).

    Article  CAS  PubMed  Google Scholar 

  35. Castanier, C. et al. MAVS ubiquitination by the E3 ligase TRIM25 and degradation by the proteasome is involved in type I interferon production after activation of the antiviral RIG-I-like receptors. BMC Biol. 10, 44 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Mori, M. et al. Identification of Ser386 of interferon regulatory factor 3 as critical target for inducible phosphorylation that determines activation. J. Biol. Chem. 279, 9698–9702 (2004).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Z. Jiang (Peking University) for Mavs−/− MEFs; D. Guo (Wuhan University) for Ddx58−/− MEFs; K.A. Fitzgerald (University of Massachusetts Medical School) for the human IFNB1 promoter reporter and its mutated reporter; and B. Sun and F. Hou (Shanghai Institutes for Biological Sciences) for the expression plasmids for TRIM30-α and MAVS(M56R). Supported by the Natural Science Foundation of China (81525012, 81273219 and 81471538 to C.G.) and the Specialized Research Fund for the Doctoral Program of Higher Education of China (20130131130010 to C.G.).

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  1. Bingyu Liu and Meng Zhang: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Immunology and Key Laboratory of Infection and Immunity of Shandong Province, Shandong University School of Basic Medical Sciences, Jinan, China

    Bingyu Liu, Honglei Chu, Honghai Zhang, Haifeng Wu, Guanhua Song, Peng Wang, Kai Zhao, Jinxiu Hou, Xueer Wang, Lei Zhang & Chengjiang Gao

  2. Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health, Qilu Hospital, Shandong University, Jinan, China

    Meng Zhang

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Contributions

C.G. designed and supervised the research; B.L. executed the biochemical, cell biological and in vitro experiments; M.Z. performed the animal experiments, viral infections, and experiments with mutant MAVS and TRIM31; H.C., H.Z., H.W., G.S., P.W., K.Z., J.H., X.W. and L.Z. contributed reagents, analytical tools and discussions; B.L. prepared figures; and B.L and C.G. analyzed the data and wrote the paper.

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Correspondence to Chengjiang Gao.

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Integrated supplementary information

Supplementary Figure 1 TRIM31 is recruited to mitochondria after infection with SeV.

(a) Confocal microscopy of TRIM31-GFP transfected into HEK293T cells for 24 h followed with SeV infection for 6 h. MitoTracker (red) were used to label mitochondria. Scale bar 10 μm. (b) Immunoblot of TRIM31 protein and TOMM20 protein in the crude mitochondrial extracts (upper) and TRIM31 protein and β-actin in the whole cell lysate (lower) prepared from peritoneal macrophages infected with SeV for indicated times. (c) Immunoblot of TRIM31 protein in mouse peritoneal macrophages infected with SeV for indicated times. (d) qPCR analysis of Trim31, Trim26, Trim30α, Ifnb1 expression in primary peritoneal macrophages transfected with mice TRIM31 siRNA, TRIM26 siRNA, TRIM30α siRNA or control siRNA for 48 h, followed with SeV infection for indicated times. mRNA results are presented relative to those of untreated cells transfected with control siRNA. (e) qPCR analysis of TRIM31 and IFNB1 mRNA in THP-1 cells transfected with human TRIM31 siRNA or control siRNA for 48 h, followed with SeV infection for indicated times. mRNA results are presented as in d. (f) qPCR analysis of IFNB1 mRNA in Hela cells transfected with Flag-TRIM31 expression plasmid or control vector for 24 h, followed with SeV infection or transfection with poly(I:C) for the indicated times. mRNA results are presented relative to those of untreated cells transfected with control plasmid. The data are representative of 3 independent experiments with three biological replicates (means ± S.D. in d-f). *p < 0.01. (two-tailed Student’s t-test).

Supplementary Figure 2 TRIM31 deficiency impairs RNA-virus-induced production of type I interferon and antiviral responses.

(a) Schematic diagram of the deletions of the Trim31 deficient mice generated by transcription activation-like effector nuclease (TALEN) technology. The deletions were confirmed by DNA sequencing analysis of genomic DNA isolated from Trim31+/+ and Trim31–/– mice tails. (b) Immunoblot of TRIM31 protein in Trim31+/+ and Trim31–/– peritoneal macrophages. (c) qPCR analysis of Ifnb1, Ccl5 and Cxcl10 mRNA in the macrophages prepared form Trim31–/– (G) mice or Trim31–/– (5) mice infected with SeV for indicated times. mRNA results are presented relative to those of untreated wild-type cells. (d,e) qPCR analysis of Ifnb1, Ccl5 and Cxcl10 mRNA in bone marrow-derived macrophages (BMDMs) (d) or MEFs (e) prepared from Trim31+/+ and Trim31–/– mice followed with infection of SeV and HSV-1 or stimulation of 5′ppp-RNA, LPS and poly(I:C) for indicated times. mRNA results are presented as in c. The data are representative of 3 independent experiments with three biological replicates (means ± S.D. in c-e). *p < 0.05 and **p < 0.01. (two-tailed Student’s t-test).

Supplementary Figure 3 Trim31-deficient mice are not impaired in their response to infection with a DNA virus.

