RESUMEN
Enterovirus A71 (EV-A71) belongs to the genus Enterovirus of the Picornaviridae family and often causes outbreaks in Asia. EV-A71 infection usually causes hand, foot, and mouth disease and can even affect the central nervous system, causing neurological complications or death. The 5'-untranslated region (5'-UTR) of EV-A71 contains an internal ribosome entry site (IRES) that is responsible for the translation of viral proteins. IRES-transacting factors can interact with the EV-A71 5'-UTR to regulate IRES activity. Heterogeneous nuclear ribonucleoprotein (hnRNP) A3 is a member of the hnRNP A/B protein family of RNA-binding proteins and is involved in RNA transport and modification. We found that hnRNP A3 knockdown promoted the replication of EV-A71 in neural calls. Conversely, increasing the expression of hnRNP A3 within cells inhibits the growth of EV-A71. HnRNP A3 can bind to the EV-A71 5'-UTR, and knockdown of hnRNP A3 enhances the luciferase activity of the EV-A71 5'-UTR IRES. The localization of hnRNP A3 shifts from the nucleus to the cytoplasm of infected cells during viral infection. Additionally, EV-A71 infection can increase the protein expression of hnRNP A3, and the protein level is correlated with efficient viral growth. Based on these findings, we concluded that hnRNP A3 plays a negative regulatory role in EV-A71 replication within neural cells.
RESUMEN
The pathogenic human enterovirus EV-A71 has raised serious public health concerns. A hallmark of EV-A71 infection is the distortion of host transcriptomes in favour of viral replication. While high-throughput approaches have been exploited to dissect these gene dysregulations, they do not fully capture molecular perturbations at the single-cell level and in a physiologically relevant context. In this study, we applied a single-cell RNA sequencing approach on infected differentiated enterocyte cells (C2BBe1), which model the gastrointestinal epithelium targeted initially by EV-A71. Our single-cell analysis of EV-A71-infected culture provided several lines of illuminating observations: 1) This systems approach demonstrated extensive cell-to-cell variation in a single culture upon viral infection and delineated transcriptomic differences between the EV-A71-infected and bystander cells. 2) By analysing expression profiles of known EV-A71 receptors and entry facilitation factors, we found that ANXA2 was closely correlated in expression with the viral RNA in the infected population, supporting its role in EV-A71 entry in the enteric cells. 3) We further catalogued dysregulated lncRNAs elicited by EV-A71 infection and demonstrated the functional implication of lncRNA CYTOR in promoting EV-A71 replication. Viewed together, our single-cell transcriptomic analysis illustrated at the single-cell resolution the heterogeneity of host susceptibility to EV-A71 and revealed the involvement of lncRNAs in host antiviral response.
Asunto(s)
Enterovirus Humano A/patogenicidad , Interacciones Huésped-Patógeno/genética , Transcriptoma , Células Cultivadas , Enterocitos/metabolismo , Enterocitos/patología , Enterocitos/virología , Enterovirus Humano A/genética , Enterovirus Humano A/inmunología , Infecciones por Enterovirus/genética , Infecciones por Enterovirus/inmunología , Infecciones por Enterovirus/patología , Infecciones por Enterovirus/virología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Interacciones Huésped-Patógeno/inmunología , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Mucosa Intestinal/virología , ARN Largo no Codificante/genética , Análisis de la Célula Individual , Replicación Viral/genéticaRESUMEN
BACKGROUND: Enterovirus A71 (EV-A71) has been noted for its tendency to lead to neurological manifestations in young children and infants. Although the alimentary tract has been identified as the primary replication site of this virus, how EV-A71 replicates in the gut and is transmitted to other organs remains unclear. METHODS: By using differentiated C2BBe1 cells as a model, we observed that intestinal epithelial cells (IECs) were permissive to EV-A71 infection, and viral particles were released in a nonlytic manner. RESULTS: The coexistence of active caspase 3 and EV-A71 protein was observed in the infected undifferentiated C2BBe1 and RD cells but not in the infected differentiated C2BBe1 cells. Furthermore, EV-A71 infection caused differentiated C2BBe1 and intestinal organoids to secrete exosomes containing viral components and have the ability to establish active infection. Inhibition of the exosome pathway decreased EV-A71 replication and release in IECs and increased the survival rates of infected animals. CONCLUSIONS: Our findings showed that EV-A71 is able to be actively replicated in enterocytes, and that the exosome pathway is involved in the nonlytic release of viral particles, which may be useful for developing antiviral strategies.
