Blocking Coxsackievirus B3 Replication And Viral Myocarditis By Cytoplasmic Fc Receptor, TRIM21

The innate response is the first line of defense against incoming pathogens and is crucial for initiating an effective adaptive response.The key events in innate immunity is recognition of pathogen-associated molecular patterns by pattern recognition receptors?PRRs?by innate macrophages and DCs,which triggers downstream signaling pathways,leading to production of anti-viral effector molecules,inflammatory cytokine and antigen presentation by APCs.Inducible type I interferon?IFN-I?response comprise the most critical event in host antiviral responses against viral infection.Transcription factor IFN regulatory factor 3?IRF3?plays an essential central role in mediating type I IFN induction in response to TLR3 and intracellular PRRs.It suggests that targeting IRF3 for ubiquitin-mediated degradation post-stimulation is a very efficient way of shutting off and limiting the type I IFN response.The tripartite motif?TRIM?proteins comprise a large protein family that comprises a RING finger domain,B-boxes,coiled-coil region,and a B30.2 domain.TRIM proteins exhibit a wide range of activities,including the regulation of cell proliferation,differentiation,development,oncogenesis,and apoptosis.TRIM proteins represent a novel and widespread class of antiviral proteins which are IFN-I-induced following infection.TRIM5?,TRIM19 and TRIM38 are capable of inhibiting the replication of retroviruses at various stages of the virus life cycle.The TRIM member,TRIM21,initially known as an autoantigen Ro52/SS-A,is a ubiquitously expressed cytosolic E3 ubiquitin ligase for IRF3 thus plays important roles in immune regulation and microbial restriction.In addition to that,TRIM21 represents as a high-affinity antibody Fc receptor and intercepts incoming antibody-opsonized virions during cellular infection,mediating efficient post-entry neutralization,the course called antibody-dependent intracellular neutralization?ADIN?,and activating innate NF-kB,AP-1,IRF3/5/7 and RIG-I signaling pathways leading to the production of IFN?/?and proinflammatory cytokines.However,one of the most complex and challenging aspects to study TRIM21 has been to elucidate how TRIM21 is activated upon virus infection and how their expression and localization is regulated in different cell types.Most of the studies on TRIMs have been performed in cell lines and by overexpression/knockdown experiments.There is still a lack of understanding of the dominant roles of TRIMs in vivo and their physiological relevance.That is why we select an RNA virus to study and answer the above scientific aspects about TRIM21.Coxsackievirus B3?CVB3?is a non-enveloped single-stranded RNA virus belonging to the family of the Picornaviridae,which infects via fecal-oral route and causes viral myocarditis?VMC?and pancreaditis in human and susceptible mice.VMC accounts for one of the main causes of sudden death among adolescents and CVB3represents the most often detected pathogen factor in such people and patients with dilated cardiomyopathy?DCM?and heart failure.Direct damage of myocytes by CVB3occurs at very early phase?day1-3?,still CVB3-induced cardiac inflammatory immune infiltration and so-caused inflammatory injury is the major cause of VMC.Our study focuses on role of TRIM21 on CVB3 replication and pathogenesis largely because:1)the initial entry site for CVB3 infection is the small intestine before viral spreading into multiple organs,however,the enteral mucosa is much less susceptible to infection while viral titers in heart and pancreas are very high.2)CVB3infection leads to severe inflammatory injury in the heart and liver while a minor one in the intestine;3)CVB3 infection up-regulated TRIM21 in the intestine and the heart;4)We find that TRIM21 is co-localized in the cytoplasm with viral RNA;5)The induced expression kinetics of TRIM21 is similar to that of CVB3 replication in vivo;6)CVB3induces intestinal IgA production,while TRIM21 is a high-affinity Ig Fc receptor;7)TRIM21 is known as a IFN-I signaling regulatory molecule while CVB3 develops mechanism to potently inhibit early IFN?production.Thus,TRIM21 may provide crucial antiviral protection against CVB3 infection and associated myocarditis.Here we investigate the antiviral activity of TRIM21 during infection of CVB3 in vitro and in vivo.We report that TRIM21 is significantly up-regulated upon CVB3 infection.TRIM21 activity provides a highly potent block to spreading CVB3 replication by activating IRF3-mediated IFN-I signaling pathway and by undergoing intestinal IgA-mediated ADIN effect.PART I Differential expression of TRIM21 under normal condition and CVB3 infection1.Establishment of murine model of CVB3-induced acute myocarditis1.5?10³ TCID₅₀ dosage of CVB3 was intraperitoneally injected into male BLAB/c mice.Total survival rate by day 7 is up to 50%with weight loss change of 20%.Massive inflammatory infiltration and myocytes necrosis in the hearts were confirmed by day 7.2.CVB3 adopted a differential replication patterns in various organsBy RT-PCR detection of viral RNA load?