Macrophage-Specific Coxsackievirus and Adenovirus Receptor Deletion Enhances Macrophage M1 Polarity in CVB3-Induced Myocarditis.

The coxsackievirus and adenovirus receptor (CAR) is very well known as an epithelial tight junction and cardiac intercalated disc protein; it mediates attachment and infection via the coxsackievirus B3 (CVB3) and type 5 adenovirus. Macrophages play important roles in early immunity during viral infections. However, the role of CAR in macrophages is not well studied in relation to CVB3 infection. In this study, the function of CAR was observed in the Raw264.7 mouse macrophage cell line. CAR expression was stimulated by treatment with lipopolysaccharide (LPS) and tumor necrosis factor-α (TNF-α). In thioglycollate-induced peritonitis, the peritoneal macrophage was activated and CAR expression was increased. The macrophage-specific CAR conditional knockout mice (KO) were generated from lysozyme Cre mice. The expression of inflammatory cytokine (IL-1ß and TNF-α) was attenuated in the KO mice's peritoneal macrophage after LPS treatment. In addition, the virus was not replicated in CAR-deleted macrophages. The organ virus replication was not significantly different in both wild-type (WT) and KO mice at days three and seven post-infection (p.i). However, the inflammatory M1 polarity genes (IL-1ß, IL-6, TNF-α and MCP-1) were significantly increased, with increased rates of myocarditis in the heart of KO mice compared to those of WT mice. In contrast, type1 interferon (IFN-α and ß) was significantly decreased in the heart of KO mice. Serum chemokine CXCL-11 was increased in the KO mice at day three p.i. compared to the WT mice. The attenuation of IFN-α and ß in macrophage CAR deletion induced higher levels of CXCL-11 and more increased CD4 and CD8 T cells in KO mice hearts compared to those of WT mice at day seven p.i. These results demonstrate that macrophage-specific CAR deletion increased the macrophage M1 polarity and myocarditis in CVB3 infection. In addition, chemokine CXCL-11 expression was increased, and stimulated CD4 and CD8 T cell activity. Macrophage CAR may be important for the regulation of innate-immunity-induced local inflammation in CVB3 infection.

Protein Kinase B2 (PKB2/AKT2) Is Essential for Host Protection in CVB3-Induced Acute Viral Myocarditis.

Protein kinase B2 (AKT2) is involved in various cardiomyocyte signaling processes, including those important for survival and metabolism. Coxsackievirus B3 (CVB3) is one of the most common pathogens that cause myocarditis in humans. The role of AKT2 in CVB3 infection is not yet well understood. We used a cardiac-specific AKT2 knockout (KO) mouse to determine the role of AKT2 in CVB3-mediated myocarditis. CVB3 was injected intraperitoneally into wild-type (WT) and KO mice. The mice's survival rate was recorded: survival in KO mice was significantly decreased compared with WT mice (WT vs. KO: 73.3 vs. 27.1%). Myocardial damage and inflammation were significantly increased in the hearts of KO mice compared with those of WT mice. Moreover, from surface ECG, AKT2 KO mice showed a prolonged atria and ventricle conduction time (PR interval, WT vs. KO: 47.27 ± 1.17 vs. 64.79 ± 7.17 ms). AKT2 deletion induced severe myocarditis and cardiac dysfunction due to CVB3 infection. According to real-time PCR, the mRNA level of IL-1, IL-6, and TNF-α decreased significantly in KO mice compared with WT mice on Days 5 after infection. In addition, innate immune response antiviral effectors, Type I interferon (interferon-α and ß), and p62, were dramatically suppressed in the heart of KO mice. In particular, the adult cardiac myocytes isolated from the heart showed high induction of TLR4 protein in KO mice in comparison with WT. AKT2 deletion suppressed the activation of Type I interferon and p62 transcription in CVB3 infection. In cardiac myocytes, AKT2 is a key signaling molecule for the heart from damage through the activation of innate immunity during acute myocarditis.

Vascular Endothelial Integrity Affects the Severity of Enterovirus-Mediated Cardiomyopathy.

