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. 2021 Feb 28;6(1):90.
doi: 10.1038/s41392-021-00477-8.

TRIM41 is required to innate antiviral response by polyubiquitinating BCL10 and recruiting NEMO

Zhou Yu #  1   2   3 , Xuelian Li #  3   4 , Mingjin Yang #  3 , Jiaying Huang  4 , Qian Fang  3 , Jianjun Jia  3 , Zheng Li  3 , Yan Gu  3 , Taoyong Chen  5 , Xuetao Cao  6   7   8   9   10
Affiliations

TRIM41 is required to innate antiviral response by polyubiquitinating BCL10 and recruiting NEMO

Zhou Yu et al. Signal Transduct Target Ther. .

Abstract

Sensing of pathogenic nucleic acids by pattern recognition receptors (PRR) not only initiates anti-microbe defense but causes inflammatory and autoimmune diseases. E3 ubiquitin ligase(s) critical in innate response need to be further identified. Here we report that the tripartite motif-containing E3 ubiquitin ligase TRIM41 is required to innate antiviral response through facilitating pathogenic nucleic acids-triggered signaling pathway. TRIM41 deficiency impairs the production of inflammatory cytokines and type I interferons in macrophages after transfection with nucleic acid-mimics and infection with both DNA and RNA viruses. In vivo, TRIM41 deficiency leads to impaired innate response against viruses. Mechanistically, TRIM41 directly interacts with BCL10 (B cell lymphoma 10), a core component of CARD proteins-BCL10 - MALT1 (CBM) complex, and modifies the Lys63-linked polyubiquitylation of BCL10, which, in turn, hubs NEMO for activation of NF-κB and TANK-binding kinase 1 (TBK1) - interferon regulatory factor 3 (IRF3) pathways. Our study suggests that TRIM41 is the potential universal E3 ubiquitin ligase responsible for Lys63 linkage of BCL10 during innate antiviral response, adding new insight into the molecular mechanism for the control of innate antiviral response.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
TRIM41 deficiency impairs innate response to pathogenic nucleic acid mimics and virus infections. a–d Trim41+/+ or Trim41–/– BMDM (1×105 cells per 24-well; a–c) or BMDC (1 × 105 cells per 24-well; d) were treated with liposome-packaged synthesized cytosolic nucleic acids sensor agonists for 4 h (a; 500 ng/ml 5′ppp-dsRNA for RIG-I, 500 ng/ml poly (I:C) for MDA5, 2 μg/ml HSV-60 or ISD for cGAS−STING), or indicated pathogens for 8 h (b–d) (MOI = 1 for VSV and SeV, MOI = 2 for LM, and MOI = 5 for HSV-1 or VACV). Amounts of IL-6, TNF, and IFNβ in supernatants were measured by ELISA (a, b and d), and the mRNA levels of ISGs were examined by Q-PCR (c). e 48 h after the transfection of Trim41+/+ or Trim41–/– BMDM cells with control or Trex1- or Adar1-specific siRNAs, the expression of indicated proteins were examined by Western blotting. f-h Cells in (e) were cultured in fresh medium for 48 h. Amounts of IL-6 and IFNβ in the supernatants were measured by ELISA (f and g), and the mRNA levels of Cxcl10 were examined by Q-PCR (h). Results are presented as mean ± SD of three biological replicates (a–d and f–h; one-way ANOVA followed by Bonferroni multiple comparison). One representative experiment of three is shown. **P < 0.01; ***P < 0.001; ****P < 0.0001
Fig. 2
Fig. 2
