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E3 ubiquitin ligase RNF128 promotes innate antiviral immunity through K63-linked ubiquitination of TBK1

Nature Immunology volume 17, pages 1342–1351 (2016)Cite this article

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Abstract

TBK1 is essential for interferon-β (IFN-β) production and innate antiviral immunity. Here we identified the T cell anergy–related E3 ubiquitin ligase RNF128 as a positive regulator of TBK1 activation. RNF128 directly interacted with TBK1 through its protease-associated (PA) domain and catalyzed the K63-linked polyubiquitination of TBK1, which led to TBK1 activation, IRF3 activation and IFN-β production. Deficiency of RNF128 expression attenuated IRF3 activation, IFN-β production and innate antiviral immune responses to RNA and DNA viruses, in vitro and in vivo. Our study identified RNF128 as an E3 ligase for K63-linked ubiquitination and activation of TBK1 and delineated a previously unrecognized function for RNF128.

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Figure 1: Viral infection induces RNF128 expression.
Figure 2: RNF128 positively regulates IFN-β signaling.
Figure 3: RNF128 targets TBK1.
Figure 4: RNF128 promotes K63-linked ubiquitination of TBK1.
Figure 5: RNF128 promotes TBK1 activation.
Figure 6: RNF128 is a general regulator of TBK1 activation.
Figure 7: RNF128 positively regulates antiviral response.

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Acknowledgements

We thank G.C. Fathman (Stanford University School of Medicine), K.A. Fitzgerald (University of Massachusetts Medical School) and H.-b. Shu (Wuhan University) for providing RNF128 expression plasmids, IFN-β reporter and TBK1 mutant plasmid S172A, respectively. We thank B. Sun (Institut Pasteur of Shanghai, Chinese Academy of Sciences) for providing TBK1-deficient MEFs, and H. Meng (Institute of Basic Medicine, Shandong Academy of Medical Sciences) for VSV and HSV-1. This work was supported in part by grants from the Natural Science Foundation of China (81525012, 81471538, 81273219) and the Specialized Research Fund for the Doctoral Program of Higher Education of China (20130131130010) to C.G. G.J. is a Taishan Scholar of Shandong, China.

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Authors and Affiliations

  1. Department of Immunology and Key Laboratory of Infection and Immunity of Shandong Province, Shandong University the School of Medicine, Jinan, China

    Guanhua Song, Bingyu Liu, Haifeng Wu, Peng Wang, Kai Zhao, Lei Zhang & Chengjiang Gao

  2. Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan, China

    Guanhua Song, Zhihui Li & Guosheng Jiang

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  1. Guanhua Song
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Contributions

C.G. conceived and supervised the study. G.S. performed most of the experiments and prepared the figures. B.L. performed part of the ubiquitination experiments. H.W., P.W., K.Z. and L.Z. provided reagents and contributed to discussions. Z.L. and G.J. provided facility for virus study. G.S. 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 Virus infection induces RNF128 expression.

(a,b) RT-PCR analysis of R nf128 (RNF128) mRNA expression in mouse peritoneal macrophages (a) and THP-1 cells (b) upon stimulation with poly(I:C) and ISD or infection with SeV and HSV-1 for indicated times. Data are representative of three independent experiments with similar results.

Supplementary Figure 2 RNF128 positively regulates IFN-β signaling.

(a) Quantitative RT-PCR analysis of Rnf128 mRNA expression (upper) and western blot analysis of RNF128 protein expression (lower) in mouse peritoneal macrophages transfected with control siRNA or mice RNF128 siRNA 1, 2 or 3 for 48 h. (b) mRNA expression of Ifnb1, Cxcl10, Mx1, Ccl5, Il6 and Tnfa in mouse peritoneal macrophages transfected with control siRNA or mice RNF128 siRNA 1, 2 or 3 and then stimulated with poly(I:C) and ISD or infected with SeV and HSV-1. (c) Quantitative RT-PCR analysis of RNF128 mRNA expression (upper) and western blot analysis of RNF128 protein expression (lower) in THP-1 cells transfected with control siRNA or human RNF128 siRNA 1, 2 or 3 for 48 h. (d) Quantitative RT-PCR analysis of IFNB1, CXCL10, MX1, CCL5, IL6 and TNFA mRNA expression in THP-1 cells transfected with control siRNA or human RNF128 siRNA 1, 2 or 3 for 48 h and then stimulated with poly(I:C) and ISD or infected with SeV and HSV-1. (e) Quantitative RT-PCR analysis of Ifnb1, Cxcl10, Mx1, Ccl5, Il6 and Tnfa mRNA expression in WT or Rnf128−/− mouse peritoneal macrophages stimulated with LPS for the indicated time. (f,g) Luciferase activity of IFN-β promoter reporter and mRNA expression of IFNB1 in HEK293 cells transfected with expression plasmids for TRIF, cGAS+STING and RIG-IN along with V5-RNF128 expression plasmid or control vector for 24 h. *p <0.05,# p <0.01 (two-tail Student's t-test). Data are from three independent experiments (a-f; mean+S.D. of triplicate assays) or are representative of three independent experiments with similar results (a,b).

