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TRIM23 mediates virus-induced autophagy via activation of TBK1


Autophagy and interferon (IFN)-mediated innate immunity are critical antiviral defence mechanisms, and recent evidence indicated that tripartite motif (TRIM) proteins are important regulators of both processes. Although the role of TRIM proteins in modulating antiviral cytokine responses has been well established, much less is known about their involvement in autophagy in response to different viral pathogens. Through a targeted RNAi screen examining the relevance of selected TRIM proteins in autophagy induced by herpes simplex virus 1 (HSV-1), encephalomyocarditis virus (EMCV) and influenza A virus (IAV), we identified several TRIM proteins that regulate autophagy in a virus-species-specific manner, as well as a few TRIM proteins that were essential for autophagy triggered by all three viruses and rapamycin, among them TRIM23. TRIM23 was critical for autophagy-mediated restriction of multiple viruses, and this activity was dependent on both its RING E3 ligase and ADP-ribosylation factor (ARF) GTPase activity. Mechanistic studies revealed that unconventional K27-linked auto-ubiquitination of the ARF domain is essential for the GTP hydrolysis activity of TRIM23 and activation of TANK-binding kinase 1 (TBK1) by facilitating its dimerization and ability to phosphorylate the selective autophagy receptor p62. Our work identifies the TRIM23-TBK1-p62 axis as a key component of selective autophagy and further reveals a role for K27-linked ubiquitination in GTPase-dependent TBK1 activation.

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  1. 1.

    Ozato, K., Shin, D. M., Chang, T. H. & Morse III, H. C. TRIM family proteins and their emerging roles in innate immunity. Nat. Rev. Immunol. 8, 849–860 (2008).

  2. 2.

    Versteeg, G. A. et al. The E3-ligase TRIM family of proteins regulates signaling pathways triggered by innate immune pattern-recognition receptors. Immunity 38, 384–398 (2013).

  3. 3.

    Levine, B. & Kroemer, G. Autophagy in the pathogenesis of disease. Cell 132, 27–42 (2008).

  4. 4.

    Stolz, A., Ernst, A. & Dikic, I. Cargo recognition and trafficking in selective autophagy. Nat. Cell Biol. 16, 495–501 (2014).

  5. 5.

    Randow, F. & Youle, R. J. Self and nonself: how autophagy targets mitochondria and bacteria. Cell Host Microbe 15, 403–411 (2014).

  6. 6.

    Tal, M. C. & Iwasaki, A. Autophagy and innate recognition systems. Curr. Top. Microbiol. Immunol. 335, 107–121 (2009).

  7. 7.

    Levine, B., Mizushima, N. & Virgin, H. W. Autophagy in immunity and inflammation. Nature 469, 323–335 (2011).

  8. 8.

    Deretic, V., Saitoh, T. & Akira, S. Autophagy in infection, inflammation and immunity. Nat. Rev. Immunol. 13, 722–737 (2013).

  9. 9.

    Sharma, S. et al. Triggering the interferon antiviral response through an IKK-related pathway. Science 300, 1148–1151 (2003).

  10. 10.

    Pilli, M. et al. TBK-1 promotes autophagy-mediated antimicrobial defense by controlling autophagosome maturation. Immunity 37, 223–234 (2012).

  11. 11.

    Richter, B. et al. Phosphorylation of OPTN by TBK1 enhances its binding to Ub chains and promotes selective autophagy of damaged mitochondria. Proc. Natl Acad. Sci. USA 113, 4039–4044 (2016).

  12. 12.

    Lei, Y. et al. The mitochondrial proteins NLRX1 and TUFM form a complex that regulates type I interferon and autophagy. Immunity 36, 933–946 (2012).

  13. 13.

    Allen, I. C. et al. NLRX1 protein attenuates inflammatory responses to infection by interfering with the RIG-I-MAVS and TRAF6-NF-κB signaling pathways. Immunity 34, 854–865 (2011).

  14. 14.

    Mandell, M. A. et al. TRIM proteins regulate autophagy and can target autophagic substrates by direct recognition. Dev. Cell 30, 394–409 (2014).

  15. 15.

    Orvedahl, A. et al. HSV-1 ICP34.5 confers neurovirulence by targeting the Beclin 1 autophagy protein. Cell Host Microbe 1, 23–35 (2007).

  16. 16.

    Kanai, R. et al. Effect of γ4.5 deletions on oncolytic herpes simplex virus activity in brain tumors. J. Virol. 86, 4420–4431 (2012).

  17. 17.

    Kuma, A. et al. The role of autophagy during the early neonatal starvation period. Nature 432, 1032–1036 (2004).

  18. 18.

    Meza-Carmen, V. et al. Regulation of growth factor receptor degradation by ADP-ribosylation factor domain protein (ARD) 1. Proc. Natl Acad. Sci. USA 108, 10454–10459 (2011).

  19. 19.

    Bjorkoy, G. et al. p62/SQSTM1 forms protein aggregates degraded by autophagy and has a protective effect on huntingtin-induced cell death. J. Cell Biol. 171, 603–614 (2005).

  20. 20.

