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. 2018 Mar 20:4:13.
doi: 10.1038/s41421-018-0010-9. eCollection 2018.

TRIM29 negatively controls antiviral immune response through targeting STING for degradation

Qijie Li #  1 , Liangbin Lin #  1 , Yanli Tong #  1 , Yantong Liu  2 , Jun Mou  1 , Xiaodong Wang  1 , Xiuxuan Wang  3 , Yanqiu Gong  3 , Yi Zhao  1 , Yi Liu  1 , Bo Zhong  4 , Lunzhi Dai  3 , Yu-Quan Wei  2 , Huiyuan Zhang  1 , Hongbo Hu  1
Affiliations

TRIM29 negatively controls antiviral immune response through targeting STING for degradation

Qijie Li et al. Cell Discov. .

Erratum in

Abstract

Innate immune system is armed by several lines of pattern recognition receptors to sense various viral infection and to initiate antiviral immune response. This process is under a tight control and the negative feedback induced by infection and/or inflammation is critical to maintain immune homoeostasis and to prevent autoimmune disorders, however, the molecular mechanism is not fully understood. Here we report TRIM29, a ubiquitin E3 ligase, functions as an inducible negative regulator of innate immune response triggered by DNA virus and cytosolic DNA. DNA virus and cytosolic DNA stimulation induce TRIM29 expression robustly in macrophages and dendritic cells, although the basal level of TRIM29 is undetectable in those cells. TRIM29 deficiency elevates IFN-I and proinflammatory cytokine production upon viral DNA and cytosolic dsDNA stimulation. Consistently, in vivo experiments show that TRIM29-deficient mice are more resistant to HSV-1 infection than WT controls, indicated by better survival rate and reduced viral load in organs. Mechanism studies suggest that STING-TBK1-IRF3 signaling pathway in TRIM29 KO cells is significantly enhanced and the degradation of STING is impaired. Furthermore, we identify that TRIM29 targets STING for K48 ubiquitination and degradation. This study reveals TRIM29 as a crucial negative regulator in immune response to DNA virus and cytosolic DNA, preventing potential damage caused by overcommitted immune responses.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. TRIM29 responses to cytosolic viral DNA stimulation and negatively regulates cytosolic DNA-induced cytokine production.
Mouse BMDM a and BMDC b, human monocyte cell line THP-1 c and PBMC-derived DC d were stimulated with dsDNA90, VACV-70, HSV-60, LPS, and poly (I:C) for 12 h. The expression of TRIM29 mRNA was detected by real-time PCR and β-actin served as the reference gene. e BMDMs were treated with dsDNA90, VACV-70, HSV-60, cGAMP, LPS, and poly (I:C) for 24 h and protein level of TRIM29 was detected by IB using anti-TRIM29 antibody, β-actin served as the loading control. f BMDMs were treated with HSV-60 or cGAMP for 0, 2, or 6 h. The expression of TRIM29 mRNA was detected by real-time PCR and β-actin served as the reference gene. Two TRIM29-targeting shRNA (shRNA#1 and shRNA#2) and scramble shRNA (ctrl) were induced to mouse BMDM to knockdown TRIM29. The efficiency of knockdown was detected by real-time PCR g. BMDMs transfected with scramble shRNA or two TRIM29-targeting shRNA were stimulated with VACV-70, HSV-60, and cGAMP for 12 h, followed by real-time PCR assay h and ELISA i to detect the cytokine production. Data in all panels are representative of two to three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 (Student’s t-test). Error bars are s.d.
Fig. 2
Fig. 2. TRIM29 regulates host immune response against viral infection in vivo.
Real-time PCR assay a, c and ELISA b were used to detect the expression of cytokine, chemokine and antiviral protein in WT and Trim29−/− BMDMs stimulated with HSV-60, VACV-70, cGAMP, and DNA90 for 12 h. d WT and Trim29-/- mice (n = 8 per strain) were infected by intravenous injected HSV-1 (2 × 107 PFU per mouse) and monitored daily for 2 weeks for survival rate. e Viral titers of brains, livers and spleens from WT and Trim29−/− mice (n = 3 per strain) 2 and 4 days after HSV-1 infection. f ELISA analysis of IFN-α, IFN-β, TNF-α and IL-6 in sera from WT and Trim29−/− mice (n = 3 per strain) after HSV-1 infection. Data in all panels are representative of three independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001 (Student’s t-test). Error bars are s.d.