+strand?and replication level?-strand?,we found a similar viral replication kinetic in the heart,liver and the intestine which showed a peak level on day 3 p.i.and a robust decline by day7.The viral titer in hearts was the highest while much lower in the small intestine.3.CVB3 infection induced up-regulation of TRIM21 in the heartWe first determined the effect of CVB3 on the TRIM21 level in various susceptible organs of mice.Total RNA from organs?heart,pancreas,lung,liver,small intestine?were harvested at various time points after infection,significantly increased mRNA levels of TRIM21 were observed at day 3 and day7 p.i.,from heart,liver and the intestine,and up-regulation of TRIM21 gene expression was most evident in the small intestine.The peak expression of TRIM21 was at 3 days after infection.4.Cytoplasmic TRIM21 was co-localization with viral RNA in cardiomyocytesTo examine the subcellular localization of TRIM21 during normal conditions,as well as after CVB3 infection,Hela,a human cell line and primary cardiomyocytes were infected with CVB3 and investigated by confocal microscopy and histochemistry.Under normal conditions,TRIM21was cytoplasmic and was rarely detected in the nucleolus.Protein expression of TRIM21 was significantly up-regulated by infection,and notably,was-co-localized with CVB3 RNA.Part II TRIM21 blocked CVB3 replication by positive regulating IFN I signaling pathway through a RING domainCVB3 is a replicating cytopathic virus which infects the cardiomyocyte,pancreas islet cells,intestinal epithelial cells and lymphoid cells.In the innate antiviral response,TLR3 is an important RNA sensor that triggers a type I interferon response via the signaling adaptor IRF3.In order to define the role of TRIM21 in host defense to CVB3in vitro and explore the modulation of IRF3,the key signaling molecule in IFN-I pathway by TRIM21,we performed the in vitro CVB3 infection experiment and luciferase reporter assay after over-expression and down-regulation of TRIM21.1.TRIM21 potently blocked CVB3 replication in vitroHuman TRIM21 was cloned into the pcDNA3.1 vector,resulting in a TRIM21-over expressing plasmid.Hela cells were transfected with pTRIM21 or siRNA for 24 hrs then infected with CVB3?MOI=5?,we used quantitative PCR?qPCR?assay,western Blot,FACS cytometry and TCID50 assay to measure in vitro replication of CVB3.It was found that over-expression of TRIM21 significantly reduced the RNA level,VP1 protein expression?>50%,p<0.05?,frequency of GFP+CVB3+cells?7.8%to4.2%,p<0.01?as well as the TCID50 titer?185000 to 72000,p<0.05?of CVB3 in Hela cells 24 hrs p.i..In consistent with that,knock-down of TRIM21 resulted in a>1,000-fold decrease in the RNA level and TCID50 titer of CVB3 in Hela cells.Our data indicate that TRIM21 has significant blocking impact on CVB3 replication.2.TRIM21 activated IRF3-mediated antiviral responsesRNA virus infection induces p-IRF3 and IFN-?expression in Hela cells.To explore the role of TRIM21 in regulating the type I interferon pathway,we transfected TRIM21 into Hela or HEK293 cells followed by CVB3 treatment,the production of IFN-?mRNA and IFN-?luciferase activity,the p-IRF3 level were measured.1)Over-expression of hTRIM21 induced IFN-a/?transcription after CVB3infection.Human Hela cells were transfected with hTRIM21 plasmid for 24 hrs,infected with CVB3 at an MOI of 5 and harvested at various time intervals of 0-36 hrs.IFN-?mRNA levels were significantly increased in response to CVB3 infection after over-expression of TRIM21.2)Over-expression of TRIM21 resulted in more transcription of IFN-?to CVB3293 cells were co-transfected with IFN-?-luciferase promoter plasmid and phTRIM21 plasmid for 24 hrs and infected with CVB3 at MOI 5 for 0-36 h.Luciferase activity revealed that over-expression of TRIM21 resulted in more transcription of IFN-?in response to CVB3 in a dose-dependent manner until 36 h.These data suggested that TRIM21 positively regulated IFN-?production upon CVB3 infection.3)TRIM21 over-expression increased CVB3-induced phosphorylation of IRF3IRF3 is a key transcriptional factor downstream of all the sensors of DNA and RNA involved in inducing type I interferon responses.After stimulation with CVB3 for0-6 hrs,there was more-rapid and robust up-regulation of the phosphorylation of IRF3in Trim21-over-expressed 293T cells than in wild-type cells.3.The RING domain is required to facilitate the IFN?/?-mediated anti-viral activity of TRIM21.TRIM21 contains a RING finger domain,a B box,CCD and a B30.2 domain.To map critical domain for its anti-CVB3 and IFN-I signaling activating function,a series of domain truncated mutants of TRIM21were constructed and individually transfected into HEK293T cells.Cells with?RING TRIM21 transfection showed remarkably increased CVB3 replication,demonstrating that the RING domain was responsible for the antiviral function of TRIM21.By IFN-?