(a) qPCR analyses of Ifnb1 mRNA in brains and ELISA analysis of IFN-β production in sera of Trim31+/+ and Trim31–/– mice intraperitoneally infected with HSV-1 (2×107 PFU/mouse)for 24 h. mRNA results are presented relative to those of untreated wild-type cells. (b) qPCR analysis of HSV-1 genomic DNA and plaque assays of HSV-1 titer in brains of Trim31+/+ and Trim31–/– mice as treated in (a). (c) Survival of Trim31+/+ and Trim31–/– mice (n=7) after intraperitoneally injection of HSV-1 (1×108 pfu/mouse). The data are representative of 3 independent experiments with three biological replicates (means ± S.D. in a, b). *p < 0.01 (two-tailed Student’s t-test(a,b) or two-way analysis of variance (ANOVA) (c)).

Supplementary Figure 4 TRIM31 targets MAVS.

(a) Luciferase activity in HEK293T cells transfected with IFNB1 luciferase reporter and expression plasmids for RIG-IN, MDA5, MAVS, TBK1, IKKɛ and IRF3 5D together with Flag-TRIM31 expression plasmid for 24 h. (b) Luciferase activity in HEK293T cells transfected with control siRNA or TRIM31 siRNA and IFNB1 luciferase reporter or ISRE luciferase reporter together with expression plasmids for RIG-IN, MAVS and TBK1 for 24 h. (c) Coimmunoprecipitation analysis of Flag-TRIM31 interaction with Myc-IRF3 or Myc-STING in HEK293T cells. The data are representative of 3 independent experiments with three biological replicates (means ± S.D. in a, b). *p < 0.01. (two-tailed Student’s t-test).

Supplementary Figure 5 TRIM31 catalyzes K63-linked polyubiquitination of MAVS.

(a) Coimmunoprecipitation analysis of RIG-I ubiquitination in HEK293T cells transfected with Myc-RIG-I and HA-Ubiquitin together with control plasmid, Flag-TRIM31 plasmid or Flag-TRIM25 plasmid. (b) Luciferase activity in HEK293T cells transfected with IFNB1 luciferase reporter or ISRE luciferase reporter together with Flag-TRIM31-WT plasmid or Flag-TRIM31-C53/56A plasmid for 24 h followed with SeV infection for indicated times. (c) qPCR analysis of IFNB1 mRNA and VSV RNA in HEK293T cells transfected with control plasmid, Flag-TRIM31-WT plasmid or Flag-TRIM31-C53/56A plasmid for 24 h followed with VSV infection (MOI, 0.1) for indicated times. mRNA results are presented relative to those of untreated cells transfected with control plasmid. (d) Immunoblot of phosphorylated and total IRF3 protein in lysates of HEK293T cells transfected with Flag-TRIM31-WT or Flag-TRIM31-C53/56A plasmid for 24 h followed with SeV infection for indicated times. The data are representative of 3 independent experiments with three biological replicates (means ± S.D. in b, c). *p < 0.01. (two-tailed Student’s t-test).

Supplementary Figure 6 TRIM31 promotes the formation of MAVS aggregates.

(a) SDD-AGE analysis of MAVS aggregation in Trim31+/+ and Trim31–/– MEFs infected with SeV for indicated times (upper). SDS-PAGE analysis of MAVS protein and TRIM31 protein in the whole cell lysates prepared above (lower). (b) SDD-AGE analysis of MAVS aggregation in HEK293T cells transfected with Flag-TRIM31 expression plasmid and Myc-MAVS for 24 h. (c) Immunoblot of TRIM31 protein in Trim31–/– MEFs reconstituted with mTRIM31-WT or mTRIM31-C52/55A. TRIM31+/+ MEFs were used as controls. qPCR analysis of Ifnb1, Ccl5 and Cxcl10 mRNA in TRIM31+/+ or Trim31–/– MEFs reconstituted with mTRIM31-WT or mTRIM31-C52/55A followed with SeV infection for 12 h. mRNA results are presented relative to those of untreated wide-type cells transfected with control plasmid. (d) qPCR analysis of Ifnb1 mRNA in Mavs–/– MEFs transfected with MAVS expression plasmid or control plasmid for 24 h followed with SeV infection for 12 h. mRNA results are presented relative to those of untreated cells transfected with control plasmid. (e) Luciferase activity in Mavs–/– MEFs transfected with IFN-β or ISRE luciferase reporter and expression plasmids for MAVS-WT, MAVS-K10R, MAVS-K10/311R, MAVS-K10/311/461R along with Flag-TRIM31 expression plasmid for 24 h followed with SeV infection for 12 h. (f) qPCR analysis of Ifnb1 mRNA and SDD-AGE analysis of MAVS aggregation in Mavs–/– MEFs transfected with MAVS-WT, MAVS-K10R, MAVS-K311R and MAVS-K461R along with Flag-TRIM31 expression plasmid or control plasmid for 24 h followed with SeV infection for 12 h. (g) qPCR analysis of Ifnb1 mRNA and SDD-AGE analysis of MAVS aggregation in Mavs–/– MEFs transfected with MAVS-WT or MAVS-W56R along with Flag-TRIM31 expression plasmid or control plasmid for 24 h followed with SeV infection for 6 h. mRNA results are presented as in d. The data are representative of 3 independent experiments with three biological replicates (means ± S.D. in c-g). *p < 0.01. (two-tailed Student’s t-test).

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Liu, B., Zhang, M., Chu, H. et al. The ubiquitin E3 ligase TRIM31 promotes aggregation and activation of the signaling adaptor MAVS through Lys63-linked polyubiquitination. Nat Immunol 18, 214–224 (2017). https://doi.org/10.1038/ni.3641

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