Asunto(s)
Enterovirus Humano A/fisiología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Exosomas/metabolismo , Animales , Diferenciación Celular , Enterovirus , Enterovirus Humano A/genética , Enterovirus Humano A/crecimiento & desarrollo , Infecciones por Enterovirus/virología , Humanos , Ratones Transgénicos , ARN Viral/genética , ARN Viral/metabolismo , Replicación ViralRESUMEN
Enterovirus 71 (EV71), a single-stranded RNA virus, is one of the most serious neurotropic pathogens in the Asia-Pacific region. Through interactions with host proteins, the 5' untranslated region (5'UTR) of EV71 is important for viral replication. To gain a protein profile that interact with the EV71 5'UTR in neuronal cells, we performed a biotinylated RNA-protein pull-down assay in conjunction with LC-MS/MS analysis. A total of 109 proteins were detected and subjected to Database for Annotation, Visualization and Integrated Discovery (DAVID) analyses. These proteins were found to be highly correlated with biological processes including RNA processing/splicing, epidermal cell differentiation, and protein folding. A protein-protein interaction network was constructed using the STRING online database to illustrate the interactions of those proteins that are mainly involved in RNA processing/splicing or protein folding. Moreover, we confirmed that the far-upstream element binding protein 3 (FBP3) was able to bind to the EV71 5'UTR. The redistribution of FBP3 in subcellular compartments was observed after EV71 infection, and the decreased expression of FBP3 in host neuronal cells markedly inhibited viral replication. Our results reveal various host proteins that potentially interact with the EV71 5'UTR in neuronal cells, and we found that FBP3 could serve as a positive regulator in host cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Enterovirus Humano A/fisiología , Infecciones por Enterovirus/metabolismo , Neuronas/virología , ARN Viral/metabolismo , Factores de Transcripción/metabolismo , Replicación Viral , Regiones no Traducidas 5' , Línea Celular , Enterovirus Humano A/genética , Infecciones por Enterovirus/patología , Humanos , Neuronas/metabolismo , Neuronas/patología , ARN Viral/genéticaRESUMEN
Enterovirus 71 (EV71) is associated with severe neurological disorders in children, and has been implicated as the infectious agent in several large-scale outbreaks with mortalities. Upon infection, the viral RNA is translated in a cap-independent manner to yield a large polyprotein precursor. This mechanism relies on the presence of an internal ribosome entry site (IRES) element within the 5'-untranslated region. Virus-host interactions in EV71-infected cells are crucial in assisting this process. We identified a novel positive IRES trans-acting factor, far upstream element binding protein 1 (FBP1). Using binding assays, we mapped the RNA determinants within the EV71 IRES responsible for FBP1 binding and mapped the protein domains involved in this interaction. We also demonstrated that during EV71 infection, the nuclear protein FBP1 is enriched in cytoplasm where viral replication occurs. Moreover, we showed that FBP1 acts as a positive regulator of EV71 replication by competing with negative ITAF for EV71 IRES binding. These new findings may provide a route to new anti-viral therapy.
Asunto(s)
Regiones no Traducidas 5' , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Enterovirus Humano A/genética , Biosíntesis de Proteínas , Proteínas de Unión al ARN/metabolismo , Animales , Unión Competitiva , Línea Celular , Citoplasma/metabolismo , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/química , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Enterovirus Humano A/crecimiento & desarrollo , Enterovirus Humano A/fisiología , Técnicas de Silenciamiento del Gen , Humanos , Dominios y Motivos de Interacción de Proteínas , ARN Viral/química , ARN Viral/metabolismo , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/química , Transactivadores/metabolismo , Replicación ViralRESUMEN
Mesenchymal stem cells (MSCs) comprise a primitive cell population and reside in various tissues and organs. These cells exhibit immunomodulatory activity and are effective in treating respiratory viral infections. The activation of type I and III interferons, which protect cells against viral infections, can be induced after pattern recognition receptors (PRRs) recognize viral nucleic acid species. Although certain viruses can upregulate IFN-ß expression in MSCs, the underlying mechanisms and responsiveness to different IFNs are unclear. We found that foreskin-derived fibroblast-like stromal cells (FDSCs), a kind of functional MSC, were permissive to IAV PR8, HCoV-229E, and EV-D68. Infection by IAV PR8 and HCoV-229E increased the expression of IFN-ß and IFN-λ species in FDSCs in an IRF-3-dependent manner. RIG-I was critical for detecting IAV PR8 in FDSCs, and IAV PR8 infection induced a significant increase in the expression of interferon signaling genes (ISGs). Interestingly, only IFN-ß, but not IFN-λ species, could induce the expression of ISGs, a finding supported by our observation that only IFN-ß induced STAT1 and STAT2 phosphorylation in FDSCs. We also proved that treatment with IFN-ß suppressed the propagation of IAV PR8 and promoted the survival of virus-infected FDSCs. Respiratory viruses could infect FDSCs and induce the expression of IFN-ß and IFN-λ1, but only IFN-ß could protect FDSCs against viral infection.