Fig. 3
Fig. 3. TRIM29 negatively regulates dsDNA-activated signaling pathway.
a WT and Trim29−/− BMDMs were stimulated with cGMAP and HSV-60 for indicated time, the cells were lysed and subjected to IB assay to detect phosphorylation of TBK1, IRF3, IκBα, and the degradation of STING. The total proteins or β-actin served as loading control. b WT, Trim29-/-, and TRIM29-reconstructed MEFs were stimulated as above for indicated time, and IB was performed to detect TRIM29, STING, phosphorylation of TBK1, IRF3. c These cells were stimulated as above for 12 h, and IFN-α, IFN-β, TNF-α, and IL-6 was detected by real-time PCR assay
Fig. 4
Fig. 4. TRIM29 interacts with STING and mediates K48 linked ubiquitination and degradation of STING.
a TRIM29-interacting proteins were identified by mass spectrum. Interaction between TRIM29 and STING was detected by co-immunoprecipitation (co-IP) in HSV-60 or cGMAP-stimulated BMDMs b and in the HEK 293T cell overexpressed TRIM29 and STING c. d Confocal microscopy of HEK 293T cells co-expressed Flag-TRIM29 and STING, non-treated and stimulated with HSV-60 for 6 h, followed by staining for Flag-TRIM29 (green) and STING (red). The bar in the picture stood for 1 μm. e HEK 293T cells were transfected with STING with or without TRIM29, treated with DMSO or MG132 for 12 h. STING and TRIM29 protein level was detected by IB. f HEK 293T cells were transfected with STING, TRIM29 and HA-tagged WT or mutant ubiquitin and subjected to ubiquitin assay. The ubiquitination of STING was detected by IB using anti-HA antibody. g WT and Trim29-/- macrophages were stimulated with HSV-60 for indicated time. The cells were lysed and subjected to IP with anti-STING antibody, the ubiquitination of STING was detected by IB using anti-ubiquitin and anti-K48 ubiquitin antibody. Data in all panels (except panel a) are representative of three independent experiments
Fig. 5
Fig. 5. Identification of critical domains mediating TRIM29–STING interaction.
a The schematic structure of TRIM29. b Full-length TRIM29 and different truncated TRIM29 mutants were co-expressed with STING in HEK 293T cells, and interaction between STING with WT or truncated TRIM29 was detected using IP and IB with indicated antibodies. c The schematic structure of STING. d The full-length STING and different truncated STING mutants were co-expressed with TRIM29, the interaction between TRIM29 with WT or truncated STING was detected by IP and IB with indicated antibodies. e The full-length STING and different truncated STING mutants were co-expressed with TRIM29 in HEK 293T cells and the protein level of STING (FL and truncated) and TRIM29 was detected with IB. f The Lys residues in STING were identified as potential targets of TRIM29 using IP-MS, highlighted in red. The number was frequency of specific peptides identified in IP product. g WT and mutated STING was co-expressed with TRIM29 and ubiquitin, the ubiquitination of STING was detected by IP and IB, and the protein level of STING was detected by IB, using indicated antibodies h
Fig. 6
Fig. 6. The schematic mechanism of TRIM29 regulating anti-DNA virus immune response through targeting STING.
Viral dsDNA, or self-dsDNA, is sensed by cGAS and other DNA sensors such as DDX41 and IFI16. cGAS catalyzes the synthesis of cGAMP, which functions as a second messenger to activate STING–TBK1–IRF3 pathway, leading to IFN-I production. NF-κB pathway is also activated by TBK1 to trigger proinflammatory cytokine production. These cytokines are crucial to mediate an efficient antiviral immune response. TRIM29 is induced by DNA virus and cytosolic DNA stimulation, and targets STING for K48 ubiquitination and degradation, therefore downregulates STING-dependent signaling pathway, to prevent the overcommitted immune response
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