RESUMEN

Introduction: Coxsackievirus A6 (CV-A6) has emerged as the predominant epidemic strain responsible for hand, foot and mouth disease (HFMD). CV-A6 infection can result in severe clinical manifestations, including encephalitis, meningitis, and potentially life-threatening central nervous system disorders. Our previous research findings demonstrated that neonatal mice infected with CV-A6 exhibited limb weakness, paralysis, and ultimately succumbed to death. However, the underlying mechanism of CV-A6-induced nervous system injury remains elusive. Numerous reports have highlighted the pivotal role of miRNAs in various viral infections. Methods: Separately established infection and control groups of mice were used to create miRNA profiles of the brain tissues before and after CV-A6 transfection, followed by experimental verification, prediction, and analysis of the results. Results: At 2 days post-infection (dpi), 4 dpi, and 2dpi vs 4dpi, we identified 175, 198 and 78 significantly differentially expressed miRNAs respectively using qRT-PCR for validation purposes. Subsequently, we predicted target genes of these differentially expressed miRNAs and determined their potential targets through GO (Gene Ontology) enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis. Finally, we verified the miRNA-mRNA pairing via double luciferase experiments while confirming functional enrichment of target genes through Western Blotting analyses. Discussion: The results from this study suggest that transcriptional regulation, neuronal necrosis, pro-inflammatory cytokine release, and antiviral immunity are all implicated in the pathogenesis of central nervous system injury in mice infected with CV-A6. Brain injury resulting from CV-A6 infection may involve multiple pathways, including glial cell activation, neuronal necrosis, synaptic destruction, degenerative diseases of the nervous system. It can even encompass destruction of the blood-brain barrier, leading to central nervous system injury. The dysregulated miRNAs and signaling pathways discovered in this study provide valuable insights for further investigations into the pathogenesis of CV-A6.

Asunto(s)

Modelos Animales de Enfermedad , MicroARNs , Animales , MicroARNs/genética , MicroARNs/metabolismo , Ratones , Encéfalo/virología , Encéfalo/patología , Encéfalo/metabolismo , Infecciones por Coxsackievirus/virología , Infecciones por Coxsackievirus/genética , Lesiones Encefálicas/virología , Lesiones Encefálicas/genética , Perfilación de la Expresión Génica , Enterovirus Humano A/genética , Enterovirus Humano A/patogenicidad , Enterovirus/genética , Enterovirus/patogenicidad , Enfermedad de Boca, Mano y Pie/virología

RESUMEN

Myocarditis is an inflammatory disease that may lead to dilated cardiomyopathy. Viral infection of the myocardium triggers immune responses, which involve, among others, macrophage infiltration, oxidative stress, expression of pro-inflammatory cytokines, and microRNAs (miRNAs). The cardioprotective role of estrogen in myocarditis is well documented; however, sex differences in the miRNA expression in chronic myocarditis are still poorly understood, and studying them further was the aim of the present study. Male and female ABY/SnJ mice were infected with CVB3. Twenty-eight days later, cardiac tissue from both infected and control mice was used for real-time PCR and Western blot analysis. NFκB, IL-6, iNOS, TNF-α, IL-1ß, MCP-1, c-fos, and osteopontin (OPN) were used to examine the inflammatory state in the heart. Furthermore, the expression of several inflammation- and remodeling-related miRNAs was analyzed. NFκB, IL-6, TNF-α, IL-1ß, iNOS, and MCP-1 were significantly upregulated in male mice with CVB3-induced chronic myocarditis, whereas OPN mRNA expression was increased only in females. Further analysis revealed downregulation of some anti-inflammatory miRNA in male hearts (let7a), with upregulation in female hearts (let7b). In addition, dysregulation of remodeling-related miRNAs (miR27b and mir199a) in a sex-dependent manner was observed. Taken together, the results of the present study suggest a sex-specific expression of pro-inflammatory markers as well as inflammation- and remodeling-related miRNAs, with a higher pro-inflammatory response in male CVB3 myocarditis mice.

Asunto(s)

Infecciones por Coxsackievirus , Modelos Animales de Enfermedad , MicroARNs , Miocarditis , Animales , Miocarditis/metabolismo , Miocarditis/virología , Miocarditis/genética , MicroARNs/genética , MicroARNs/metabolismo , Femenino , Masculino , Ratones , Infecciones por Coxsackievirus/metabolismo , Infecciones por Coxsackievirus/genética , Infecciones por Coxsackievirus/virología , Enterovirus Humano B , Biomarcadores/metabolismo , Caracteres Sexuales , Citocinas/metabolismo , Citocinas/genética , Miocardio/metabolismo , Miocardio/patología , Inflamación/genética , Inflamación/metabolismo , Factores Sexuales , Regulación de la Expresión Génica