Coxsackievirus and adenovirus receptor (CAR) is present in epithelial and vascular endothelial cell junctions. We have previously shown a hemorrhagic phenotype in germ-line CAR knock-out mouse embryos; we have also found that CAR interacts with ZO-1 and ß-catenin. However, the role of CAR in vascular endothelial junction permeability has not been proven. To understand the roles of CAR in the vascular endothelial junctions, we generated endothelium-specific CAR knockout (CAR-eKO) mice. In the absence of CAR, the endothelial cell layer showed an increase in transmembrane electrical resistance (TER, Ω) and coxsackievirus permeability. Evans blue dye and 70 kDa dextran-FITC were delivered by tail vein injection. We observed increased vascular permeability in the hearts of adult CAR-eKO mice compare with wild-type (WT) mice. There was a marked increase in monocyte and macrophage penetration into the peritoneal cavity caused by thioglycolate-induced peritonitis. We found that CAR ablation in endothelial cells was not significantly increased coxsackievirus B3 (CVB3) induced myocarditis in murine model. However, tissue virus titers were significantly higher in CAR-eKO mice compared with WT. Moreover, CVB3 was detected in the brain of CAR-eKO mice. Endothelial CAR deletion affects the expression of major endothelial junction proteins, such as cadherin and platelet endothelial cell adhesion molecule-1 (PECAM-1) in the cultured endothelial cells as well as liver vessel. We suggest that CAR expression is required for normal vascular permeability and endothelial tight junction homeostasis. Furthermore, CVB3 organ penetration and myocarditis severities were dependent on the endothelial CAR level.

Inhibition of RNA Helicase Activity Prevents Coxsackievirus B3-Induced Myocarditis in Human iPS Cardiomyocytes.

AIMS: Coxsackievirus B3 (CVB3) is known to be an important cause of myocarditis and dilated cardiomyopathy. Enterovirus-2C (E2C) is a viral RNA helicase. It inhibits host protein synthesis. Based on these facts, we hypothesize that the inhibition of 2C may suppress virus replication and prevent enterovirus-mediated cardiomyopathy. METHODS AND RESULTS: We generated a chemically modified enterovirus-2C inhibitor (E2CI). From the in vitro assay, E2CI was showed strong antiviral effects. For in vivo testing, mice were treated with E2CI intraperitoneally injected daily for three consecutive days at a dose of 8mg/kg per day, after CVB3 post-infection (p.i) (CVB3 + E2CI, n = 33). For the infected controls (CVB3 only, n = 35), mice were injected with PBS (phosphate buffered saline) in a DBA/2 strain to establish chronic myocarditis. The four-week survival rate of E2CI-treated mice was significantly higher than that of controls (92% vs. 71%; p < 0.05). Virus titers and myocardial damage were significantly reduced in the E2CI treated group. In addition, echocardiography indicated that E2CI administration dramatically maintained mouse heart function compared to control at day 28 p.i chronic stage (LVIDD, 3.1 ± 0.08 vs. 3.9 ± 0.09, p < 0.01; LVDS, 2.0 ± 0.07 vs. 2.5 ± 0.07, p < 0.001; FS, 34.8 ± 1.6% vs. 28.5 ± 1.5%; EF, 67. 9 ± 2.9% vs. 54.7 ± 4.7%, p < 0.05; CVB3 + E2CI, n = 6 vs. CVB3, n = 4). Moreover, E2CI is effectively worked in human iPS (induced pluripotent stem cell) derived cardiomyocytes. CONCLUSION: Enterovirus-2C inhibitor (E2CI) was significantly reduced viral replication, chronic myocardium damage, and CVB3-induced mortality in DBA/2 mice. These results suggested that E2CI is a novel therapeutic agent for the treatment of enterovirus-mediated diseases.

Elevation of Serum APE1/Ref-1 in Experimental Murine Myocarditis.

