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iRhom2 is essential for innate immunity to DNA viruses by mediating trafficking and stability of the adaptor STING

Nature Immunology volume 17, pages 1057–1066 (2016)Cite this article

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Abstract

STING is a central adaptor in the innate immune response to DNA viruses. However, the manner in which STING activity is regulated remains unclear. We identified iRhom2 ('inactive rhomboid protein 2') as a positive regulator of DNA-virus-triggered induction of type I interferons. iRhom2 deficiency markedly impaired DNA-virus- and intracellular-DNA-induced signaling in cells, and iRhom2-deficient mice were more susceptible to lethal herpes simplex virus type 1 (HSV-1) infection. iRhom2 was constitutively associated with STING and acted in two distinct processes to regulate STING activity. iRhom2 recruited the translocon-associated protein TRAPβ to the STING complex to facilitate trafficking of STING from the endoplasmic reticulum to perinuclear microsomes. iRhom2 also recruited the deubiquitination enzyme EIF3S5 to maintain the stability of STING through removal of its K48-linked polyubiquitin chains. These results suggest that iRhom2 is essential for STING activity, as it regulates TRAPβ-mediated translocation and EIF3S5-mediated deubiquitination of STING.

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Figure 1: Identification of iRhom2 as a positive regulator of DNA-virus-triggered signaling.
Figure 2: iRhom2 is essential for DNA-virus-triggered induction of downstream antiviral genes.
Figure 3: iRhom2 is essential for host defense against HSV-1 infection in mice.
Figure 4: iRhom2 acts at the level of STING.
Figure 5: iRhom2 facilitates the trafficking of STING.
Figure 6: iRhom2 maintains the stability of STING.
Figure 7: iRhom2-EIF3S5 axis deubiquitinates STING.
Figure 8: iRhom2-mediated translocation and stability of STING are two independent processes.

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References

  1. Akira, S., Uematsu, S. & Takeuchi, O. Pathogen recognition and innate immunity. Cell 124, 783–801 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  3. Barbalat, R., Ewald, S.E., Mouchess, M.L. & Barton, G.M. Nucleic acid recognition by the innate immune system. Annu. Rev. Immunol. 29, 185–214 (2011).

    Article  CAS  Google Scholar 

  4. Lund, J., Sato, A., Akira, S., Medzhitov, R. & Iwasaki, A. Toll-like receptor 9-mediated recognition of Herpes simplex virus-2 by plasmacytoid dendritic cells. J. Exp. Med. 198, 513–520 (2003).

    Article  CAS  Google Scholar 

  5. Bürckstümmer, T. et al. An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome. Nat. Immunol. 10, 266–272 (2009).

    Article  Google Scholar 

  6. Takaoka, A. et al. DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature 448, 501–505 (2007).

    Article  CAS  Google Scholar 

  7. Chiu, Y.H., Macmillan, J.B. & Chen, Z.J. RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway. Cell 138, 576–591 (2009).

    Article  CAS  Google Scholar 

  8. Ablasser, A. et al. RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate. Nat. Immunol. 10, 1065–1072 (2009).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Zhang, Z. et al. The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells. Nat. Immunol. 12, 959–965 (2011).

    Article  CAS  Google Scholar 

  11. Li, Y. et al. LSm14A is a processing body-associated sensor of viral nucleic acids that initiates cellular antiviral response in the early phase of viral infection. Proc. Natl. Acad. Sci. USA 109, 11770–11775 (2012).

    Article  CAS  Google Scholar 

  12. Paludan, S.R. & Bowie, A.G. Immune sensing of DNA. Immunity 38, 870–880 (2013).

    Article  CAS  Google Scholar 

  13. 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).

    Article  CAS  Google Scholar 

  14. Li, X.D. et al. Pivotal roles of cGAS-cGAMP signaling in antiviral defense and immune adjuvant effects. Science 341, 1390–1394 (2013).

    Article  CAS  Google Scholar 

  15. Wu, J. et al. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339, 826–830 (2013).

    Article  CAS  Google Scholar 

  16. Ishikawa, H. & Barber, G.N. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 455, 674–678 (2008).

    Article  CAS  Google Scholar 

  17. Ishikawa, H., Ma, Z. & Barber, G.N. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature 461, 788–792 (2009).

    Article  CAS  Google Scholar 

  18. Zhong, B. et al. The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation. Immunity 29, 538–550 (2008).

    Article  CAS  Google Scholar 

  19. Tsuchida, T. et al. The ubiquitin ligase TRIM56 regulates innate immune responses to intracellular double-stranded DNA. Immunity 33, 765–776 (2010).

    Article  CAS  Google Scholar 

  20. Zhang, J., Hu, M.M., Wang, Y.Y. & Shu, H.B. TRIM32 protein modulates type I interferon induction and cellular antiviral response by targeting MITA/STING protein for K63-linked ubiquitination. J. Biol. Chem. 287, 28646–28655 (2012).