RESUMEN

Acute pancreatitis (AP) is an inflammatory disease initiated by the death of exocrine acinar cells, but its pathogenesis remains unclear. Signal transducer and activator of transcription 3 (STAT3) is a multifunctional factor that regulates immunity and the inflammatory response. The protective role of STAT3 is reported in Coxsackievirus B3 (CVB3)-induced cardiac fibrosis, yet the exact role of STAT3 in modulating viral-induced STAT1 activation and type I interferon (IFN)-stimulated gene (ISG) transcription in the pancreas remains unclarified. In this study, we tested whether STAT3 regulated viral-induced STAT1 translocation. We found that CVB3, particularly capsid VP1 protein, markedly upregulated the phosphorylation and nuclear import of STAT3 (p-STAT3) while it significantly impeded the nuclear translocation of p-STAT1 in the pancreases and hearts of mice on day 3 postinfection (p.i.). Immunoblotting and an immunofluorescent assay demonstrated the increased expression and nuclear translocation of p-STAT3 but a blunted p-STAT1 nuclear translocation in CVB3-infected acinar 266-6 cells. STAT3 shRNA knockdown or STAT3 inhibitors reduced viral replication via the rescue of STAT1 nuclear translocation and increasing the ISRE activity and ISG transcription in vitro. The knockdown of STAT1 blocked the antiviral effect of the STAT3 inhibitor. STAT3 inhibits STAT1 activation by virally inducing a potent inhibitor of IFN signaling, the suppressor of cytokine signaling-3 ((SOCS)-3). Sustained pSTAT1 and the elevated expression of ISGs were induced in SOCS3 knockdown cells. The in vivo administration of HJC0152, a pharmaceutical STAT3 inhibitor, mitigated the viral-induced AP and myocarditis pathology via increasing the IFNß as well as ISG expression on day 3 p.i. and reducing the viral load in multi-organs. These findings define STAT3 as a negative regulator of the type I IFN response via impeding the nuclear STAT1 translocation that otherwise triggers ISG induction in infected pancreases and hearts. Our findings identify STAT3 as an antagonizing factor of the IFN-STAT1 signaling pathway and provide a potential therapeutic target for viral-induced AP and myocarditis.

Asunto(s)

Enterovirus Humano B , Miocarditis , Pancreatitis , Factor de Transcripción STAT1 , Factor de Transcripción STAT3 , Replicación Viral , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT1/genética , Miocarditis/virología , Miocarditis/metabolismo , Miocarditis/patología , Miocarditis/genética , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genética , Animales , Pancreatitis/metabolismo , Pancreatitis/virología , Pancreatitis/patología , Pancreatitis/genética , Enterovirus Humano B/fisiología , Ratones , Humanos , Infecciones por Coxsackievirus/metabolismo , Infecciones por Coxsackievirus/virología , Infecciones por Coxsackievirus/patología , Infecciones por Coxsackievirus/genética , Núcleo Celular/metabolismo , Masculino , Transporte Activo de Núcleo Celular , Regulación de la Expresión Génica , Enfermedad Aguda , Línea Celular , Transducción de Señal

RESUMEN

Viral myocarditis is characterized by infiltration of mononuclear cells essential for virus elimination. GPR15 has been identified as a homing receptor for regulatory T cells in inflammatory intestine diseases, but its role in inflammatory heart diseases is still elusive. Here we show that GPR15 deficiency impairs coxsackievirus B3 elimination, leading to adverse cardiac remodeling and dysfunction. Delayed recruitment of regulatory T cells in GPR15-deficient mice was accompanied by prolonged persistence of cytotoxic and regulatory T cells. In addition, RNA sequencing revealed prolonged inflammatory response and altered chemotaxis in knockout mice. In line, we identified GPR15 and its ligand GPR15L as an important chemokine receptor-ligand pair for the recruitment of regulatory and cytotoxic T cells. In summary, the insufficient virus elimination might be caused by a delayed recruitment of T cells as well as delayed interferon-γ expression, resulting in a prolonged inflammatory response and an adverse outcome in GPR15-deficient mice.

Asunto(s)

Infecciones por Coxsackievirus , Modelos Animales de Enfermedad , Enterovirus Humano B , Ratones Noqueados , Miocarditis , Receptores Acoplados a Proteínas G , Animales , Miocarditis/inmunología , Miocarditis/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/deficiencia , Receptores Acoplados a Proteínas G/inmunología , Infecciones por Coxsackievirus/inmunología , Infecciones por Coxsackievirus/genética , Enterovirus Humano B/inmunología , Ratones Endogámicos C57BL , Linfocitos T Reguladores/inmunología , Enfermedad Aguda , Interferón gamma/metabolismo , Ratones , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Masculino , Quimiotaxis de Leucocito/genética , Quimiotaxis de Leucocito/inmunología , Miocardio/metabolismo , Miocardio/inmunología , Miocardio/patología , Transducción de Señal

RESUMEN

Major 5'-terminally deleted (5'TD) RNA forms of group-B coxsackievirus (CVB-5'TD) has been associated with myocarditis in both mice and humans. Although it is known that interferon-ß (IFN-ß) signaling is critical for an efficient innate immune response against CVB-induced myocarditis, the link between CVB-5'TD RNA forms and type I IFN signaling in cardiomyocytes remains to be explored. In a mouse model of CVB3/28-induced myocarditis, major early-emerging forms of CVB-5'TD RNA have been characterized as replicative viral populations that impair IFN-ß production in the heart. Synthetic CVB3/28 RNA forms mimicking each of these major 5'TD virus populations were transfected in mice and have been shown to modulate innate immune responses in the heart and to induce myocarditis in mice. Remarkably, transfection of synthetic viral RNA with deletions in the secondary structures of the 5'-terminal CVB3 RNA domain I, modifying stem-loops "b", "c" or "d", were found to impair IFN-ß production in human cardiomyocytes. In addition, the activation of innate immune response by Poly(I:C), was found to restore IFN-ß production and to reduce the burden of CVB-5'TD RNA-forms in cardiac tissues, thereby reducing the mortality rate of infected mice. Overall, our results indicate that major early-emerging CVB3 populations deleted in the domain I of genomic RNA, in the 5' noncoding region, modulate the activation of the type I IFN pathway in cardiomyocytes and induce myocarditis in mice. These findings shed new light on the role of replicative CVB-5'TD RNA forms as key pathophysiological factors in CVB-induced human myocarditis.