Myocarditis is an inflammatory disease of the myocardium that causes cardiogenic shock and death. However, endomyocardial biopsy that is, the gold standard for a diagnosis is limited. Apurinic/apyrimidinic endonuclease 1/redox effector factor-1 (APE1/Ref-1) is a multifunctional protein, which is involved in DNA-based excision repair pathway, and in redox signaling, its changes are observed in various cardiovascular diseases including hypertension and coronary artery disease. We analyzed serum APE1/Ref-1 in experimental murine myocarditis. To induce myocarditis, coxsackievirus B3 was injected intraperitoneally to BALB/c mice. The serum APE1/Ref-1, N-terminal pro-B-type natriuretic peptide (NT-proBNP) and troponin I were measured. The histology and virus titers measurements were performed. The troponin I and inflammation were significantly elevated at day 3, peaked to day 7 and decreased at day 10. The NT-proBNP and virus titers were significantly peaked at day 3, and dropped at day 7 and 10. The serum APE1/Ref-1 was gradually raised and its elevation is still maintained until a later time, namely day 10. Also, its level was positively correlated with myocardial inflammation, reflecting severity of myocardial injury. We suggest that serum APE1/Ref-1 can be used to assess for myocardial injury in viral myocarditis without endomyocardial biopsy.

A Novel Angiotensin Type I Receptor Antagonist, Fimasartan, Prevents Doxorubicin-induced Cardiotoxicity in Rats.

Angiotensin receptor blockers (ARBs) have organ-protective effects in heart failure and may be also effective in doxorubicin-induced cardiomyopathy (DOX-CMP); however, the efficacy of ARBs on the prevention of DOX-CMP have not been investigated. We performed a preclinical experiment to evaluate the preventive effect of a novel ARB, fimasartan, in DOX-CMP. All animals underwent echocardiography and were randomly assigned into three groups: treated daily with vehicle (DOX-only group, n=22), 5 mg/kg of fimasartan (Low-fima group, n=22), and 10 mg/kg of fimasartan (High-fima group, n=19). DOX was injected once a week for six weeks. Echocardiography and hemodynamic assessment was performed at the 8th week using a miniaturized conductance catheter. Survival rate of the High-fima group was greater (100%) than that of the Low-fima (75%) and DOX-only groups (50%). Echocardiography showed preserved left ventricular (LV) ejection fraction in the High-fima group, but not in the DOX-only group (P=0.002). LV dimensions increased in the DOX-only group; however, remodeling was attenuated in the Low-fima and High-fima groups. Hemodynamic assessment showed higher dP/dt in the High-fima group compared with the DOX-only group. A novel ARB, fimasartan, may prevent DOX-CMP and improve survival rate in a dose-dependent manner in a rat model of DOX-CMP and could be a treatment option for the prevention of DOX-CMP.

ORI2 inhibits coxsackievirus replication and myocardial inflammation in experimental murine myocarditis.

We purified ORI2 [3-(3,4-dihydroxyphenyl)acrylic acid 1-(3,4-dihydroxyphenyl)-2-methoxycarbonylethyl ester] from an extract of the plant Isodon excisus. We tested the antiviral effect of ORI2 in a coxsackievirus-induced myocarditis model. Coxsackievirus B3 (CVB3) is a common cause of myocarditis and dilated cardiomyopathy. Activation of extracellular signal-regulated kinase (ERK) and Akt signaling in virus-infected cells is essential for CVB3 replication. Antiviral compounds were screened by HeLa cell survival assay. Several purified natural compounds were added to HeLa cells cultured in 96-well plates for 30 min after 1 multiplicity of infection (m.o.i) CVB3 infection. ORI2 significantly improved HeLa cell survival in a dose-dependent manner. For in vivo studies, BALB/c mice (n=20) were infected with CVB3, then 10 of the mice were treated by daily intraperitoneal injections of ORI2 (100 mM) for 3 consecutive days. ORI2 treatment significantly improved early survival in the treated mice compared to untreated mice (85% vs. 50%, respectively). Organ virus titers and myocardial damage were significantly lower in the ORI2-treated mice than in untreated mice. These results demonstrate that ORI2, delivered by intraperitoneal injection after CVB3 infection, has a significant antiviral effect by markedly inhibiting virus replication, resulting in a decrease in organ virus titer and myocardial damage. ORI2 may be developed as a potential therapeutic agent for the treatment of CVB3 infections.

