RESUMEN
In enterovirus-induced cardiomyopathy, information regarding the detailed impact of natural killer (NK) cells on the outcome of the disease is limited. We therefore hypothesized that NK cells and certain NK cell receptors determine the different outcome of coxsackievirus B3 (CVB3) myocarditis. Here, we demonstrate in murine models that resistance to chronic CVB3 myocarditis in immunocompetent C57BL/6 mice is characterized by significantly more mature CD11b(high) NK cells, the presence of NKG2D on NK cells, and enhanced NKG2D-dependent cytotoxicity compared to CVB3-susceptible A.BY/SnJ mice. The highly protective role of NKG2D in myocarditis was further proven by in vivo neutralization of NKG2D as well as in NKG2D-deficient mice but was shown to be independent of CD8(+) T-cell-dependent immunity. Moreover, the adoptive transfer of immunocompetent C57BL/6 NK cells pre- (day -1) as well as post-infectionem (day +2) displayed the potential to prevent permissive A.BY/SnJ mice from a progressive outcome of CVB3 myocarditis reflected by significantly improved cardiopathology and heart function. Altogether, our results provide firm evidence for a protective role of NKG2D-activated NK cells in CVB3 myocarditis leading to an effective virus clearance, thus offering novel therapeutic options in the treatment of virus-induced myocarditis.
Asunto(s)
Cardiomiopatías/virología , Enterovirus , Células Asesinas Naturales/metabolismo , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Animales , Linfocitos T CD8-positivos/inmunología , Cardiomiopatías/inmunología , Cardiomiopatías/patología , Infecciones por Coxsackievirus/prevención & control , Enterovirus/inmunología , Inflamación/inmunología , Células Asesinas Naturales/inmunología , Masculino , Ratones Endogámicos C57BL , Miocarditis/etiología , Miocarditis/inmunología , Miocardio/patología , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunologíaRESUMEN
BACKGROUND: Heme oxygenase-1 (HO-1), which is suggested to play a role in defending the organism against oxidative stress-mediated injuries, can be induced by diverse factors including viruses and iron. As coxsackievirus B3 (CVB3)-infected SWR/J mice susceptible for chronic myocarditis were found to have a significant iron incorporation and HO-1 upregulation in the myocardium, we aimed to investigate the molecular interplay between HO-1 expression and iron homeostasis in the outcome of viral myocarditis. METHODS AND RESULTS: In susceptible SWR/J mice, but not in resistant C57BL/6 mice, we observed at later stages of CVB3 myocarditis significant iron deposits in macrophages and also in cardiomyocytes, which were spatially associated with oxidative stress, upregulation of HO-1 and caspase-3 activation. HO-1, which is also expressed in cultivated RAW 264.7 macrophages upon incubation with iron and/or CVB3, could be downregulated by inhibition of NO/iNOS using L-NAME. Moreover, specific inhibition of HO-1 by tin mesoporphyrin revealed a suppression of superoxide production in iron and/or CVB3-treated macrophages. The molecular relationship of HO-1 and caspase-3 activation was proven by downregulation with HO-1 siRNA in iron- and/or CVB3-treated cultivated cells. Importantly, iron was found to increase viral replication in vitro. CONCLUSION: These results indicate that HO-1 induces a paracrine signalling in macrophages via reactive oxygen species production, mediating apoptosis of heart muscle cells at later stages of myocarditis. Notably, in genetically susceptible mice iron potentiates the detrimental effects of CVB3 by the NO/HO-1 pathway, thus increasing cardiac pathogenicity.
Asunto(s)
Apoptosis , Infecciones por Coxsackievirus/enzimología , Enterovirus Humano B/fisiología , Hemo-Oxigenasa 1/metabolismo , Miocarditis/enzimología , Estrés Oxidativo , Animales , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Técnicas de Cocultivo , Infecciones por Coxsackievirus/patología , Activación Enzimática/efectos de los fármacos , Inducción Enzimática/efectos de los fármacos , Silenciador del Gen/efectos de los fármacos , Células HeLa , Hemo-Oxigenasa 1/genética , Humanos , Hierro/metabolismo , Hierro/farmacología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Miocarditis/patología , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Óxido Nítrico/biosíntesis , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Estrés Oxidativo/efectos de los fármacos , Células RAW 264.7 , ARN Mensajero/genética , ARN Mensajero/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Infections with coxsackieviruses of type B (CVBs), which are known to induce severe forms of acute and chronic myocarditis, are often accompanied by ventricular arrhythmias and sudden cardiac death. The mechanisms underlying the development of virus-induced, life-threatening arrhythmias, which are phenotypically similar to those observed in patients having functionally impaired cardiac ion channels, remain, however, enigmatic. In the present study, we show, for the first time, modulating time-dependent effects of CVB3 on the cardiac ion channels KCNQ1, hERG1, and Cav1.2 in heterologous expression. Channel protein abundance in cellular plasma membrane and patterns of their subcellular distribution were altered in infected murine hearts. The antiviral compound AG7088 did not prevent these effects on channels. In silico analyses of infected human myocytes suggest pronounced alterations of electrical and calcium signaling and increased risk of arrhythmogenesis. These modifications are attenuated by the common Asian polymorphism KCNQ1 P448R, a genetic determinant preventing coxsackievirus-induced effects in vitro. This study provides a previously unknown explanation for the development of arrhythmias in enteroviral myocarditis, which will help to develop therapeutic strategies for arrhythmia treatment.