TRIM41 deficiency impairs innate response and exaggerates virus propagation in vivo. a-e Trim41+/+ or Trim41–/– mice were intraperitoneally injected with 100 μl of PBS or PBS containing 5 × 106 PFUs of VSV or LM, or 2 × 108 PFUs of HSV-1 as indicated. The survival of mice (n = 10 per group) was monitored by Kaplan and Meier method and analyzed by Log-rank test (a). Otherwise, 12 h after pathogen infections, serum levels of IL-6 (b) and IFNβ (c) was measured by ELISA (n = 5 mice per group). Alternatively, 24 h after pathogen infections, the microbe titers in the spleen and liver (three biological samples derived from each mouse; n = 5 per group) were determined by plaque formation assays (d, e). f, g Trim41+/+ or Trim41–/– mice were intravenously injected with 100 μl of PBS or PBS containing 2 × 108 PFUs of HSV-1 as indicated. 72 h after treatments, the replication status of HSV-1 in the brain was evaluated by IHC staining of HSV1 (representative images are shown; f) or by Q-PCR assays of Icp0 mRNA (three biological samples derived from each mouse; n = 3 mice for the PBS group, and n = 5 mice for the HSV-1 group; g). Results are presented as mean ± SE (b–e and g, one-way ANOVA followed by Bonferroni multiple comparison). One representative experiment of three is shown. One representative experiment of three is shown. *P < 0.05; **P < 0.01; ****P < 0.0001
Fig. 3
Fig. 3
TRIM41 potentiates the activation of both NF-κB and IRF3 during virus infection. a–e HEK293T cells were cotransfected with mock or Flag-tagged TRIM41 (10, 50, or 100 ng) vectors, vectors for PRR adaptors (100 ng MAVS or STING), and reporter vectors. 48 h later, the expression of TRIM41-Flag and STING-Myc was examined by Western blotting (a). Then the cells were infected with (e) or without (b–d) VSV (MOI = 1) or HSV-1 (MOI = 5) for 4 h, and the luciferase activity was determined (b-e). f Trim41+/+ or Trim41–/– BMDM were infected with VSV (MOI = 1) or HSV-1 (MOI = 5) viruses as indicated. The activation of the signaling mediators were examined by Western blotting. Results (b–e) are presented as mean ± SD of four biological samples (one-way ANOVA followed by Bonferroni multiple comparison). One representative experiment of three is shown. ns, not significant; *P < 0.05; ***P < 0.001; ****P < 0.0001
Fig. 4
Fig. 4
The PRY-SPRY domain of TRIM41 directly binds the CARD domain of BCL10. a Wild type BMDM were infected with VSV (MOI = 1) or HSV-1 (MOI = 5) viruses for 2 h as indicated. Then whole cell extracts (WCE) were immunoprecipitated with anti-TRIM41 antibody plus protein A/G beads. Components in the TRIM41 complex were examined by Western blotting. b 1 μg GST or GST-TRIM41 was coincubated with 1 μg recombinant CARD9, CARD11, or BCL10 for 1 h. Then sepharose 4B beads were used to pull down the GST complex. Components pulled down by the beads were examined by Western blotting. c Schematic illustration of domains in full-length (FL) or fragments (F) of TRIM41-Flag or BCL10-Myc. R, ring finger domain; B, B-box domain; CC, coiled-coil domain; CTD, C-terminal domain. Numerical numbers indicate the site of amino acids. d, e HEK293T cells were transiently transfected with Flag-tagged TRIM41 (or mutants) and Myc-tagged BCL10 (or mutants) as indicated for 48 h. Whole cell extracts (WCE) were immunoprecipitated with anti-Flag Sepharose Beads (d) or anti-Myc Sepharose Beads (e), and the associated BCL10 or TRIM41 was examined by Western blotting. f, g RAW264.7 cells grown on cover slides were transiently transfected with green fluorescent protein (GFP)-tagged TRIM41 and red fluorescent protein (RFP)-tagged BCL10 for 48 h, and then infected with VSV (MOI = 1) or HSV-1 (MOI = 5) viruses for 2 h. After counterstained with DAPI, cells were examined by confocal microscope (f; Scale bar, 100 nm). Ten double-positive randomly-selected cells on cover slides were measured for fluorescence colocalization, and the data are presented as mean ± SD (g)
Fig. 5
Fig. 5