    Pankiv, S. et al. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J. Biol. Chem. 282, 24131–24145 (2007).

  21. 21.

    Arimoto, K. et al. Polyubiquitin conjugation to NEMO by triparite motif protein 23 (TRIM23) is critical in antiviral defense. Proc. Natl Acad. Sci. USA 107, 15856–15861 (2010).

  22. 22.

    Laurent-Rolle, M. et al. The interferon signaling antagonist function of yellow fever virus NS5 protein is activated by type I interferon. Cell Host Microbe 16, 314–327 (2014).

  23. 23.

    Orvedahl, A. et al. Autophagy protects against Sindbis virus infection of the central nervous system. Cell Host Microbe 7, 115–127 (2010).

  24. 24.

    Montespan, C. et al. Multi-layered control of Galectin-8 mediated autophagy during adenovirus cell entry through a conserved PPxY motif in the viral capsid. PLoS Pathog. 13, e1006217 (2017).

  25. 25.

    Vitale, N., Moss, J. & Vaughan, M. ARD1, a 64-kDa bifunctional protein containing an 18-kDa GTP-binding ADP-ribosylation factor domain and a 46-kDa GTPase-activating domain. Proc. Natl Acad Sci USA 93, 1941–1944 (1996).

  26. 26.

    Vichi, A., Payne, D. M., Pacheco-Rodriguez, G., Moss, J. & Vaughan, M. E3 ubiquitin ligase activity of the trifunctional ARD1 (ADP-ribosylation factor domain protein 1). Proc. Natl Acad. Sci. USA 102, 1945–1950 (2005).

  27. 27.

    Mevissen, T. E. et al. OTU deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis. Cell 154, 169–184 (2013).

  28. 28.

    Cherfils, J. Arf GTPases and their effectors: assembling multivalent membrane-binding platforms. Curr. Opin. Struct. Biol. 29, 67–76 (2014).

  29. 29.

    D’Souza-Schorey, C. & Chavrier, P. ARF proteins: roles in membrane traffic and beyond. Nat. Rev. Mol. Cell Biol. 7, 347–358 (2006).

  30. 30.

    Vitale, N., Horiba, K., Ferrans, V. J., Moss, J. & Vaughan, M. Localization of ADP-ribosylation factor domain protein 1 (ARD1) in lysosomes and Golgi apparatus. Proc. Natl Acad. Sci. USA 95, 8613–8618 (1998).

  31. 31.

    Kuma, A., Mizushima, N., Ishihara, N. & Ohsumi, Y. Formation of the approximately 350-kDa Apg12-Apg5·Apg16 multimeric complex, mediated by Apg16 oligomerization, is essential for autophagy in yeast. J. Biol. Chem. 277, 18619–18625 (2002).

  32. 32.

    Thurston, T. L. et al. Recruitment of TBK1 to cytosol-invading Salmonella induces WIPI2-dependent antibacterial autophagy. EMBO J. 35, 1779–1792 (2016).

  33. 33.

    Clark, K., Plater, L., Peggie, M. & Cohen, P. Use of the pharmacological inhibitor BX795 to study the regulation and physiological roles of TBK1 and IκB kinase epsilon: a distinct upstream kinase mediates Ser-172 phosphorylation and activation. J. Biol. Chem. 284, 14136–14146 (2009).

  34. 34.

    Matsumoto, G., Wada, K., Okuno, M., Kurosawa, M. & Nukina, N. Serine 403 phosphorylation of p62/SQSTM1 regulates selective autophagic clearance of ubiquitinated proteins. Mol. Cell 44, 279–289 (2011).

  35. 35.

    Soulat, D. et al. The DEAD-box helicase DDX3X is a critical component of the TANK-binding kinase 1-dependent innate immune response. EMBO J. 27, 2135–2146 (2008).

  36. 36.

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

  37. 37.

    Tu, D. et al. Structure and ubiquitination-dependent activation of TANK-binding kinase 1. Cell Rep. 3, 747–758 (2013).

  38. 38.

    Ma, X. et al. Molecular basis of Tank-binding kinase 1 activation by transautophosphorylation. Proc. Natl Acad. Sci. USA 109, 9378–9383 (2012).

  39. 39.

    Kimura, T. et al. TRIM-mediated precision autophagy targets cytoplasmic regulators of innate immunity. J. Cell Biol. 210, 973–989 (2015).

  40. 40.

    Kimura, T. et al. Dedicated SNAREs and specialized TRIM cargo receptors mediate secretory autophagy. EMBO J. 36, 42–60 (2017).

  41. 41.

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

  42. 42.

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

  43. 43.

    Narayan, K. et al. TRIM13 is a negative regulator of MDA5-mediated type I interferon production. J. Virol. 88, 10748–10757 (2014).

  44. 44.

    Matsumoto, G., Shimogori, T., Hattori, N. & Nukina, N. TBK1 controls autophagosomal engulfment of polyubiquitinated mitochondria through p62/SQSTM1 phosphorylation. Hum. Mol. Genet. 24, 4429–4442 (2015).

  45. 45.