Asunto(s)
Enterovirus Humano B/clasificación , Enterovirus Humano B/fisiología , Regulación de la Expresión Génica/fisiología , Canales Iónicos/metabolismo , Miocitos Cardíacos/metabolismo , Transporte de Proteínas/fisiología , Animales , Simulación por Computador , Células HEK293 , Humanos , Ratones , Modelos Biológicos , Miocitos Cardíacos/virología , Oocitos , Polimorfismo Genético , XenopusRESUMEN
Human cytomegalovirus (HCMV) remains the leading viral cause of birth defects and life-threatening disease in transplant recipients. All approved antiviral drugs target the viral DNA polymerase and are associated with severe toxicity issues and the emergence of drug resistance. Attempts to discover improved anti-HCMV drugs led to the identification of the small-molecular-weight compound AIC246 (Letermovir). AIC246 exhibits outstanding anti-HCMV activity in vitro and in vivo and currently is undergoing a clinical phase IIb trial. The initial mode-of-action studies suggested that the drug acts late in the HCMV replication cycle via a mechanism distinct from that of polymerase inhibitors. Here, we extend our mode-of-action analyses and report that AIC246 blocks viral replication without inhibiting the synthesis of progeny HCMV DNA or viral proteins. The genotyping of mutant viruses that escaped AIC246 inhibition uncovered distinct point mutations in the UL56 subunit of the viral terminase complex. Marker transfer analyses confirmed that these mutations were sufficient to mediate AIC246 resistance. The mapping of drug resistance to open reading frame UL56 suggests that viral DNA processing and/or packaging is targeted by AIC246. In line with this, we demonstrate that AIC246 affects the formation of proper unit-length genomes from viral DNA concatemers and interferes with virion maturation. However, since AIC246-resistant viruses do not exhibit cross-resistance to previously published terminase inhibitors, our data suggest that AIC246 interferes with HCMV DNA cleavage/packaging via a molecular mechanism that is distinct from that of other compound classes known to target the viral terminase.
Asunto(s)
Antivirales/farmacología , Citomegalovirus/efectos de los fármacos , Endodesoxirribonucleasas/metabolismo , Proteínas Estructurales Virales/metabolismo , Ensamble de Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Análisis Mutacional de ADN , ADN Viral/metabolismo , Farmacorresistencia Viral , Endodesoxirribonucleasas/genética , Humanos , Mutación Missense , Proteínas Estructurales Virales/genéticaRESUMEN
Enteroviruses, such as coxsackieviruses of group B (CVB), are able to induce a chronic inflammation of the myocardium, which may finally lead to the loss of functional tissue, remodeling processes and the development of fibrosis, thus affecting the proper contractile function of the heart. In other fibrotic diseases like scleroderma, the prostacyclin agonist iloprost was found to inhibit the extracellular signal-regulated kinase (ERK, p44/42 MAPK), a mitogen-activated protein kinase, and consecutively, the expression of the profibrotic cytokine connective tissue growth factor (CTGF), thereby preventing the development of fibrosis. As CTGF was found to mediate fibrosis in chronic CVB3 myocarditis as well, we evaluated whether the in vivo application of iloprost is capable to reduce the development of ERK/CTGF-mediated fibrosis in enteroviral myocarditis. Unexpectedly, the application of iloprost resulted in a prolonged myocardial inflammation and an aggravated fibrosis and failed to reduce activation of ERK and expression of CTGF at later stages of the disease. In addition, viral replication was found to be increased in iloprost-treated mice. Notably, the expression of cardiac inducible nitric oxide synthase (iNOS), which is known to aggravate myocardial damage in CVB3-infected mice, was strongly enhanced by iloprost. Using cultivated bone marrow macrophages (BMM), we confirmed these results, proving that iloprost potentiates the expression of iNOS mRNA and protein in CVB3-infected and IFN-gamma stimulated BMM. In conclusion, these results suggest a critical reflection of the clinical use of iloprost, especially in patients possibly suffering from an enteroviral myocarditis.