Hypermorphic mutation of the voltage-gated sodium channel encoding gene Scn10a causes a dramatic stimulus-dependent neurobehavioral phenotype.

The voltage-gated sodium channel Na(v)1.8 is known to function in the transmission of pain signals induced by cold, heat, and mechanical stimuli. Sequence variants of human Na(v)1.8 have been linked to altered cardiac conduction. We identified an allele of Scn10a encoding the α-subunit of Na(v)1.8 among mice homozygous for N-ethyl-N-nitrosourea-induced mutations. The allele creates a dominant neurobehavioral phenotype termed Possum, characterized by transient whole-body tonic immobility induced by pinching the skin at the back of the neck ("scruffing"). The Possum mutation enhanced Na(v)1.8 sodium currents and neuronal excitability and heightened sensitivity of mutants to cold stimuli. Striking electroencephalographic changes were observed concomitant with the scruffing-induced behavioral change. In addition, electrocardiography demonstrated that Possum mice exhibited marked sinus bradycardia and R-R variability upon scruffing, abrogated by infusion of atropine. However, atropine failed to prevent or mitigate the tonic immobility response. Hyperactive sodium conduction via Na(v)1.8 thus leads to a complex neurobehavioral phenotype, which resembles catatonia in schizophrenic humans and tonic immobility in other mammals upon application of a discrete stimulus; no other form of mechanosensory stimulus could induce the immobility phenotype. Our data confirm the involvement of Na(v)1.8 in transducing pain initiated by cold and additionally implicate Na(v)1.8 in previously unknown functions in the central nervous system and heart.

Caboxamycin Inhibits Heart Inflammation in a Coxsackievirus B3-Induced Myocarditis Mouse Model.

Coxsackievirus B3 (CVB3) is a positive single-strand RNA genome virus which belongs to the enterovirus genus in the picornavirus family, like poliovirus. It is one of the most prevalent pathogens that cause myocarditis and pancreatitis in humans. However, a suitable therapeutic medication and vaccination have yet to be discovered. Caboxamycin, a benzoxazole antibiotic isolated from the culture broth of the marine strain Streptomyces sp., SC0774, showed an antiviral effect in CVB3-infected HeLa cells and a CVB3-induced myocarditis mouse model. Caboxamycin substantially decreased CVB3 VP1 production and cleavage of translation factor eIF4G1 from CVB3 infection. Virus-positive and -negative strand RNA was dramatically reduced by caboxamycin treatment. In addition, the cleavage of the pro-apoptotic molecules BAD, BAX, and caspase3 was significantly inhibited by caboxamycin treatment. In animal experiments, the survival rate of mice was improved following caboxamycin treatment. Moreover, caboxamycin treatment significantly decreased myocardial damage and inflammatory cell infiltration. Our study showed that caboxamycin dramatically suppressed cardiac inflammation and mouse death. This result suggests that caboxamycin may be suitable as a potential antiviral drug for CVB3.

Assessing the impact of mRNA vaccination in chronic inflammatory murine model.

The implications of administration of mRNA vaccines to individuals with chronic inflammatory diseases, including myocarditis, rheumatoid arthritis (RA), and inflammatory bowel disease (IBD), are unclear. We investigated mRNA vaccine effects in a chronic inflammation mouse model implanted with an LPS pump, focusing on toxicity and immunogenicity. Under chronic inflammation, mRNA vaccines exacerbated cardiac damage and myocarditis, inducing mild heart inflammation with heightened pro-inflammatory cytokine production and inflammatory cell infiltration in the heart. Concurrently, significant muscle damage occurred, with disturbances in mitochondrial fusion and fission factors signaling impaired muscle repair. However, chronic inflammation did not adversely affect muscles at the vaccination site or humoral immune responses; nevertheless, it partially reduced the cell-mediated immune response, particularly T-cell activation. These findings underscore the importance of addressing mRNA vaccine toxicity and immunogenicity in the context of chronic inflammation, ensuring their safe and effective utilization, particularly among vulnerable populations with immune-mediated inflammatory diseases.

