Journal: Frontiers in Immunology

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

doi: 10.3389/fimmu.2018.02479

Figure Lengend Snippet: TRIM21 is up-regulated in heart tissues of CVB3-induced VMC mice. Male BALB/c mice were intraperitoneally injected with 1000 TCID 50 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 TCID 50 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) (E) .TRIM21 mRNA level were analyzed by Q-PCR. Data were presented as mean ± SEM of three representative independent experiments. The GAPDH expression level of heart tissues was set as 1.0 (F) . ** p < 0.01; *** p < 0.001.

Article Snippet: HEK293 cells were transfected with plasmids containing human or murine TRIM21 (1 μg) using the Lipofectamine Plus reagent (Invitrogen) for 24 h, and then infected by CVB3 (MOI = 5) for 18 h. Samples were resuspended in sample lysis buffer (Bio-Rad).

Techniques: Injection, Staining, Expressing, Infection, Microscopy

Journal: Frontiers in Immunology

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

doi: 10.3389/fimmu.2018.02479

Figure Lengend Snippet: 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 TCID 50 (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 TCID 50 (E) . VP1caspid protein of virus were analyzed by Western blot (F) . * p < 0.05; ** p < 0.01.

Article Snippet: HEK293 cells were transfected with plasmids containing human or murine TRIM21 (1 μg) using the Lipofectamine Plus reagent (Invitrogen) for 24 h, and then infected by CVB3 (MOI = 5) for 18 h. Samples were resuspended in sample lysis buffer (Bio-Rad).

Techniques: Infection, In Vitro, Transfection, Western Blot, Virus, Negative Control, Expressing

Journal: Frontiers in Immunology

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

doi: 10.3389/fimmu.2018.02479

Figure Lengend Snippet: 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 determined. Data were presented as mean ± SEM of three representative independent experiments. (C) HeLa cells were transfected with TRIM21 expression or vector plasmids for 24 h before infection with CVB3 (MOI = 5). The mRNA levels of endogenous ISG15 and ISG54 were detected by Q-PCR assay. (D) HEK293 cells were co-transfected with the IRF3 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 and data were presented as mean ± SEM of three representative independent experiments. (E,F) HeLa cells were transfected with TRIM21 expression or vector plasmids for 24 h before infection with CVB3 (MOI = 5). Cell lysates were subjected to probe withanti-IRF3, anti-pIRF3 and anti-GAPDH antibodies by Western blotting and Native page for the detection of IRF3 dimerization. (G) HeLa cells were transfected with Flag-MAVS plasmids as indicated, 24 h later cells were infected with CVB3 (MOI = 5). Cellular lysates were immunoprecipitated with anti-Flag. Immunoprecipitates were analyzed by WB with anti-Flag and anti-TRIM21. (H) HeLa cells were co-transfected with TRIM21, Flag-MAVS, and HA-K27Ub for 24 h and treated with CVB3 for additional 12 h. Ubiquitination and immunoblotting assays were performed with indicated antibodies. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: HEK293 cells were transfected with plasmids containing human or murine TRIM21 (1 μg) using the Lipofectamine Plus reagent (Invitrogen) for 24 h, and then infected by CVB3 (MOI = 5) for 18 h. Samples were resuspended in sample lysis buffer (Bio-Rad).

Techniques: Over Expression, Virus, Phospho-proteomics, Transfection, Expressing, Plasmid Preparation, Infection, Luciferase, Activity Assay, Western Blot, Clear Native PAGE, Immunoprecipitation, Ubiquitin Proteomics

Journal: Frontiers in Immunology

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

doi: 10.3389/fimmu.2018.02479

Figure Lengend Snippet: 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.

Article Snippet: HEK293 cells were transfected with plasmids containing human or murine TRIM21 (1 μg) using the Lipofectamine Plus reagent (Invitrogen) for 24 h, and then infected by CVB3 (MOI = 5) for 18 h. Samples were resuspended in sample lysis buffer (Bio-Rad).

Techniques: Transfection, Expressing, Plasmid Preparation, Infection, Western Blot, Luciferase, Activity Assay

Journal: Frontiers in Immunology

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

doi: 10.3389/fimmu.2018.02479

Figure Lengend Snippet: 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 1000TCID 50 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 TCID 50 assay. Data represent mean values of CVB3 PFU per gram of the heart tissues. Results are presented as mean ± SEM; Data pooled from 3 independent experiments. * p < 0.05; ** p < 0.01. (H) Relative mRNA level of IFN-β (day 3 and 7p.i.) was detected by Q-PCR. Data were normalized to GAPDH expression and presented as mean ± SEM of three representative independent. (I) Hearts of mice at day 3 and 7 p.i. were OCT-embedded and cyrosections (5 μM) were subjected to fluorescent staining. Composite confocal represented images show dsRNA (red, anti-dsRNA Ab) and nuclear (blue, DAPI).Low magnification (magnification, × 100) and higher magnification of the boxed areas (magnification, × 200) are shown. The number of red-stained viral-infected cells in the heart sections of mice were numerated. Data are expressed as mean ± SEM from three repeated experiments ( n = 3). *** p < 0.001.