TRIM41 mediates Lys63-linked polyubiquitination of BCL10. a-c Trim41+/+ or Trim41–/– BMDM were infected with VSV (MOI = 1) or HSV-1 (MOI = 5) viruses for 2 h. Then whole-cell extracts (WCE) heated in buffer containing 1% SDS were immunoprecipitated (IP) with anti-BCL10 (a), anti-CARD9 (b), or anti-CARD11 (c) antibody plus protein A/G beads. Polyubiquitination of BCL10 (a), CARD9 (b), and CARD11 (c) was examined by Western blotting. d HEK293T cells were transiently transfected with Flag-tagged TRIM41 (or mutant) and Myc-tagged BCL10 vectors as indicated for 48 h. Then polyubiquitination of BCL10 was examined by Western blotting after immunoprecipitations with anti-Myc Sepharose Beads. e HEK293T cells were transiently transfected with Flag-tagged TRIM41, Myc-tagged BCL10, and HA-tagged Ub (mutants) vectors as indicated for 48 h. Then polyubiquitination of BCL10 was examined by Western blotting after immunoprecipitations with anti-Myc Sepharose Beads. f After incubation for 30 min, the in vitro polyubiquitination system was boiled for 5 min, and then polyubiquitinated BCL10 was examined by Western blotting against Ub after immunoprecipitations. g HEK293T cells were transiently transfected with Flag-tagged TRIM41 and Myc-tagged BCL10 (mutant) vectors as indicated for 48 h. Then polyubiquitination of BCL10 was examined by Western blotting after immunoprecipitations with anti-Myc Sepharose Beads. One representative experiment of three is shown
Fig. 6
Fig. 6
Lys63-linkage of BCL10 by TRIM41 hubs NEMO for activation of virus-triggered innate signaling and response. a, b Trim41+/+ or Trim41–/– BMDM cells were infected with VSV (MOI = 1) or HSV-1 (MOI = 5) viruses as indicated. Then whole-cell extracts were immunoprecipitated with anti-MAVS (a) or anti-STING (b) antibody plus protein A/G beads. Components in the immune complex were examined by Western blotting. c Parental (+/+) or knockout (–/–; for Bcl10 or Nemo) RAW264.7 cells were transiently transfected with mock, wild type BCL10 or NEMO, or mutated BCL10 or NEMO vectors as indicated for 48 h. The amounts of BCL10 and NEMO were examined by Western blotting. d-f Bcl10+/+ or Bcl10–/– RAW264.7 cells rescued with mock, WT BCL10 or mutated BCL10 vectors for 48 h as indicated were infected with VSV (MOI = 1) or HSV-1 (MOI = 5) viruses for 8 h (d) or as indicated (e and f). Levels of IL-6, TNF, and IFNβ in supernatants were measured by ELISA (d). Otherwise, whole-cell extracts were immunoprecipitated with anti-MAVS (e) or anti-STING (f) antibody plus protein A/G beads. Components in the immune complex were examined by Western blotting. Results are presented as mean ± SD of triplicates biological replicates (d) (One-way ANOVA followed by Bonferroni multiple comparison). One representative experiment of three is shown. ns, not significant; *P < 0.05; ****P < 0.0001
Fig. 7
Fig. 7
Role of TRIM41 in innate antiviral signaling requires both Lys63-linkage of BCL10 and ubiquitin-binding capacity of NEMO. a, b Bcl10+/+ or Bcl10–/– RAW264.7 cells were rescued with mock, WT BCL10, or mutated BCL10 vectors for 48 h as indicated. Then cells were transfected with NF-κB (a) or Ifnb (b) reporters and mock or TRIM41 vectors for 48 h. After infection with VSV (MOI = 1) or HSV-1 (MOI = 5) viruses for 4 h, the luciferase activity was determined. c, d Nemo+/+ or Nemo–/– RAW264.7 cells rescued with mock, WT NEMO, or mutated NEMO vectors were transfected with NF-κB (c) or Ifnb (d) reporters and mock or TRIM41 vectors for 48 h. After infection with VSV (MOI = 1) or HSV-1 (MOI = 5) viruses for 4 h, the luciferase activity was determined. Results are presented as mean ± SD of four biological replicates (a–d) (One-way ANOVA followed by Bonferroni multiple comparison). One representative experiment of three is shown. UT, untreated; HSV, HSV-1; ns, not significant; ****P < 0.0001
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