Supplementary Figure 3 RNF128 promotes IRF3 activation.

(a) Luciferase activity in HEK293 cells transfected with IFN-β, PRDI-III, PRDII, PRDIV luciferase reporter and RNF128 expression plasmid together with expression plasmids for TRIF, MAVS, cGAS+STING or control plasmid. (b) Western blot analysis of phosphorylated-IRF3 in HEK293 cells transfected with RNF128 expression plasmid or control plasmid, along with expression plasmids for TRIF, MAVS, cGAS+STING or control plasmid. (c) Western blot analysis of phosphorylated-IRF3 protein in HEK293 cells transfected with expression plasmids for MAVS and increased amount of V5-RNF128 plasmid. *p <0.05,** p <0.01 (two-tail Student's t-test). Data are from three independent experiments (a; mean±S.D. of triplicate assays) or are representative of three independent experiments with similar results (b,c).

Supplementary Figure 4 RNF128 targets TBK1.

(a) Coimmunoprecipitation analysis of RNF128 interaction with TBK1 in THP-1 cells infected with SeV or HSV-1 for 8 h. (b) ISRE luciferase activity in HEK293 cells transfected with V5-RNF128 expression plasmid or mutant plasmids together with expression plasmids as indicated. (c) Western blot analysis of phosphorylated-IRF3 protein in HEK293 cells transfected with expression plasmids for RNF128 expression plasmid or the mutants and MAVS or cGAS and STING expression plasmids. (d) Coimmunoprecipitation analysis of MAVS ubiquitination in primary macrophages from WT and Rnf128−/− mice were infected with SeV. (e) Coimmunoprecipitation analysis of STING ubiquitination in primary macrophages from WT and Rnf128−/− mice were infected with HSV-1. **p <0.01 (two-tail Student's t-test). Data are representative of three independent experiments with similar results (a,c-e) or are from three independent experiments (b; mean±S.D. of triplicate assays).

Supplementary Figure 5 RNF128 positively regulates TBK1 kinase activity.

(a) TBK1kinase activity assays in HEK293 cells transfected with the RNF128 expression plasmid and then infected with SeV for 6 h. (b) TBK1 kinase activity assays in peritoneal macrophages transfected with mice RNF128 siRNA, then the cells were infected with SeV for 6 h. (c) Quantitative RT-PCR analysis of mRNA expression of Ifnb1 (upper) and western blot analysis of phosphorylated-TBK1 protein (lower) in TBK1 deficient MEFs transfected with expression plasmids for Myc-TBK1-WT or Myc-TBK1-S172A along with V5-RNF128 plasmids. *p <0.05, **p <0.01, ns, not significant (two-tail Student's t-test). Data are from three independent experiments (a,b; mean±S.D. of triplicate assays) or are representative of three independent experiments with similar results (c-h).

Supplementary Figure 6 RNF128 positively regulates antiviral response.

(a) VSV titers and VSV-G protein in HEK293 (2×105) transfected with the V5-RNF128-WT, mutant V5-RNF128-H2N2 and V5-RNF128-△PA expression plasmid and then infected with VSV (MOI, 0.1). (b) VSV titers and VSV-G protein in mouse peritoneal macrophages transfected with control siRNA or RNF128 siRNA and then infected with VSV (MOI, 0.1). (c) HSV-1 genomic DNA and titers in mouse primary macrophages transfected with control siRNA or RNF128 siRNA and then infected by HSV-1 (MOI, 10). (d) VSV titers and VSV-G protein in THP-1 cells transfected with control siRNA or RNF128 siRNA and then infected with VSV (MOI, 0.1). (e) HSV-1 titers and genomic DNA in THP-1 cells transfected with control siRNA or RNF128 siRNA and then infected by HSV-1 (MOI, 10). *p <0.05, **p <0.01 (two-tail Student's t-test). Data are from three independent experiments (a-e; mean±S.D. of triplicate assays) or are representative of three independent experiments with similar results (a,b,d).

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Song, G., Liu, B., Li, Z. et al. E3 ubiquitin ligase RNF128 promotes innate antiviral immunity through K63-linked ubiquitination of TBK1. Nat Immunol 17, 1342–1351 (2016). https://doi.org/10.1038/ni.3588

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