Asunto(s)

Infecciones por Coxsackievirus , Enterovirus Humano B , Interferón Tipo I , Miocarditis , Miocitos Cardíacos , ARN Viral , Miocarditis/virología , Miocarditis/inmunología , Miocarditis/genética , Animales , Miocitos Cardíacos/virología , Miocitos Cardíacos/metabolismo , Ratones , Enterovirus Humano B/inmunología , Infecciones por Coxsackievirus/inmunología , Infecciones por Coxsackievirus/virología , Infecciones por Coxsackievirus/genética , Interferón Tipo I/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Humanos , Inmunidad Innata , Transducción de Señal , Interferón beta/metabolismo , Interferón beta/genética , Interferón beta/inmunología , Masculino , Regiones no Traducidas 5'

RESUMEN

AIMS: Virus infection triggers inflammation and, may impose nutrient shortage to the heart. Supported by type I interferon (IFN) signalling, cardiomyocytes counteract infection by various effector processes, with the IFN-stimulated gene of 15 kDa (ISG15) system being intensively regulated and protein modification with ISG15 protecting mice Coxsackievirus B3 (CVB3) infection. The underlying molecular aspects how the ISG15 system affects the functional properties of respective protein substrates in the heart are unknown. METHODS AND RESULTS: Based on the protective properties due to protein ISGylation, we set out a study investigating CVB3-infected mice in depth and found cardiac atrophy with lower cardiac output in ISG15-/- mice. By mass spectrometry, we identified the protein targets of the ISG15 conjugation machinery in heart tissue and explored how ISGylation affects their function. The cardiac ISGylome showed a strong enrichment of ISGylation substrates within glycolytic metabolic processes. Two control enzymes of the glycolytic pathway, hexokinase 2 (HK2) and phosphofructokinase muscle form (PFK1), were identified as bona fide ISGylation targets during infection. In an integrative approach complemented with enzymatic functional testing and structural modelling, we demonstrate that protein ISGylation obstructs the activity of HK2 and PFK1. Seahorse-based investigation of glycolysis in cardiomyocytes revealed that, by conjugating proteins, the ISG15 system prevents the infection-/IFN-induced up-regulation of glycolysis. We complemented our analysis with proteomics-based advanced computational modelling of cardiac energy metabolism. Our calculations revealed an ISG15-dependent preservation of the metabolic capacity in cardiac tissue during CVB3 infection. Functional profiling of mitochondrial respiration in cardiomyocytes and mouse heart tissue by Seahorse technology showed an enhanced oxidative activity in cells with a competent ISG15 system. CONCLUSION: Our study demonstrates that ISG15 controls critical nodes in cardiac metabolism. ISG15 reduces the glucose demand, supports higher ATP production capacity in the heart, despite nutrient shortage in infection, and counteracts cardiac atrophy and dysfunction.

Asunto(s)

Infecciones por Coxsackievirus , Citocinas , Metabolismo Energético , Glucólisis , Mitocondrias Cardíacas , Miocitos Cardíacos , Ubiquitinas , Animales , Humanos , Masculino , Infecciones por Coxsackievirus/metabolismo , Infecciones por Coxsackievirus/virología , Infecciones por Coxsackievirus/genética , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Enterovirus Humano B/patogenicidad , Enterovirus Humano B/metabolismo , Interacciones Huésped-Patógeno , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias Cardíacas/metabolismo , Mitocondrias Cardíacas/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/virología , Miocitos Cardíacos/patología , Procesamiento Proteico-Postraduccional , Transducción de Señal , Ubiquitinas/metabolismo , Ubiquitinas/genética

RESUMEN

Coxsackievirus B3 (CVB3) is known to cause acute myocarditis and pancreatitis in humans. We investigated the microRNAs (miRNAs) that can potentially govern the viral life cycle by binding to the untranslated regions (UTRs) of CVB3 RNA. MicroRNA-22-3p was short-listed, as its potential binding site overlapped with the region crucial for recruiting internal ribosome entry site trans-acting factors (ITAFs) and ribosomes. We demonstrate that miR-22-3p binds CVB3 5' UTR, hinders recruitment of key ITAFs on viral mRNA, disrupts the spatial structure required for ribosome recruitment, and ultimately blocks translation. Likewise, cells lacking miR-22-3p exhibited heightened CVB3 infection compared to wild type, confirming its role in controlling infection. Interestingly, miR-22-3p level was found to be increased at 4 hours post-infection, potentially due to the accumulation of viral 2A protease in the early phase of infection. 2Apro enhances the miR-22-3p level to dislodge the ITAFs from the SD-like sequence, rendering the viral RNA accessible for binding of replication factors to switch to replication. Furthermore, one of the cellular targets of miR-22-3p, protocadherin-1 (PCDH1), was significantly downregulated during CVB3 infection. Partial silencing of PCDH1 reduced viral replication, demonstrating its proviral role. Interestingly, upon CVB3 infection in mice, miR-22-3p level was found to be downregulated only in the small intestine, the primary target organ, indicating its possible role in influencing tissue tropism. It appears miR-22-3p plays a dual role during infection by binding viral RNA to aid its life cycle as a viral strategy and by targeting a proviral protein to restrict viral replication as a host response.IMPORTANCECVB3 infection is associated with the development of end-stage heart diseases. Lack of effective anti-viral treatments and vaccines for CVB3 necessitates comprehensive understanding of the molecular players during CVB3 infection. miRNAs have emerged as promising targets for anti-viral strategies. Here, we demonstrate that miR-22-3p binds to 5' UTR and inhibits viral RNA translation at the later stage of infection to promote viral RNA replication. Conversely, as host response, it targets PCDH1, a proviral factor, to discourage viral propagation. miR-22-3p also influences CVB3 tissue tropism. Deciphering the multifaced role of miR-22-3p during CVB3 infection unravels the necessary molecular insights, which can be exploited for novel intervening strategies to curb infection and restrict viral pathogenesis.