Asunto(s)
Enterovirus Humano B , Infecciones por Enterovirus/virología , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Iloprost/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Miocarditis/virología , Miocardio/patología , Óxido Nítrico Sintasa de Tipo II/genética , Replicación Viral/efectos de los fármacos , Animales , Colágeno Tipo I/genética , Cadena alfa 1 del Colágeno Tipo I , Factor de Crecimiento del Tejido Conjuntivo/genética , Infecciones por Enterovirus/metabolismo , Infecciones por Enterovirus/patología , Fibrosis , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Miocarditis/metabolismo , Miocarditis/patologíaRESUMEN
The glycoprotein gO (UL74) of human cytomegalovirus (HCMV) forms a complex with gH/gL. Virus mutants with a deletion of gO show a defect in secondary envelopment with the consequence that virus spread is restricted to a cell-associated pathway. Here we report that the positional homolog of HCMV gO, m74 of mouse CMV (MCMV), codes for a glycosylated protein which also forms a complex with gH (M75). m74 knockout mutants of MCMV show the same spread phenotype as gO knockout mutants of HCMV, namely, a shift from supernatant-driven to cell-associated spread. We could show that this phenotype is due to a reduction of infectious virus particles in cell culture supernatants. m74 knockout mutants enter fibroblasts via an energy-dependent and pH-sensitive pathway, whereas in the presence of an intact m74 gene product, entry is neither energy dependent nor pH sensitive. This entry phenotype is shared by HCMV expressing or lacking gO. Our data indicate that the m74 and UL74 gene products both codetermine CMV spread and CMV entry into cells. We postulate that MCMV, like HCMV, expresses alternative gH/gL complexes which govern cell-to-cell spread of the virus.
Asunto(s)
Glicoproteínas de Membrana/fisiología , Muromegalovirus/fisiología , Proteínas del Envoltorio Viral/fisiología , Internalización del Virus , Animales , Células Cultivadas , Medios de Cultivo , Citomegalovirus/genética , Citomegalovirus/fisiología , Fibroblastos/virología , Técnicas de Inactivación de Genes , Humanos , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Muromegalovirus/genética , Unión Proteica , Multimerización de Proteína , Proteínas del Envoltorio Viral/genética , Carga Viral , Ensayo de Placa ViralRESUMEN
The human cytomegalovirus (HCMV) glycoproteins gH (UL75) and gL (UL115) can form complexes with gO (UL74) or with proteins of the UL128-UL131A locus. Deletion of gO abolishes cell-free virus transmission and renders cell-associated virus transmission in fibroblasts more sensitive to inhibition by human anti-HCMV serum. To test whether the latter effect is specific for gO, we compared mutants with deletions in UL74, UL99 and the UL128-131A locus regarding their sensitivity to anti-HCMV antibodies. UL74 deletion mutants were more sensitive to a further restriction by polyspecific or gH-specific antibodies than control mutants, showing that gO specifically protects focal growth against inhibitory antibodies. This effect was not confined to gH-specific antibodies, as UL74 deletion mutants were also inhibited by an anti-gB antibody. In conclusion, gO specifically promotes focal spread in the presence of gH and gB antibodies, thus contributing to the ability of HCMV to resist the host's immune response.
Asunto(s)
Citomegalovirus/inmunología , Citomegalovirus/patogenicidad , Glicoproteínas de Membrana/inmunología , Proteínas del Envoltorio Viral/inmunología , Anticuerpos Antivirales , Especificidad de Anticuerpos , Secuencia de Bases , Células Cultivadas , Cromosomas Artificiales Bacterianos/genética , Citomegalovirus/genética , Citomegalovirus/ultraestructura , Infecciones por Citomegalovirus/inmunología , Infecciones por Citomegalovirus/virología , ADN Viral/genética , Fibroblastos/virología , Genes Virales , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Glicoproteínas de Membrana/genética , Microscopía Electrónica de Transmisión , Datos de Secuencia Molecular , Mutación , Eliminación de Secuencia , Proteínas del Envoltorio Viral/genética , Virulencia/genética , Virulencia/inmunologíaRESUMEN
The glycoprotein (g) complex gH/gL represents an essential part of the herpesvirus fusion machinery mediating entry of cell-free virions and cell-associated viral spread. In some herpesviruses additional proteins are associated with gH/gL contributing to the cell tropism of the respective virus. Human cytomegalovirus (HCMV) gH/gL forms complexes with either gO (UL74) or proteins of the UL128-131A gene locus. While a contribution of UL128-131A to endothelial cell tropism is known, the role of gO is less clear. We studied the role of gH/gL-associated proteins in HCMV replication in human foreskin fibroblasts (HFF) and human umbilical vein endothelial cells (HUVEC). Deletions of UL74 alone or in combination with mutations of the UL128-131A gene region were introduced into bacterial artificial chromosome vectors derived from the endotheliotropic strain TB40/E. Deletion of UL74 caused a profound defect regarding virus release from infected HFF and HUVEC. Large numbers of capsids accumulated in the cytoplasm of infected HFF but failed to acquire an envelope. Clear cell type differences were observed in the cell-associated spread of the UL74-defective virus. In HFF, focal growth was severely impaired, whereas it was normal in HUVEC. Deletion of UL131A abolished focal growth in endothelial cells. UL74/UL128-131A dual mutants showed severely impaired reconstitution efficiency. Our data suggest that gO plays a critical role in secondary envelopment and release of cell-free virions independent of the cell type but affects cell-associated growth specifically in HFF, whereas UL128-131A contributes to cell-associated spread in HFF and HUVEC.