-luciferase promoter-phTRIM21 co-transfection and Luciferase activity assay,we confirmed that TRIM21-enhancing IFN-?transcriptional activation in response to CVB3 was dependent on RING,but not SPRY or B-Box domain.Taken together,over expression or knockdown of TRIM21 could respectively enhance or impair IRF3-mediated IFN I genes expression as well as the anti-viral effect.Over-expression of TRIM21 resulted in up-regulation of p-IRF3 and IFN-?in CVB3-infected cells.Our results provide biological evidence that TRIM21 serves as a positive regulator of IRF3 signaling and thereby activating the induction of IFN?response upon CVB3 infection.Part III TRIM21 restricted the CVB3 replication and acute myocarditis in vivoThe function of TRIM21 in host defense to viral infection in vivo has remained elusive.We next evaluated the importance of TRIM21 in vivo in facilitating effective host defense against CVB3 infection.1.In vivo over-expression of TRIM21 significantly reduced CVB3 replicationMurine TRIM21 was cloned into a plasmid as pCDNA3.1-Flag-mTRIM21.We facilitated the in vivo over-expression by InvivoJET-PEI delivery system.We injected mice with InvivoJET-PEI-pmTRIM21 on day-1 and+1,and intraperitoneally infected with HSV-1 on day 0,then measured viral titers in hearts by plaque assay 3 days after infection.We detected significantly less CVB3 in Trim21-overexpressed mice than in control mice?titers 7x10⁵ to 2x10⁵,P<0.05?;Meanwhile,a significantly enhanced production of IFN?was observed in the hearts of mice at day3 p.i..2.In vivo over-expression of TRIM21 reduced severity of acute myocarditisBy evaluating the cardiac histopathology using HE staining,we found that the CVB3-induced cardiac immune infiltration and cardiomyocyte injuries were significantly decreased,together with significantly reduced TNF?,IL--6production in the hearts of mice.These data indicated an important role for TRIM21 in regulating the protective innate host immune defense to RNA virus,CVB3.PART IV TRIM21 and intestinal Ig A synergistically mediated intracellular neutralization of CVB3TRIM21 is the highest affinity human Fc receptor.After binding incoming virus-antibody complexes in the cytoplasm,TRIM21 targets virions for proteasome and VCP-dependent degradation in a process known as ADIN.We tried to clarify whether TRIM21 have IgA-mediated ADIN effect on intestinal CVB.1.CVB3 infection induced production of intestinal IgAFirst,we measured the Ab induction kinetic of serum IgG and fecal IgA induced by CVB3 infection,we found a increased Ab response which peaked on 14 days after infection.The 14 p.i.serum and fecal IgA samples were collected and purified.In the intestine mucosa,IgA rather than IgG antibodies dominate the antibody repertoire.2.TRIM21 expression was up-regulated in primary MEF cellsIn order to study the possible TRIM21-mediated IgA-CVB3 ADIN effect,we isolated and cultured primary mouse embryonic fibroblast?MEF?cells from mice fetus to represent the intestinal epithelial cells and confirmed that upon CVB3 infection,the TRIM21 expression was significantly up-regulated.3.Intestinal IgA-intracellular neutralization of CVB3 required TRIM21To test the possible ADIN effect of TRIM21,we pre-incubated CVB3 with specific serum IgG or fecal IgA for 1 hrs then added the virions to cultured MEF cells.Then we tested the effect of fecal IgA on CVB3 infection.We found that there was a robust reduction of viral RNA and titers?>90%?after pre-treated with serum IgG or fecal IgA-bound virus.To demonstrate the neutralization of virus by Ig A/G virus requires TRIM21,functional TRIM21 inhibitor,DbeQ,was pre-incubated with MEF cells before treatment with IgG/IgA-CVB3.Strikingly,DbeQ significantly reversed TRIM21 neutralization of CVB3 infectivity?SIgA,80-100%reduction;IgG,20-50%reduction,p<0.001?.These data indicate that intestinal IgA?more potently?/serum IgG and TRIM21operate synergistically to neutralize CVB3 infection.It can be speculated that TRIM21-mediated immunity could be more effective in the early stages of intestinal CVB3 infection.This is the first report showing the role of TRIM21 in modulating the type I interferon response upon CVB3 infection in mice.TRIM21 was significantly up-regulated by CVB3 infection.Silencing of TRIM21 leads to downregulation of CVB3-mediated activation of IRF-3,downstream IFN-?production and increase in viral replication;while over-expression of TRIM21 in vivo results in facilitation of CVB3-mediated activation of IRF-3 and restriction of viral replication and acute myocarditis via up regulation of IFN-?production.We thereby report the activating role of TRIM21 on IFN-?production during CVB3 infection by modulating IRF3phosphorylation.We also confirmed that cytoplasmic TRIM21 recognize intestinal antiviral IgA and facilitate rapid destruction of virus via ADIN mechanism.