Asunto(s)

Regiones no Traducidas 5' , Infecciones por Coxsackievirus , Enterovirus Humano B , Interacciones Microbiota-Huesped , MicroARNs , Biosíntesis de Proteínas , ARN Viral , Animales , Humanos , Ratones , Regiones no Traducidas 5'/genética , Antivirales/metabolismo , Infecciones por Coxsackievirus/genética , Infecciones por Coxsackievirus/virología , Enterovirus Humano B/genética , Enterovirus Humano B/patogenicidad , Enterovirus Humano B/fisiología , Células HeLa , Intestino Delgado/metabolismo , Intestino Delgado/virología , MicroARNs/genética , MicroARNs/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Tropismo Viral/genética , Replicación Viral/genética , Cisteína Endopeptidasas/metabolismo , Protocadherinas/deficiencia , Protocadherinas/genética , Miocarditis , Interacciones Microbiota-Huesped/genética

RESUMEN

Calcium/calmodulin-dependent protein kinase II (CaMKII) has been demonstrated to be aberrantly activated in viral myocarditis (VMC), but the role of its subtype CaMKIIδ in VMC remains unclear.VMC mice and cardiomyocytes models were induced by Coxsackievirus B3 (CVB3) treatment. Mice that underwent sham surgery and saline-treated cardiomyocytes served as controls. Body weight, survival, left ventricular ejection fraction (LVEF), and fractional shortening (LVFS) were measured, and HE staining was performed to evaluate heart function in VMC mice model and sham control. Inflammation factors in serum or cell supernatant were detected by ELISA. Expressions of CaMKIIδ, Toll/interleukin-1 receptor domain containing adaptor protein (TIRAP), insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2), nuclear factor NF-kappaB (NF-κB) signals, and inflammation factors were examined by quantitative real time polymerase chain reaction (qRT-PCR) or western blot. CCK-8, EdU, and flow cytometry were used to evaluate cell behaviors. Co-immunoprecipitation (Co-IP), RNA immunoprecipitation (RIP), and RNA pull-down were utilized to validate molecule interaction. Methylated RNA immunoprecipitation (MeRIP) was performed to measure N6-methyladenosine (m6A) level of specific molecule.CaMKIIδ was upregulated in VMC mice and CVB3-treated primary cardiomyocytes, of which knockdown improved cell viability, proliferation, and suppressed cell apoptosis in vitro, thereby alleviating myocarditis in vivo. The stability of CaMKIIδ was attributed to the presence of IGF2BP2 through m6A modification. Loss of CaMKIIδ repressed NF-κB pathway via negatively and directly regulating TIRAP to be involved in inflammatory damage.CaMKIIδ, stabilized by m6A reader IGF2BP2, modulated NF-κB pathway via interacting with TIRAP to alter cell viability, proliferation, and apoptosis, thereby affecting VMC outcome.

Asunto(s)

Apoptosis , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Infecciones por Coxsackievirus , Modelos Animales de Enfermedad , Miocarditis , Miocitos Cardíacos , FN-kappa B , Proteínas de Unión al ARN , Receptores de Interleucina-1 , Transducción de Señal , Animales , Masculino , Ratones , Adenosina/análogos & derivados , Adenosina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proliferación Celular , Células Cultivadas , Infecciones por Coxsackievirus/metabolismo , Infecciones por Coxsackievirus/genética , Infecciones por Coxsackievirus/enzimología , Infecciones por Coxsackievirus/virología , Infecciones por Coxsackievirus/patología , Enterovirus Humano B/patogenicidad , Mediadores de Inflamación/metabolismo , Ratones Endogámicos BALB C , Miocarditis/metabolismo , Miocarditis/genética , Miocarditis/patología , Miocarditis/virología , Miocarditis/enzimología , Miocitos Cardíacos/enzimología , Miocitos Cardíacos/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/virología , FN-kappa B/metabolismo , Receptores de Interleucina-1/metabolismo , Receptores de Interleucina-1/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Función Ventricular Izquierda

RESUMEN

BACKGROUND: Dysregulated cell death machinery and an excessive inflammatory response in Coxsackievirus B3(CVB3)-infected myocarditis are hallmarks of an abnormal host response. Complement C4 and C3 are considered the central components of the classical activation pathway and often participate in the response process in the early stages of virus infection. METHODS: In our study, we constructed a mouse model of CVB3-related viral myocarditis via intraperitoneal injection of Fer-1 and detected myocarditis and ferroptosis markers in the mouse myocardium. Then, we performed co-IP and protein mass spectrometry analyses to explore which components interact with the ferroptosis gene transferrin receptor (TFRC). Finally, functional experiments were conducted to verify the role of complement components in regulating ferroptosis in CVB3 infection. RESULTS: It showed that the ferroptosis inhibitor Fer-1 could alleviate the inflammation in viral myocarditis as well as ferroptosis. Mechanistically, during CVB3 infection, the key factor TFRC was activated and inhibited by Fer-1. Fer-1 effectively prevented the consumption of complement C3 and overload of the complement product C4b. Interestingly, we found that TFRC directly interacts with complement C4, leading to an increase in the product of C4b and a decrease in the downstream complement C3. Functional experiments have also confirmed that regulating the complement C4/C3 pathway can effectively rescue cell ferroptosis caused by CVB3 infection. CONCLUSIONS: In this study, we found that ferroptosis occurs through crosstalk with complement C4 in viral myocarditis through interaction with TFRC and that regulating the complement C4/C3 pathway may rescue ferroptosis in CVB3-infected cardiomyocytes.