Development of a enterovirus diagnostic assay system for diagnosis of viral myocarditis in humans.

The coxsackieviruses type B3 (CVB3) are members of the genus Enterovirus of the family Picornaviridae. They are the commonest cause of chronic myocarditis and dilated cardiomyopathy. However, there is still no effective method for diagnosing CVB3 infection in humans. Here, a fast and accurate system that uses a capsid-protein-specific peptide sequence to detect CVB3 in the sera of patients with viral myocarditis was established. The peptide sequence was selected from the whole CVB3 capsid protein sequence by computationally predicting fragments with high antigenicity and low hydrophobicity. Two of eight possible peptide sequences were selected and commercially synthesized. The synthesized peptides encoded either the VP2 or VP1 capsid protein and induced immunoglobulin G antibody expression in immunized rabbits. Anti-VP2 and anti-VP1 sera detected the viral proteins extracted from CVB3-infected HeLa cells. The newly synthesized peptides successfully induced antibody production. These peptides, applied in an ELISA system, detected anti-CVB3 antibodies in virus-infected mouse serum. Moreover, an ELISA system based on the VP2 peptide detected CVB3 infection in patients with positively identified CVB3-induced fulminant myocarditis. These results indicate that these new peptides specifically interact with anti-CVB3 IgG antibodies in mouse and human sera. This ELISA system should be useful for the clinical diagnosis of enterovirus-induced myocarditis.

Antiviral activity of coxsackievirus B3 3C protease inhibitor in experimental murine myocarditis.

BACKGROUND: We investigated the efficacy of a 3C protease inhibitor (3CPI) in a murine coxsackievirus B3 (CVB3) myocarditis model. CVB3 is a primary cause of viral myocarditis. The CVB3 genome encodes a single polyprotein that undergoes a series of proteolytic events to produce several viral proteins. Most of this proteolysis is catalyzed by the 3C protease (3CP). METHODS AND RESULTS: By way of a micro-osmotic pump, each mouse received 50 mM 3CPI in 100 µL of 100% dimethyl sulfoxide (DMSO) during a 72-hour period. On the day of pump implantation, mice (n = 40) were infected intraperitoneally with 10(6) plaque-forming units of CVB3. For the infected controls (n = 50), the pump was filled with 100% DMSO without 3CPI. The 3-week survival rate of 3CPI-treated mice was significantly higher than that of controls (90% vs 22%; P < .01). Myocardial inflammation, viral titers, and viral RNA levels were also reduced significantly in the 3CPI-treated group compared with these measures in the controls. CONCLUSIONS: The protein-based drug 3CPI inhibited the activity of 3CP of CVB3, significantly inhibited viral proliferation, and attenuated myocardial inflammations, subsequent fibrosis, and CVB3-induced mortality in vivo. Thus, this CVB3 3CPI has the potential to be a novel therapeutic agent for the treatment of acute viral myocarditis during the viremic phase.

Development of a Universal Cloning System for Reverse Genetics of Human Enteroviruses.