    Article  CAS  Google Scholar 

  21. Wang, Q. et al. The E3 ubiquitin ligase AMFR and INSIG1 bridge the activation of TBK1 kinase by modifying the adaptor STING. Immunity 41, 919–933 (2014).

    Article  CAS  Google Scholar 

  22. Zhong, B. et al. The ubiquitin ligase RNF5 regulates antiviral responses by mediating degradation of the adaptor protein MITA. Immunity 30, 397–407 (2009).

    Article  CAS  Google Scholar 

  23. Qin, Y. et al. RNF26 temporally regulates virus-triggered type I interferon induction by two distinct mechanisms. PLoS Pathog. 10, e1004358 (2014).

    Article  Google Scholar 

  24. Ahn, J., Gutman, D., Saijo, S. & Barber, G.N. STING manifests self DNA-dependent inflammatory disease. Proc. Natl. Acad. Sci. USA 109, 19386–19391 (2012).

    Article  CAS  Google Scholar 

  25. Jeremiah, N. et al. Inherited STING-activating mutation underlies a familial inflammatory syndrome with lupus-like manifestations. J. Clin. Invest. 124, 5516–5520 (2014).

    Article  Google Scholar 

  26. Liu, Y. et al. Activated STING in a vascular and pulmonary syndrome. N. Engl. J. Med. 371, 507–518 (2014).

    Article  CAS  Google Scholar 

  27. Freeman, M. Rhomboid proteases and their biological functions. Annu. Rev. Genet. 42, 191–210 (2008).

    Article  CAS  Google Scholar 

  28. Adrain, C., Zettl, M., Christova, Y., Taylor, N. & Freeman, M. Tumor necrosis factor signaling requires iRhom2 to promote trafficking and activation of TACE. Science 335, 225–228 (2012).

    Article  CAS  Google Scholar 

  29. McIlwain, D.R. et al. iRhom2 regulation of TACE controls TNF-mediated protection against Listeria and responses to LPS. Science 335, 229–232 (2012).

    Article  CAS  Google Scholar 

  30. Bergbold, N. & Lemberg, M.K. Emerging role of rhomboid family proteins in mammalian biology and disease. Biochim. Biophys. Acta 1828, 2840–2848 (2013).

    Article  CAS  Google Scholar 

  31. Issuree, P.D. et al. iRHOM2 is a critical pathogenic mediator of inflammatory arthritis. J. Clin. Invest. 123, 928–932 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. Prantner, D. et al. 5,6-Dimethylxanthenone-4-acetic acid (DMXAA) activates stimulator of interferon gene (STING)-dependent innate immune pathways and is regulated by mitochondrial membrane potential. J. Biol. Chem. 287, 39776–39788 (2012).

    Article  CAS  Google Scholar 

  33. Gao, P. et al. Structure-function analysis of STING activation by c[G(2′,5′)pA(3′,5′)p] and targeting by antiviral DMXAA. Cell 154, 748–762 (2013).

    Article  CAS  Google Scholar 

  34. Shu, H.B. & Wang, Y.Y. Adding to the STING. Immunity 41, 871–873 (2014).

    Article  CAS  Google Scholar 

  35. Saitoh, T. et al. Atg9a controls dsDNA-driven dynamic translocation of STING and the innate immune response. Proc. Natl. Acad. Sci. USA 106, 20842–20846 (2009).

    Article  CAS  Google Scholar 

  36. Konno, H., Konno, K. & Barber, G.N. Cyclic dinucleotides trigger ULK1 (ATG1) phosphorylation of STING to prevent sustained innate immune signaling. Cell 155, 688–698 (2013).

    Article  CAS  Google Scholar 

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Acknowledgements

Supported by the Ministry of Science and Technology of China (2014CB910103 and 2012CB910201) and the National Natural Science Foundation of China (31521091 and 91429304).

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Authors and Affiliations

  1. College of Life Sciences, Wuhan University, Wuhan, China

    Wei-Wei Luo, Shu Li, Chen Li, Qing Yang, Bo Zhong & Hong-Bing Shu

  2. Medical Research Institute, Collaborative Innovation Center for Viral Immunology, School of Medicine, Wuhan University, Wuhan, China

    Huan Lian, Bo Zhong & Hong-Bing Shu

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Contributions

H.-B.S., S.L. and W.-W.L. conceived and designed the study. S.L. performed the human cDNA expression plasmid screens. W.-W.L., C.L., H.L. and Q.Y. performed the majority of experiments. H.B.S., S.L., W.-W.L., C.L., H.L. and B.Z. analyzed the data. H.-B.S. and W.-W.L. wrote the manuscript. All of the authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Hong-Bing Shu.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Generation and analysis of iRhom2-deficient mice.