Asunto(s)

Infecciones por Coxsackievirus , Ferroptosis , Miocarditis , Virosis , Animales , Ratones , Miocarditis/metabolismo , Complemento C3/genética , Complemento C3/metabolismo , Complemento C3/farmacología , Infecciones por Coxsackievirus/genética , Infecciones por Coxsackievirus/metabolismo , Enterovirus Humano B/metabolismo , Miocardio/metabolismo , Factores Inmunológicos/farmacología , Complemento C4/metabolismo , Complemento C4/farmacología , Receptores de Transferrina

RESUMEN

Evidence is emerging on the roles of long noncoding RNAs (lncRNAs) as regulatory factors in a variety of viral infection processes, but the mechanisms underlying their functions in coxsackievirus group B type3 (CVB3)-induced acute viral myocarditis have not been explicitly delineated. We previously demonstrated that CVB3 infection decreases miRNA-21 expression; however, lncRNAs that regulate the miRNA-21-dependent CVB3 disease process have yet to be identified. To evaluate lncRNAs upstream of miRNA-21, differentially expressed lncRNAs in CVB3-infected mouse hearts were identified by microarray analysis and lncRNA/miRNA-21 interactions were predicted bioinformatically. MEG3 was identified as a candidate miRNA-21-interacting lncRNA upregulated in CVB3-infected mouse hearts. MEG3 expression was verified to be upregulated in HeLa cells 48 h post CVB3 infection and to act as a competitive endogenous RNA of miRNA-21. MEG3 knockdown resulted in the upregulation of miRNA-21, which inhibited CVB3 replication by attenuating P38-MAPK signaling in vitro and in vivo. Knockdown of MEG3 expression before CVB3 infection inhibited viral replication in mouse hearts and alleviated cardiac injury, which improved survival. Furthermore, the knockdown of CREB5, which was predicted bioinformatically to function upstream of MEG3, was demonstrated to decrease MEG3 expression and CVB3 viral replication. This study identifies the function of the lncRNA MEG3/miRNA-21/P38 MAPK axis in the process of CVB3 replication, for which CREB5 could serve as an upstream modulator.

Asunto(s)

Infecciones por Coxsackievirus , Enterovirus , MicroARNs , Miocarditis , ARN Largo no Codificante , Virosis , Animales , Humanos , Ratones , Infecciones por Coxsackievirus/complicaciones , Infecciones por Coxsackievirus/genética , Enterovirus/genética , Enterovirus Humano B/genética , Enterovirus Humano B/metabolismo , Células HeLa/virología , MicroARNs/genética , MicroARNs/metabolismo , Miocarditis/genética , Miocarditis/metabolismo , Miocarditis/virología , ARN Largo no Codificante/genética , Replicación Viral

RESUMEN

Viral myocarditis (VMC) is the major reason for sudden cardiac death among both children and young adults. Of these, coxsackievirus B3 (CVB3) is the most common causative agent of myocarditis. Recently, the role of signaling pathways in the pathogenesis of VMC has been evaluated in several studies, which has provided a new perspective on identifying potential therapeutic targets for this hitherto incurable disease. In the present study, in vivo and in vitro experiments showed that CVB3 infection leads to increased Bim expression and triggers apoptosis. In addition, by knocking down Bim using RNAi, we further confirmed the biological function of Bim in apoptosis induced by CVB3 infection. We additionally found that Bim and forkhead box O1 class (FOXO1) inhibition significantly increased the viability of CVB3-infected cells while blocking viral replication and viral release. Moreover, CVB3-induced Bim expression was directly dependent on FOXO1 acetylation, which is catalyzed by the co-regulation of CBP and SirTs. Furthermore, the acetylation of FOXO1 was an important step in Bim activation and apoptosis induced by CVB3 infection. The findings of this study suggest that CVB3 infection induces apoptosis through the FOXO1 acetylation-Bim pathway, thus providing new insights for developing potential therapeutic targets for enteroviral myocarditis.

Asunto(s)

Apoptosis , Proteína 11 Similar a Bcl2 , Infecciones por Coxsackievirus , Enterovirus Humano B , Proteína Forkhead Box O1 , Miocarditis , Miocitos Cardíacos , Proteína 11 Similar a Bcl2/metabolismo , Proteína 11 Similar a Bcl2/genética , Apoptosis/genética , Miocitos Cardíacos/virología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Proteína Forkhead Box O1/metabolismo , Proteína Forkhead Box O1/genética , Animales , Miocarditis/virología , Miocarditis/metabolismo , Miocarditis/genética , Miocarditis/patología , Enterovirus Humano B/fisiología , Infecciones por Coxsackievirus/genética , Infecciones por Coxsackievirus/virología , Infecciones por Coxsackievirus/metabolismo , Infecciones por Coxsackievirus/patología , Acetilación , Humanos , Masculino , Ratones , Transducción de Señal , Ratas