Enteroviruses (EVs) have been associated with several human diseases. Due to their continuous emergence and divergence, EV species have generated more than 100 types and recombinant strains, increasing the public health threat caused by them. Hence, an efficient and universal cloning system for reverse genetics (RG) of highly divergent viruses is needed to understand the molecular mechanisms of viral pathology and evolution. In this study, we generated a versatile human EV whole-genome cDNA template by enhancing the template-switching method and designing universal primers capable of simultaneous cloning and rapid amplification of cDNA ends (RACE)-PCR of EVs. Moreover, by devising strategies to overcome limitations of previous cloning methods, we simplified significant cloning steps to be completed within a day. Of note, we successfully verified our efficient universal cloning system enabling RG of a broad range of human EVs, including EV-A (EV-A71), EV-B (CV-B5, ECHO6, and ECHO30), EV-C (CV-A24), and EV-D (EV-D68), with viral titers and phenotypes comparable to those of their wild types. This rapid and straightforward universal EV cloning strategy will help us elucidate molecular characteristics, pathogenesis, and applications of a broad range of EV serotypes for further development of genetic vaccines and delivery tools using various replication systems. IMPORTANCE Due to the broad spread, incidence, and genetic divergence of enteroviruses (EVs), it has been challenging to deal with this virus that causes severe human diseases, including aseptic meningitis, myocarditis, encephalitis, and poliomyelitis. Therefore, an efficient and universal cloning system for the reverse genetics of highly divergent EVs contributes to an understanding of the viral pathology and molecular mechanisms of evolution. We have simplified the important cloning steps, hereby enhancing the template-switching method and designing universal primers, which enable the important cloning steps to be completed in a day. We have also successfully demonstrated recovery of a broad range of human EVs, including EV-A to -D types, using this advanced universal cloning system. This rapid and robust universal EV cloning strategy will aid in elucidating the molecular characteristics, pathogenesis, and applications of a wide range of EVs for further development of genetic vaccines and antiviral screening using various replication systems.

Foreign gene transfer to cardiomyocyte using a replication-defective recombinant coxsackievirus B3 without cytotoxicity.

BACKGROUND: Replication-competent coxsackievirus B3 (CVB3) has been used as a gene transfer vector for cultured cardiomyocytes and hearts in vivo. However, CVB3 induces cell lysis when it replicates in infected cells. In this study, we investigated whether a replication-defective rCVB3 vector could be generated and used as a noncytotoxic gene transfer vector for cardiomyocytes. METHODS: We generated a replication-defective luciferase-expressing CVB3 plasmid. This recombinant cDNA and pCMV-P1 plasmids were amplified and cotransfected into Hek293 cells using transfection reagents. Replication-defective rLuCVB3 virus was recovered from the cells and cell culture supernatants for 3 days after transfection. The generated rLuCVB3 viruses were concentrated on a 30% sucrose cushion and semiquantified using a luciferase assay. In addition, foreign gene delivery by the rLuCVB3 was tested in cultured cardiomyocytes and intact mouse hearts after rLuCVB3 infection. RESULTS: Luciferase was expressed in Hek293, HeLa cells and cardiomyocytes after rLuCVB3 infection. In addition, these cells did not show a significant cytopathic effect after 72 h. Luciferase protein expression or activity were detected for 3 days in the myocardium of rLuCVB3-infected mouse hearts without producing cytotoxicity or inflammation. CONCLUSION: As a proof-of-concept, these data indicate that a replication-defective rCVB3 vector can be generated and used as a novel gene transfer system to transfect exogenous genes into cardiomyocytes without generating cytotoxicity.

Role of the myristoylation site in expressing exogenous functional proteins in coxsackieviral vector.

We generated a cardiotropic replication-competent chimeric coxsackievirus B3 (CVB3) to express alcohol dehydrogenase (ADH). Although exogenously expressed ADH was found by Western blot analysis, its enzyme function was repressed. To define the factor that inhibits the enzymatic function of ADH, we introduced a site-directed mutation at the second amino acid (MGAQEF···) of the CVB3 VP0 capsid protein, effectively changing glycine to alanine. This glycine is known to be a myristoylation site during viral capsid protein maturation in infected cells. In contrast to the unmodified virus, ADH expression and enzymatic function were readily detectable in the mutated rCVB3-ADH (G2A) virus. While expression of ADH required mutation of the CVB3 VP0 myristoylation site for proper function, another chimeric virus that expresses green fluorescent protein (rCVB3-GFP (G or A)) worked independently of the myristoylation site. Indeed, infected HeLa cells displayed GFP under a fluorescent microscope. These results indicate that the myristoylation site in the VP0 capsid protein inhibited the expression of enzymatically active ADH but not GFP. VP0 myristoylation is dispensable for chimeric CVB3 virus replication.