    Zaffagnini, G. & Martens, S. Mechanisms of selective autophagy. J. Mol. Biol. 428, 1714–1724 (2016).

  46. 46.

    Li, S., Wang, L., Berman, M., Kong, Y. Y. & Dorf, M. E. Mapping a dynamic innate immunity protein interaction network regulating type I interferon production. Immunity 35, 426–440 (2011).

  47. 47.

    Davis, M. E. & Gack, M. U. Ubiquitination in the antiviral immune response. Virology 479–480, 52–65 (2015).

  48. 48.

    Ni, H. M. et al. Dissecting the dynamic turnover of GFP-LC3 in the autolysosome. Autophagy 7, 188–204 (2011).

  49. 49.

    Ding, W. X. et al. Linking of autophagy to ubiquitin-proteasome system is important for the regulation of endoplasmic reticulum stress and cell viability. Am. J. Pathol. 171, 513–524 (2007).

  50. 50.

    Feng, Y. & Longmore, G. D. The LIM protein Ajuba influences interleukin-1-induced NF-kappaB activation by affecting the assembly and activity of the protein kinase Czeta/p62/TRAF6 signaling complex. Mol. Cell. Biol. 25, 4010–4022 (2005).

  51. 51.

    Liang, C. et al. Autophagic and tumour suppressor activity of a novel Beclin1-binding protein UVRAG. Nat. Cell Biol. 8, 688–699 (2006).

  52. 52.

    Wang, L., Li, S. & Dorf, M. E. NEMO binds ubiquitinated TANK-binding kinase 1 (TBK1) to regulate innate immune responses to RNA viruses. PLoS ONE 7, e43756 (2012).

  53. 53.

    Lim, K. L. et al. Parkin mediates nonclassical, proteasomal-independent ubiquitination of synphilin-1: implications for Lewy body formation. J. Neurosci. 25, 2002–2009 (2005).

  54. 54.

    Livingston, C. M., Ifrim, M. F., Cowan, A. E. & Weller, S. K. Virus-induced chaperone-enriched (VICE) domains function as nuclear protein quality control centers during HSV-1 infection. PLoS Pathog. 5, e1000619 (2009).

  55. 55.

    Kimura, S., Noda, T. & Yoshimori, T. Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent-tagged LC3. Autophagy 3, 452–460 (2007).

  56. 56.

    Gaush, C. R. & Youngner, J. S. A tissue culture color test for measuring influenza virus and antibody. Proc. Soc. Exp. Biol. Med. 101, 853–856 (1959).

  57. 57.

    Biasini, M. et al. SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res. 42, W252–W258 (2014).

  58. 58.

    O’Neal, C. J., Jobling, M. G., Holmes, R. K. & Hol, W. G. Structural basis for the activation of cholera toxin by human ARF6-GTP. Science 309, 1093–1096 (2005).

  59. 59.

    Menetrey, J., Macia, E., Pasqualato, S., Franco, M. & Cherfils, J. Structure of Arf6-GDP suggests a basis for guanine nucleotide exchange factors specificity. Nat. Struct. Biol. 7, 466–469 (2000).

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The authors thank A. García-Sastre (Icahn School of Medicine at Mount Sinai) and J. Jung (University of Southern California) for the TRIM cDNA library, and S. Rabkin (Harvard) for providing mutant HSV-1. The authors also thank M. Ericsson (Harvard Electron Microscopy Facility) for assistance with sample preparation and S. Hwang (The University of Chicago) for discussions. This study was supported in part by the US National Institutes of Health grants R01 AI087846 and R21 AI118509 (to M.U.G.) and R01 GM112508 (to O.P.). K.M.J.S. and F.F. were supported by fellowships from the German Research Foundation (SP 1600/1-1 and FU 949/1-1, respectively). G.P.-R., J.K., J.M. and M.V. were supported by the Intramural Research Program of the NIH (National Heart, Lung, and Blood Institute). M.A.Z. received support by NIH training grant T32 GM007183.

Author information

K.M.J.S., S.G. and M.U.G. conceived and designed the experiments. K.M.J.S. and S.G. performed and analysed all experiments, except those in Supplementary Fig. 4f (M.A.Z.), Supplementary Fig. 2b,c (F.F.) and Fig. 5e (Z.M.P.). G.J.B. performed mutagenesis experiments. J.K., G.P.-R., J.M. and M.V. provided TRIM23 −/− and WT MEFs. C.L. contributed reagents, materials and analysis tools for experiments. O.P. performed the TRIM23 ARF structure modelling. K.M.J.S. and M.U.G. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

Correspondence to Michaela U. Gack.

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Fig. 1: TRIM proteins modulate viral-induced autophagy in a virus species-specific manner.
Fig. 2: TRIM23 is essential for virus-induced autophagy.
Fig. 3: K27-linked auto-ubiquitination of the ARF domain of TRIM23 is necessary for its autophagy function.
Fig. 4: ARF ubiquitination is required for the GTP hydrolysis activity of TRIM23 and its localization to autophagosomes.
Fig. 5: TRIM23 interacts with TBK1 and p62.
Fig. 6: TRIM23 GTPase activates TBK1 to phosphorylate p62.