RESUMEN

Exploring the immune mechanism of coxsackievirus B3 (CVB3)-induced myocarditis may provide a promising therapeutic strategy. Here, we investigated the regulatory role of macrophage CAPN4 in the phenotypic transformation of macrophages and NOD-like receptor protein 3 (NLRP3) inflammasome activation. We found that CAPN4 was the most upregulated subtype of the calpain family in CVB3-infected bone marrow-derived macrophages (BMDMs) and Raw 264.7 cells after CVB3 infection and was upregulated in cardiac macrophages from CVB3-infected mice. Conditional knockout of CAPN4 (CAPN4flox/flox; LYZ2-Cre, CAPN4-cKO mice) ameliorated inflammation and myocardial injury and improved cardiac function and survival after CVB3 infection. Enrichment analysis revealed that macrophage differentiation and the interleukin signaling pathway were the most predominant biological processes in macrophages after CVB3 infection. We further found that CVB3 infection and the overexpression of CAPN4 promoted macrophage M1 polarization and NLRP3 inflammasome activation, while CAPN4 knockdown reversed these changes. Correspondingly, CAPN4-cKO alleviated CVB3-induced M1 macrophage transformation and NLRP3 expression and moderately increased M2 transformation in vivo. The culture supernatant of CAPN4-overexpressing or CVB3-infected macrophages impaired cardiac fibroblast function and viability. Moreover, macrophage CAPN4 could upregulate C/EBP-homologous protein (chop) expression, which increased proinflammatory cytokine release by activating the phosphorylation of transducer of activator of transcription 1 (STAT1) and 3 (STAT3). Overall, these results suggest that CAPN4 increases M1-type and inhibits M2-type macrophage polarization through the chop-STAT1/STAT3 signaling pathway to mediate CVB3-induced myocardial inflammation and injury. CAPN4 may be a novel target for viral myocarditis treatment.

Asunto(s)

Infecciones por Coxsackievirus , Inflamasomas , Miocarditis , Animales , Ratones , Infecciones por Coxsackievirus/genética , Infecciones por Coxsackievirus/metabolismo , Enterovirus Humano B/metabolismo , Inflamasomas/metabolismo , Inflamación/genética , Inflamación/metabolismo , Macrófagos/metabolismo , Miocarditis/genética , Miocarditis/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteínas NLR/metabolismo

RESUMEN

Viral myocarditis is an inflammatory disease of the myocardium, often leads to cardiac dysfunction and death. PARKIN (PRKN) and PINK1, well known as Parkinson's disease-associated genes, have been reported to be involved in innate immunity and mitochondrial damage control. Therefore, we investigated the role of parkin and PINK1 in coxsackievirus B3 (CVB3)-induced viral myocarditis because the etiology of myocarditis is related to abnormal immune response to viral infection and mitochondrial damage. After viral infection, the survival was significantly lower and myocardial damage was more severe in parkin knockout (KO) and PINK1 KO mice compared to wild-type (WT) mice. Parkin KO and PINK1 KO showed defective immune cell recruitment and impaired production of antiviral cytokines such as interferon-gamma, allowing increased viral replication. In addition, parkin KO and PINK1 KO mice were more susceptible to CVB3-induced mitochondrial damage than WT mice, resulting in susceptibility to viral-induced cardiac damage. Finally, using publicly available RNA-seq data, we found that pathogenic mutants of the PRKN gene are more common in patients with dilated cardiomyopathy and myocarditis than in controls or the general population. This study will help elucidate the molecular mechanism of CVB3-induced viral myocarditis.

Asunto(s)

Infecciones por Coxsackievirus , Miocarditis , Virosis , Animales , Humanos , Ratones , Infecciones por Coxsackievirus/genética , Infecciones por Coxsackievirus/patología , Modelos Animales de Enfermedad , Enterovirus Humano B/genética , Ratones Noqueados , Miocarditis/genética , Miocarditis/patología , Proteínas Quinasas/genética , Ubiquitina-Proteína Ligasas/genética

RESUMEN

Coxsackievirus B3 (CVB3) is an enterovirus that causes diseases such as pancreatitis and myocarditis in humans. Approximately 10% of the CVB3 RNA genome consists of a highly structured 5' untranslated region (5' UTR) that is organized into six domains and contains a type I internal ribosome entry site (IRES). These features are common to all enteroviruses. Each RNA domain plays a vital role in translation and replication during the viral multiplication cycle. We used SHAPE-MaP chemistry to generate secondary structures of the 5' UTR from the avirulent strain CVB3/GA and the virulent strain CVB3/28. Our comparative models show how key nucleotide substitutions cause major restructuring of domains II and III of the 5' UTR in CVB3/GA. Despite these structural shifts, the molecule maintains several well-characterized RNA elements, which allows persistence of the unique avirulent strain. The results shed light on the 5' UTR regions serving as virulence determinants and those required for fundamental viral mechanisms. We used the SHAPE-MaP data to produce theoretical tertiary models using 3dRNA v2.0. These models suggest a compact conformation of the 5' UTR from the virulent strain CVB3/28 that brings critical domains into close contact. In contrast, the model of the 5' UTR from the avirulent strain CVB3/GA suggests a more extended conformation where the same critical domains are more separated. Our results suggest that the structure and orientation of RNA domains in the 5' UTR are responsible for low-efficiency translation, low viral titers, and absence of virulence observed during infection by CVB3/GA. IMPORTANCE Human enteroviruses, which include five different species and over 100 serotypes, are responsible for diseases ranging from mild respiratory infections to serious infections of pancreas, heart, and neural tissue. All enteroviral RNA genomes have a long and highly structured 5' untranslated region (5' UTR) containing an internal ribosome entry site (IRES). Major virulence determinants are located in the 5' UTR. We present RNA structure models that directly compare the 5' UTR derived from virulent and avirulent strains of the enterovirus coxsackievirus B3 (CVB3). The secondary-structure models show rearrangement of RNA domains known to be virulence determinants and conservation of structure in RNA elements known to be vital for translation and replication in the avirulent strain CVB3/GA. The tertiary-structure models reveal reorientation of RNA domains in CVB3/GA. Identifying the details of structure in these critical RNA domains will help direct antiviral approaches to this major human pathogen.