Article Snippet: HEK293 cells were transfected with plasmids containing human or murine TRIM21 (1 μg) using the Lipofectamine Plus reagent (Invitrogen) for 24 h, and then infected by CVB3 (MOI = 5) for 18 h. Samples were resuspended in sample lysis buffer (Bio-Rad).

Techniques: Over Expression, In Vivo, Injection, Staining, Infection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Expressing

Journal: Frontiers in Immunology

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

doi: 10.3389/fimmu.2018.02479

Figure Lengend Snippet: 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 TCID 50 assay. Results are presented as mean ± SEM;Data pooled from 3 independent experiments. * p < 0.05; ** p < 0.01 (G) .The mRNA and protein level of IFN-β (day 1–3 p.i.) in hearts of mice was detected by Q-PCR and ELISA. Data as mean ± SEM of three representative independent. * p < 0.05; ** p < 0.01 (H) . (I) Proposed model depicting the role of TRIM21 in positive regulation of IFN-I production during CVB3 infection. TRIM21 targets and promotes the activity of MAVS, leading to the increased phosphorylation and translocation of p-IRF3 into the nucleus, leading to enhanced transcription and production of IFNs and IFN-stimulated genes (ISGs) that limits CVB3 infection.

Article Snippet: HEK293 cells were transfected with plasmids containing human or murine TRIM21 (1 μg) using the Lipofectamine Plus reagent (Invitrogen) for 24 h, and then infected by CVB3 (MOI = 5) for 18 h. Samples were resuspended in sample lysis buffer (Bio-Rad).

Techniques: CRISPR, Western Blot, Expressing, Control, Infection, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Phospho-proteomics, Translocation Assay

Journal:

Article Title: Coxsackievirus Expression of the Murine Secretory Protein Interleukin-4 Induces Increased Synthesis of Immunoglobulin G1 in Mice

doi:

Figure Lengend Snippet: Structure of CVB3-IL-4 plasmid and cDNA sequence. (A) Outline of the plasmid construct that gives rise to the mIL-4-expressing CVB3 strains. Both plasmids encode the full ORF of mIL-4 (GenBank accession no. M25892) between the capsid coding region (P1) and the 2Apro coding region, with a repeated 2Apro cleavage site (▾) bordering the insert. (B) pCVB3/0-mIL/47 (termed 47 in the figure) has a perfect repeat of 72 bp, 24 aa; pCVB3-PL2-mIL4/46 (termed 46 in the figure) has 70% identity between the 50-nt sequence encoding a perfect 17-aa repeat 2Apro cleavage site. Due to the length of the sequence, it is presented in three contiguous segments. The CVB3 sequences of both recombinant cDNA genomes are derived from pCVB3/0 and cloned in the plasmid vector pSVN (16). The repeated sequences encoding 2Apro cleavage sites are underlined, and the restriction sites used in the construction of the plasmids are indicated. The sequence and annealing site of primers (ID9, ID10, KNIL4S, and KNIL4AS) used in the detection of chimeric and deleted genomes are indicated. N, nucleotide; AA, amino acid.

Article Snippet: Briefly, the ELISA was set up using 96-well flat-bottomed plates coated with a 1:1,000 dilution of hyperimmune horse anti-CVB3 serum (ATCC).

Techniques: Plasmid Preparation, Sequencing, Construct, Expressing, Recombinant, Derivative Assay, Clone Assay

Journal:

Article Title: Coxsackievirus Expression of the Murine Secretory Protein Interleukin-4 Induces Increased Synthesis of Immunoglobulin G1 in Mice

doi:

Figure Lengend Snippet: Characterization of mIL-4-expressing CVB3 strains in cell culture. (A) Single-step growth curves of the viruses on HeLa cells. (B) Concentration of mIL-4 in lysates as measured by ELISA. HeLa cells were inoculated with the parental virus CVB3/0 or the chimeric strains CVB3/0-mIL4-47 and CVB3-PL2-mIL4-46. Inoculated cell cultures were harvested by freeze-thaw lysis at the stated times. Error bars indicate standard error of the mean.

Article Snippet: Briefly, the ELISA was set up using 96-well flat-bottomed plates coated with a 1:1,000 dilution of hyperimmune horse anti-CVB3 serum (ATCC).

Techniques: Expressing, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Lysis

Journal:

Article Title: Coxsackievirus Expression of the Murine Secretory Protein Interleukin-4 Induces Increased Synthesis of Immunoglobulin G1 in Mice

doi:

Figure Lengend Snippet: Western blot analysis of viral proteins in inoculated HeLa cells. Proteins were harvested from HeLa cells inoculated with the parental CVB3/0 or either of the chimeric strains by lysing the cell monolayers in Laemmli loading buffer. Lysates were prepared at the times shown (in hours p.i.) and electrophoresed in SDS–14% PAGE followed by electroblotting. Capsid protein 1D was detected with a polyclonal horse anti-CVB3 neutralizing antibody followed by peroxidase-conjugated rabbit anti-horse IgG. C, purified CVB3/0; TC, uninfected HeLa cell lysate. 1D, location of the detected capsid protein at 34 kDa is shown. Lane M, size markers (in kilodaltons).