A peptide vaccine based on a B-cell epitope on the VP1 protein of enterovirus 70 induces a strong antibody response.

Enterovirus 70 (EV70) is the causative agent of acute hemorrhagic conjunctivitis (AHC), for which no effective vaccine is available. This study revealed a high reactivity of the N-terminal region of EV70 VP1 (VP1-1) with an anti-EV70 mouse serum. The analysis of overlapping synthetic peptides of VP1-1 identified a B-cell epitope in this region. The E-peptide (14-ANTVESEIKAELGVI-28) showing the highest reactivity with the anti-EV70 serum induced neutralizing antibodies in mice and reduced the virus titer in the eyes, suggesting that it is a candidate vaccine against AHC caused by EV70.

Coxsackievirus and adenovirus receptor (CAR) mediates atrioventricular-node function and connexin 45 localization in the murine heart.

The coxsackievirus and adenovirus receptor (CAR) is a transmembrane protein that belongs to the family of adhesion molecules. In the postnatal heart, it is localized predominantly at the intercalated disc, where its function is not known. Here, we demonstrate that a first degree or complete block of atrioventricular (AV) conduction developed in the absence of CAR in the adult mouse heart and that prolongation of AV conduction occurred in the embryonic heart of the global CAR-KO mouse. In the cardiac-specific CAR-KO (CAR-cKO) mouse, we observed the loss of connexin 45 localization to the cell-cell junctions of the AV node but preservation of connexin 40 and 43 in contracting myocardial cells and connexin 30.2 in the AV node. There was also a marked decrease in beta-catenin and zonula occludens-1 (ZO-1) localization to the intercalated discs of CAR-cKO mouse hearts at 8 weeks before the mice developed cardiomyopathy at 21 weeks of age. We also found that CAR formed a complex with connexin 45 via its PSD-95/DigA/ZO-1-binding (PDZ-binding) motifs. We conclude that CAR expression is required for normal AV-node conduction and cardiac function. Furthermore, localization of connexin 45 at the AV-node cell-cell junction and of beta-catenin and ZO-1 at the ventricular intercalated disc are dependent on CAR.

Corrigendum: CVB3-Mediated Mitophagy Plays an Important Role in Viral Replication via Abrogation of Interferon Pathways.

[This corrects the article DOI: 10.3389/fcimb.2021.704494.].

CVB3-Mediated Mitophagy Plays an Important Role in Viral Replication via Abrogation of Interferon Pathways.

Coxsackievirus B3 (CVB3) is a common enterovirus that causes systemic inflammatory diseases, such as myocarditis, meningitis, and encephalitis. CVB3 has been demonstrated to subvert host cellular responses via autophagy to support viral replication in neural stem cells. Mitophagy, a specialized form of autophagy, contributes to mitochondrial quality control via degrading damaged mitochondria. Here, we show that CVB3 infection induces mitophagy in human neural progenitor cells, HeLa and H9C2 cardiomyocytes. In particular, CVB3 infection triggers mitochondrial fragmentation, loss of mitochondrial membrane potential, and Parkin/LC3 translocation to the mitochondria. Rapamycin or carbonyl cyanide m-chlorophenyl hydrazone (CCCP) treatment led to increased CVB3 RNA copy number in a dose-dependent manner, suggesting enhanced viral replication via autophagy/mitophagy activation, whereas knockdown of PTEN-induced putative kinase protein 1(PINK1) led to impaired mitophagy and subsequent reduction in viral replication. Furthermore, CCCP treatment inhibits the interaction between mitochondrial antiviral signaling protein (MAVS) and TANK-binding kinase 1(TBK1), thus contributing to the abrogation of type I and III interferon (IFN) production, suggesting that mitophagy is essential for the inhibition of interferon signaling. Our findings suggest that CVB3-mediated mitophagy suppresses IFN pathways by promoting fragmentation and subsequent sequestration of mitochondria by autophagosomes.

Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens.

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.