Asunto(s)

Infecciones por Coxsackievirus , Enterovirus Humano B , ARN Viral , Humanos , Regiones no Traducidas 5' , Infecciones por Coxsackievirus/genética , Enterovirus Humano B/genética , Células HeLa , Sitios Internos de Entrada al Ribosoma , Fenotipo , ARN Viral/genética , ARN Viral/metabolismo , Virulencia , Factores de Virulencia

RESUMEN

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.

Asunto(s)

Infecciones por Coxsackievirus , Miocarditis , Ratones , Animales , Miocarditis/patología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Lipopolisacáridos/metabolismo , Enterovirus Humano B/metabolismo , Infecciones por Coxsackievirus/genética , Infecciones por Coxsackievirus/patología , Macrófagos/metabolismo , Ratones Noqueados

RESUMEN

BACKGROUND: Myocardial inflammation and apoptosis are key processes in coxsackievirus B3 (CVB3)-induced acute viral myocarditis (AVMC). Accumulating evidence reveals the essential roles of long noncoding RNAs (lncRNAs) in the pathogenesis of AVMC. Here, we aimed to evaluate the biological functions of a novel lncRNA guanylate-binding protein 9 (lncGBP9) in AVMC progression and further explore its underlying mechanisms. METHODS: Initially, mouse models of AVMC were constructed by CVB3 infection. The expression and localization of lncGBP9 in heart tissues were analyzed using RT-qPCR and FISH. Adeno-associated virus serotype 9 (AAV9)-mediated lncGBP9 knockdown was then employed to clarify its roles in survival, cardiac function, and myocardial inflammation and apoptosis. Moreover, the mRNA and protein levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1ß) were detected by RT-qPCR and ELISA, and the regulation of lncGBP9 knockdown on the NF-κB signaling pathway was investigated by Western blotting. Using an in vitro model of HL-1 cardiomyocytes exposed to CVB3 infection, the effects of lncGBP9 knockdown on cell viability, inflammation, and apoptosis were further evaluated in vitro. RESULTS: Increased lncGBP9 expression was detected in the heart tissues of AVMC mice and CVB3-infected HL-1 cells, and was mainly located in the cytoplasm. Knockdown of lncGBP9 remarkably alleviated the severity of AVMC in CVB3-infected mice, as verified by improved cardiac function, and reduced myocardial inflammation and apoptosis. Additionally, lncGBP9 knockdown suppressed the NF-κB signaling pathway and consequently reduced productions of pro-inflammatory cytokines in vivo. In vitro functional assays further confirmed that lncGBP9 knockdown promoted cell viability, inhibited cell apoptosis, and reduced pro-inflammatory cytokines release in CVB3-infected HL-1 cells through suppressing NF-κB activation. CONCLUSIONS: Collectively, lncGBP9 knockdown exerts anti-inflammatory and anti-apoptotic effects in CVB3-induced AVMC, which may be mediated in part via NF-κB signaling pathway. These findings highlight lncGBP9 as an attractive target for therapeutic interventions.

Asunto(s)

Infecciones por Coxsackievirus , Miocarditis , Ratones , Animales , Miocarditis/genética , FN-kappa B/metabolismo , Enterovirus Humano B/metabolismo , Infecciones por Coxsackievirus/genética , Infecciones por Coxsackievirus/metabolismo , Infecciones por Coxsackievirus/patología , Transducción de Señal , Inflamación/metabolismo , Apoptosis , Citocinas/metabolismo , Ratones Endogámicos BALB C

RESUMEN

CVB3 is a single positive-strand enterovirus, and a common pathogen in myocarditis etiology. Although a number of antiviral candidates are under development, specific targeted therapy is not available for CVB3. Ferroptosis is a new type of regulatory cell death discovered in recent years. In this study, our team provided the first evidence that ferroptosis existed in CVB3 infection in vivo and in vitro by iron overload, and massive accumulation of lipid peroxides. Mechanistically, we construct a classical model of HeLa cells following a time-course infection (6, 12, 24, 36, 48 h) with CVB3 (MOI = 10). We demonstrated that the TFRC gene plays an important role in promoting ferroptosis in CVB3 infection and downregulation of TFRC attenuated the ferroptosis. Interestingly, we observed that TFRC was nuclear translocation induced by the CVB3, which was predominantly localized in the cell membrane, but redistributed to the nucleus during CVB3 infection. Moreover, we found that the transcription factor Sp1 was an essential factor that could bind to the TFRC promoter and upregulate the TFRC transcription. Collectively, these results suggest that the Sp1/TFRC/Fe axis may provide a new target for the development of therapies against CVB3 infection.