Article Snippet: Briefly, the ELISA was set up using 96-well flat-bottomed plates coated with a 1:1,000 dilution of hyperimmune horse anti-CVB3 serum (ATCC).

Techniques: Western Blot, Purification

Journal:

Article Title: Coxsackievirus Expression of the Murine Secretory Protein Interleukin-4 Induces Increased Synthesis of Immunoglobulin G1 in Mice

doi:

Figure Lengend Snippet: RT-PCR analysis of mIL-4 insert stability following passage of chimeric viruses in HeLa cells. Amplimers were obtained and analyzed on 1.5% agarose gels following RT-PCR of viral RNA isolated from sequentially passaged CVB3-PL2-mIL4/46 (A and C) and CVB3/0-mIL4/47 (B and D). ELISA detection of mIL-4 protein is also shown for comparison (C and D). Viruses were passaged 10 times in HeLa cells as described in the text. Amplification of the region containing the insert in the viral RNA populations was accomplished with the primers ID9 and ID10 (A and B); the intact mIL-4 insert was detected as an 836-bp (or 830-bp) product and as a 344-bp product when the insert was deleted. Specific detection of the mIL-4-containing insert was accomplished using primers KNIL4S and KNIL4AS (C and D); the presence intact insert was detected as a 414-bp product, whereas deletion of the product resulted in no amplimer produced. mIL-4 ELISA (C and D), concentration of mIL-4 protein as detected by ELISA for each pass (5 × 105 HeLa cells infected at an MOI of ≥10 with designated viral passage stock, harvested at 6 h p.i.). Specific activities of mIL-4 ranged between 0.13 and 0.61 U/pg. Lanes P2 to P10, results from passages 2 to 10, respectively; lane M, 100-bp ladder (Life Technologies); lane PC, positive control amplification using pCVB3-PL2-mIL4/46 DNA; lane −, negative control (no DNA added) amplification; lane 0, PCR of CVB3/0 virus stock. Gel was stained with Cyber Green (FMC, Philadelphia, Pa.); images were captured using a Nucleo Vision gel documentation system (Nucleo Tech Corp.).

Article Snippet: Briefly, the ELISA was set up using 96-well flat-bottomed plates coated with a 1:1,000 dilution of hyperimmune horse anti-CVB3 serum (ATCC).

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Enzyme-linked Immunosorbent Assay, Amplification, Produced, Concentration Assay, Infection, Positive Control, Negative Control, Staining

Journal:

Article Title: Coxsackievirus Expression of the Murine Secretory Protein Interleukin-4 Induces Increased Synthesis of Immunoglobulin G1 in Mice

doi:

Figure Lengend Snippet: Detection of CVB3-associated mIL-4 insert at 14 days p.i. in mice. Amplimers were obtained and analyzed on 1.5% agarose gels following RT-PCR of viral RNA in total tissue RNA isolated from hearts (A), pancreas (B), and liver (C) of mice inoculated 14 days previously with 5 × 105 TCID50 of CVB3-PL2-mIL4/46. cDNA was made from RNA of tissues from five individual mice. Amplification of the region containing the insert in the viral RNA populations was accomplished using the CVB3-based primers ID9 and ID10. The intact mIL-4 insert was detected as an 830-bp product and as a 344-bp product when the insert was deleted. Lanes 1 to 5, PCR from cDNA of individual mouse tissues; lane 46, PCR from CVB3-PL2-mIL4/46 virus stock; lane 0, PCR from CVB3/0 virus stock; lane M, 100-bp ladder (Life Technologies). Gel was stained with Cyber Green (FMC) and images were captured using a Nucleo Vision gel documentation system (Nucleo Tech Corp.).

Article Snippet: Briefly, the ELISA was set up using 96-well flat-bottomed plates coated with a 1:1,000 dilution of hyperimmune horse anti-CVB3 serum (ATCC).

Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Amplification, Staining

Journal:

Article Title: Coxsackievirus Expression of the Murine Secretory Protein Interleukin-4 Induces Increased Synthesis of Immunoglobulin G1 in Mice

doi:

Figure Lengend Snippet: Increase in CVB3-binding IgG1 in mice inoculated with CVB3-PL2-mIL4/46 compared to CVB3/0. Quantity of CVB3-binding IgG1 (A) or IgG2a (B) in individual mouse serum samples from mice inoculated 14 days previously with CVB3-PL2-mIL4/46 (46) or CVB3-0 (0) or cell culture medium (control). CVB3-binding isotype was detected by ELISA using peroxidase activity of bound goat anti-mouse IgG1 or bound goat anti-mouse IgG2a and quantitated by absorbance at 405 nm.

Article Snippet: Briefly, the ELISA was set up using 96-well flat-bottomed plates coated with a 1:1,000 dilution of hyperimmune horse anti-CVB3 serum (ATCC).

Techniques: Binding Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Activity Assay