TRIM21 Restricts Coxsackievirus B3 Replication, Cardiac and Pancreatic Injury via Interacting With MAVS and Positively Regulating IRF3-Mediated Type-I Interferon Production

Figure 1. TRIM21 is up-regulated in heart tissues of CVB3-induced VMC mice. Male BALB/c mice were intraperitoneally injected with 1000 TCID50 dose of CVB3 and the tissues were collected at the indicated time. (A) Paraffin sections of heart tissues were prepared and subjected to H&E staining (200 × magnifications). (B) The viral titers were analyzed by TCID50 assay. Data were presented as mean ± SEM of three representative independent experiments. (C) TRIM21 protein level in heart tissues was evaluated by IHC assay. Five photomicrographs were captured at each time under high power fields (400× magnifications) randomly and one representative image was shown. (D) TRIM21 mRNA level was analyzed by Q-PCR. Data were presented as mean ± SEM of three representative independent experiments. The GAPDH expression levels of heart tissues was set 1.0. (E,F) Primary cardiomyocytes of mice were mock infected or infected by CVB3 (MOI = 5) for 24 h. CVB3 dsRNA, endogenous TRIM21 protein and nucleus were stained with anti-dsRNA antibody (green), anti-TRIM21Ab (red) and DAPI dye (blue) and observed under confocal microscope. Photomicrographs were captured under high power fields (100 × magnifications)…

Figure 2. TRIM21 inhibits CVB3 infection in vitro. (A) HeLa cells were transiently transfected with human TRIM21-flag, or mock plasmids and 24 h later cells were subjected to Q-PCR and Western blot for detecting TRIM21 mRNA and protein levels. (B–D) HeLa cells were transiently transfected with human TRIM21-flag, or mock plasmids and 24 h later cells were infected with CVB3 at an MOI of 5 for the indicated time. Supernatants were collected and subjected to TCID50 (B).Caspid protein of virus was analyzed by Western blot (C). (D) HeLa cells were transfected with negative-control siRNA or TRIM21 siRNAs. 24 h later, cells were collected and subjected to Q-PCR and Western blot for analysis of TRIM21 expression.(E,F) HeLa cells were transfected with negative-control siRNA or TRIM21 siRNA and 24 h later, cells were infected with CVB3 (MOI = 5) for the indicated times. Supernatants were collected and subjected to TCID50 (E). VP1caspid protein of virus were analyzed by Western blot (F). *p < 0.05; **p < 0.01.

Figure 3. Overexpression of TRIM21 activates the virus-induced type I IFN signaling via catalyzing the K27-linked polyubiquitination of MAVS and increases IRF3 phosphorylation. (A) HeLa cells were transfected with TRIM21 expression or vector plasmids. 24 h after transfection, cells were infected with CVB3 (MOI = 5) for the indicated time. The mRNA levels of endogenous IFN-β and IFN-α were detected by Q-PCR assay. Data were presented as mean ± SEM of three representative independent experiments. (B) HEK293 cells were co-transfected with the IFN-β promoter reporter plasmids and the indicated amounts of TRIM21 expression plasmid or 100 ng vector plasmid. 24 h later, the luciferase activity was determined. HEK293 cells were co-transfected with the IFN-β promoter reporter plasmids and TRIM21 expression plasmid or mock vector. 24 h later, cells were infected with CVB3 (MOI = 5) for 12 h and the luciferase activity was determined. HEK293 cells were co-transfected with the IFN-β promoter reporter plasmids and TRIM21 expression plasmid or mock vector, together with MAVS or MDA5 expression plasmids. 24 h later, cells were infected with CVB3 (MOI = 5) for 12 h and the luciferase activity was…

Figure 4. The RING domain and PRY-SPRY domain are essential for its antiviral effect against CVB3. (A) Schematic of domain organization and deletion mutants of TRIM21. Approximate amino acid positions of domains are shown at the top. Various domains are boxed and discontinuous lines represent deletion of those regions. (B) HeLa cells were transiently transfected with TRIM21 expression plasmid, or indicated domain deletion mutants and 24 h later cells were infected with CVB3 (MOI = 5) for 24 h. The expression efficiency of domain deletion mutants and VP-1 production were analyzed by Western blot. (C) HEK293 cells were transfected with the IFN-β or IRF3 promoter reporter plasmids, together with TRIM21 expression plasmid or the indicated domain deletion mutants. The luciferase activity was determined after 24 h and data were presented as mean ± SEM of three representative independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5. Overexpression of TRIM21 in vivo significantly reduces viral load and alleviates CVB3-induced viral myocarditis. (A) Male BALB/c mice were retroorbitally injected 50 μg TRIM21 plasmids using in vivo-jet PEI and were sacrificed daily till day 4. Protein level of cardiac TRIM21was measured by IHC assay. (B) Mice were injected retroordibitally with 2 doses of 50 μg PEI-packaged mock or TRIM21 plasmids on day −2 and 1 and subjected to 1000TCID50 CVB3 on day 0(n = 6). The survival rate (C) and body weight change (D) were monitored daily until day 7 p.i. (E) Representative image of HE-staining hearts of CVB3-infected mice (day 7 p.i.) treated with PEI-TRIM21 or PEI-vector, showing intra-cardiac immune infiltrates (marked with arrows). Scale bar: 100 μm. Pathological scores of the heart of mice are shown. Results are presented as mean ± SEM; *p < 0.05. (F) Protein levels of inflammatory cytokines in the homogenates of heart were measured by ELISA. Data were presented as mean ± SEM of three representative independent. (G) The cardiac CVB3 titer at day 3 p.i. were determined by TCID50 assay. Data represent mean values of CVB3 PFU per gram of the heart tissues. Results are presented as…

Figure 6. TRIM21 deficient mice increases CVB3 replication in organs and aggravates pancreatic acinar cell necrosis as well as myocarditis. (A) Schematic diagram of deficient mice construction by CRISPR-CAS9 strategy. (B,C) Q-PCR and Western blot analysis of TRIM21 expression from tissues and BM cells of wild-type (WT) and TRIM21-/- mice, GAPDH was used as a loading control. (D-H) WT and TRIM21-deficient mice were infected i.p. with CVB3 (n = 6).The body weight change were monitored daily until day 7 p.i. (D). Representative image of HE-staining hearts and pancreas of CVB3-infected WT or TRIM21-/-mice (day 7 p.i.), showing intra-cardiac immune infiltrates or intactpancreatic acini (marked with arrows). Scale bar: 100 μm. Pathological scores of the heart and pancreas of mice are shown. Results are presented as mean ± SEM; Data pooled from 3 independent experiments (E).The mRNA levels of inflammatory cytokines in the homogenates of heart (day 7 p.i.) were measured by Q-PCR. Data were presented as mean ± SEM of three representative independent (F).Viral loadin pancreas and hearts of mice(day 3p.i.) was assessed by TCID50 assay. Results are presented as mean ± SEM;Data pooled from 3…