Asunto(s)

Infecciones por Coxsackievirus , Enterovirus Humano B , Ferroptosis , Factor de Transcripción Sp1 , Antígenos CD/genética , Antígenos CD/metabolismo , Núcleo Celular/metabolismo , Infecciones por Coxsackievirus/genética , Infecciones por Coxsackievirus/metabolismo , Células HeLa , Humanos , Receptores de Transferrina/genética , Receptores de Transferrina/metabolismo , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismo , Activación Transcripcional , Regulación hacia Arriba

RESUMEN

Coxsackievirus B3 (CVB3) is a positive single-strand RNA virus causing myocarditis, pancreatitis and meningitis. During CVB3 infection, various host cellular components, including proteins and non-coding RNAs, interact with the virus and affect viral infection. Poly(rC) binding protein 1 (PCBP1) is a multifunctional RNA binding protein regulating transcription, translation and mRNA stability of a variety of genes. In this study, we observed a significant reduction of PCBP1 protein during CVB3 infection. By bioinformatic prediction and luciferase-assay verification, we confirmed that the expression of PCBP1 was directly inhibited by miR-21, a microRNA upregulated during CVB3 infection. Furthermore, we found that overexpression of PCBP1 promoted CVB3 infection and knocking down of PCBP1 inhibited it. In the subsequent mechanism study, our results revealed that PCBP1 blocked the translation of p62/SQSTM1 (sequestosome 1), an autophagy-receptor protein suppressing CVB3 replication, by interacting with the cis-element in the 5' untranslational region (5' UTR) of p62/SQSTM1. In summary, our studies have identified PCBP1 as a beneficial factor for CVB3 infection. These findings may deepen the understanding of host-virus interactions and provide a potential target for intervention of CVB3 infection.

Asunto(s)

Infecciones por Coxsackievirus , Enterovirus Humano B , Regiones no Traducidas 5' , Proteínas Portadoras/genética , Infecciones por Coxsackievirus/genética , Proteínas de Unión al ADN/metabolismo , Enterovirus Humano B/genética , Células HeLa , Humanos , Poli A/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteína Sequestosoma-1/genética , Proteína Sequestosoma-1/metabolismo , Replicación Viral/genética

RESUMEN

Group B coxsackieviruses (CVBs) are the main cause of virus-induced myocarditis. CVBs use coxsackievirus and adenovirus receptor (CAR) for infection and targeting CAR has been shown to ameliorate CVBs-induced myocarditis. Ligand-of-Numb protein X1 (LNX1) is an E3 ubiquitin ligase that was shown to interact with CAR. However, the precise effect of LNX1 on CAR and the roles of LNX1 on CVBs-induced myocarditis remain unknown. In the present study, we generated mice deficient in LNX1 in the heart and evaluated the symptoms of myocarditis after CVB3 infection. We also monitored the expression and ubiquitination of CAR in LNX1-deficient cardiomyocytes after CVBs infection. We found that CVBs infection decreased CAR expression while promoted the expression of LNX1. Mice with deficiency of LNX1 in the heart had normal myocardial development while had deteriorated myocarditis symptoms after CVB3 infection. In LNX1-deficient cardiomyocytes, decreased ubiquitination of CAR and upregulation of CAR were observed after CVB3 infection. In summary, LNX1 controls CVB3-induced myocarditis by regulating the expression of CAR.

Asunto(s)

Infecciones por Coxsackievirus , Enterovirus , Miocarditis , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Infecciones por Coxsackievirus/genética , Enterovirus Humano B/fisiología , Ligandos , Proteínas de la Membrana , Ratones , Miocarditis/genética , Miocarditis/metabolismo , Proteínas del Tejido Nervioso , Receptores Virales

RESUMEN

Sporadic occurrences and outbreaks of hand, foot, and mouth disease (HFMD) caused by Coxsackievirus A2 (CVA2) have frequently reported worldwide recently, which pose a great challenge to public health. Epidemiological studies have suggested that the main cause of death in critical patients is pulmonary edema. However, the pathogenesis of this underlying comorbidity remains unclear. In this study, we utilized the 5-day-old BALB/c mouse model of lethal CVA2 infection to evaluate lung damage. We found that the permeability of lung microvascular was significantly increased after CVA2 infection. We also observed the direct infection and apoptosis of lung endothelial cells as well as the destruction of tight junctions between endothelial cells. CVA2 infection led to the degradation of tight junction proteins (e.g., ZO-1, claudin-5, and occludin). The gene transcription levels of von Willebrand factor (vWF), endothelin (ET), thrombomodulin (THBD), granular membrane protein 140 (GMP140), and intercellular cell adhesion molecule-1 (ICAM-1) related to endothelial dysfunction were all significantly increased. Additionally, CVA2 infection induced the increased expression of inflammatory cytokines (IL-6, IL-1ß, and MCP-1) and the activation of p38 mitogen-activated protein kinase (MAPK). In conclusion, the disruption of the endothelial barrier contributes to acute lung injury induced by CVA2 infection; targeting p38-MAPK signaling may provide a therapeutic approach for pulmonary edema in critical infections of HFMD.

Asunto(s)

Lesión Pulmonar Aguda/genética , Infecciones por Coxsackievirus/genética , Enfermedad de Boca, Mano y Pie/genética , Edema Pulmonar/genética , Lesión Pulmonar Aguda/complicaciones , Lesión Pulmonar Aguda/patología , Lesión Pulmonar Aguda/virología , Animales , Apoptosis/genética , Claudina-5/genética , Infecciones por Coxsackievirus/complicaciones , Infecciones por Coxsackievirus/patología , Infecciones por Coxsackievirus/virología , Citocinas/genética , Modelos Animales de Enfermedad , Células Endoteliales/patología , Células Endoteliales/virología , Enfermedad de Boca, Mano y Pie/complicaciones , Enfermedad de Boca, Mano y Pie/patología , Enfermedad de Boca, Mano y Pie/virología , Humanos , Ratones , Ocludina/genética , Edema Pulmonar/complicaciones , Edema Pulmonar/patología , Edema Pulmonar/virología , Uniones Estrechas/genética , Uniones Estrechas/patología , Proteína de la Zonula Occludens-1/genética , Proteínas Quinasas p38 Activadas por Mitógenos/genética