(a) Schematic representation of wild-type iRhom2 locus shows its exons (boxes) and introns (lines). The iRhom2 locus was disrupted by the targeting vector and the positions of the primers for genotyping are shown. The splice acceptor (SA) sequence in the LacZ cassette interrupts the normal splicing of iRhom2 gene, resulting in the loss of iRhom2 expression.

(b) Genotyping of iRhom2 mutant mice.

(c) qPCR analysis of iRhom2 mRNA abundance in MEFs, BMDMs and BMDCs of various genotypes.

(d) Genotypes of the offsprings from the breeding of iRhom2 heterozygous mice.

(e) Cells from thymus, spleen and peripheral lympho nodes were analyzed by FACS after staining with the indicated antibodies.

Data are representative of three (b,c) or two (e) independent experiments.

Supplementary Figure 2 Effects of iRhom2 deficiency on mRNA and protein abundance of TNF.

(a) ELISA analysis of secreted IFN-β and TNF from iRhom2+/+ and iRhom2−/− MEFs un-treated or treated with LPS for 4 h.

(b) The mRNA or secreted protein of TNF in iRhom2+/+ and iRhom2−/− MEFs infected with HSV-1 for the indicated times was quantitated by qPCR (left histograph) or ELISA (right histograph) analysis respectively.

**P < 0.01 (unpaired t-test). Data are representative of three experiments with similar results (mean and s.d. in a,b).

Supplementary Figure 3 Effects of iRhom2 deficiency on cGAMP- and DMXAA-induced signaling.

(a) Immunoblot analysis of the indicated proteins in iRhom2+/+ and iRhom2−/− MEFs transfected with cGAMP for the indicated times.

(b) qPCR analysis of mRNA abundance of the indicated genes in iRhom2+/+ and iRhom2−/− MEFs treated with DMXAA for the indicated times.

*P < 0.05; **P < 0.01 (unpaired t-test). Data are representative of three experiments with similar results (mean and s.d. in b).

Supplementary Figure 4 Colocalization of iRhom2 with various components.

Confocal microscopy of HeLa cells transfected with iRhom2-Cherry and the indicated Flag-tagged components. Data are representative of three experiments with similar results. Scale bars, 20 μm.

Supplementary Figure 5 Domain-mapping analysis of the interaction between iRhom2 and TRAPβ.

HEK293 cells were transfected with the indicated plasmids before coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies. The top part shows schematic representations of iRhom2 truncations. Data are representative of three experiments with similar results.

Supplementary Figure 6 iRhom2 regulates the protein abundance and K48-linked polyubiquitination of STING.

(a) Immunoblot analysis of STING in iRhom2+/+ and iRhom2−/− BMDMs infected with HSV-1 for the indicated times.

(b) Immunoblot analysis of STING, TBK1 and iRhom2 in iRhom2-knockdown THP-1 cells infected with HSV-1 for the indicated times.

(c) qPCR analysis of STING mRNA in HEK293 cells transfected with an iRhom2-RNAi plasmid for 24 h and then selected with puromycin for 24 h.

(d) Immunoblot analysis of the indicated proteins in HEK293 cells transfected with iRhom2 for 20 h and then treated with cycloheximide for the indicated times.

(e) HEK293 cells were transfected with iRhom2, STING-Flag, and HA-Ub or its mutants as indicated for 24 h before coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies.

Data are representative of three experiments with similar results.

Supplementary Figure 7 Identification of EIF3S5 as a deubiquitinase for STING.

(a-c) HEK293 cells were transfected with the indicated plasmids for 24 h before coimmunoprecipitation and immunoblot analysis were performed with the indicated antibodies. EV, empty vector.

(d) Immunostaining showing localization of EIF3S5 (red) in HeLa cells transfected with Myc-tagged EIF3S5 and GFP-tagged Rab9, GM130 or Atg12a respectively and infected with HSV-1 for 6 h. Scale bars, 20 μm.

(e) Immunostaining showing colocalization of EIF3S5 and STING in HeLa cells transfected with EIF3S5-Myc and STING-GFP or iRhom2-Cherry respectively for 24 h and then infected with HSV-1 for 6 h. Scale bars, 20 μm.

(f) qPCR analysis of IFNB1, IL6 and EIF3S5 mRNAs in HFFs transfected with EIF3S5-siRNA for 2 days and then retransfected with ISD for 6 h.

**P < 0.01 (unpaired t-test). Data are representative of three experiments with similar results (mean and s.d. in f).

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Luo, WW., Li, S., Li, C. et al. iRhom2 is essential for innate immunity to DNA viruses by mediating trafficking and stability of the adaptor STING. Nat Immunol 17, 1057–1066 (2016). https://doi.org/10.1038/ni.3510

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