Asunto(s)
Células Madre Mesenquimatosas , Virosis , Virus , Humanos , Interferones/genética , Interferones/metabolismo , Interferón lambda , Transducción de Señal , Virus/metabolismo , Células Madre Mesenquimatosas/metabolismo , Factor 3 Regulador del Interferón/metabolismoRESUMEN
Enterovirus A71 (EV-A71) is an emerging enterovirus that can cause neurological complications. Enhanced serum IL-1ß levels were observed in EV-A71 patients with severe neurological symptoms. However, the roles of sensors in enterovirus-induced IL-1ß production are unclear. In this study, we identified that pattern recognition receptors, including RIG-I, TLR3, and TLR8, are implicated in EV-A71-triggered IL-1ß release in human macrophages. EV-A71 infection results in caspase-1 and caspase-8, which act as regulators of EV-A71-induced NLRP3 and RIG-I inflammasome activation. Moreover, knockdown of the expression of TLR3 and TLR8 decreased the released IL-1ß in an NLRP3-dependent manner. Since TLR3 and TLR8 ligands promote NLRP3 inflammasome activation via caspase-8, the alternative pathway may be involved. In summary, these results indicate that activation of the NLRP3 and RIG-I inflammasomes in EV-A71-infected macrophages is mediated by caspase-1 and caspase-8 and affected by TLRs, including TLR3 and TLR8.
Asunto(s)
Infecciones por Enterovirus , Enterovirus , Humanos , Antígenos Virales , Caspasa 1 , Caspasa 8 , Inflamasomas , Macrófagos , Receptor Toll-Like 3RESUMEN
Enterovirus A71 (EV-A71), which belongs to the family Picornaviridae, can invade the central nervous system (CNS) and cause severe CNS complications or death. The EV-A71 antigen has been detected in the neurons in the brains of humans who died from EV-A71 infection. However, the effect of EV-A71 infection on human neuronal cells remains poorly understood. Human neural stem cells (NSCs) and IMR-32 neuroblastoma cells were differentiated into neuronal cells for this study. Although the neuronal cells were permissive to EV-A71 infection, EV-A71 infection did not induce an obvious cytopathic effect on the neuronal cells. EV-A71 infection did not induce apoptosis in neuronal cells. However, autophagy and autophagic flux were induced in EV-A71-infected neuronal cells. The production of autophagosomes was shown to be important for EV-A71 viral RNA (vRNA) replication in neuronal cells.
Asunto(s)
Enterovirus Humano A/fisiología , Enterovirus Humano A/patogenicidad , Neuronas/virología , Autofagosomas/virología , Autofagia/fisiología , Caspasas/metabolismo , Diferenciación Celular , Células Cultivadas , Efecto Citopatogénico Viral/fisiología , Enterovirus Humano A/genética , Activación Enzimática , Interacciones Microbiota-Huesped/fisiología , Humanos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Células-Madre Neurales/virología , Neuronas/metabolismo , Neuronas/patología , ARN Viral/biosíntesis , ARN Viral/genética , Replicación Viral/fisiologíaRESUMEN
BACKGROUND: Neural stem cells (NSCs) residing in the central nervous system play an important role in neurogenesis. Several viruses can infect these neural progenitors and cause severe neurological diseases. The innate immune responses against the neurotropic viruses in these tissue-specific stem cells remain unclear. METHODS: Human NSCs were transfected with viral RNA mimics or infected with neurotropic virus for detecting the expression of antiviral interferons (IFNs) and downstream IFN-stimulated antiviral genes. RESULTS: NSCs are able to produce interferon-ß (IFN-ß) (type I) and λ1 (type III) after transfection with poly(I:C) and that downstream IFN-stimulated antiviral genes, such as ISG56 and MxA, and the viral RNA sensors RIG-I, MDA5, and TLR3, can be expressed in NSCs under poly(I:C) or IFN-ß stimulation. In addition, our results show that the pattern recognition receptors RIG-I and MDA5, as well as the endosomal pathogen recognition receptor TLR3, but not TLR7 and TLR8, are involved in the activation of IFN-ß transcription in NSCs. Furthermore, NSCs infected with the neurotropic viruses, Zika and Japanese encephalitis viruses, are able to induce RIG-I-mediated IFN-ß expression. CONCLUSION: Human NSCs have the ability to activate IFN signals against neurotropic viral pathogens.
Asunto(s)
Interferón Tipo I/inmunología , Células-Madre Neurales/inmunología , Células-Madre Neurales/virología , Infección por el Virus Zika/inmunología , Línea Celular , Células Cultivadas , Proteína 58 DEAD Box/genética , Proteína 58 DEAD Box/inmunología , Virus de la Encefalitis Japonesa (Subgrupo)/inmunología , Encefalitis Japonesa/genética , Encefalitis Japonesa/inmunología , Humanos , Inmunidad Innata , Interferón Tipo I/biosíntesis , Interferón beta/biosíntesis , Interferón beta/genética , Interferón beta/inmunología , Interferones/genética , Interferones/inmunología , Células-Madre Neurales/patología , Receptores Inmunológicos , Transcripción Genética , Transfección , Virus Zika/inmunología , Infección por el Virus Zika/genética , Infección por el Virus Zika/patología , Interferón lambdaRESUMEN
Enterovirus 71 (EV71) can invade the central nervous system (CNS) and cause neurological disease. Accumulating evidence indicates that EV71 can directly infect neurons in the CNS. Innate immune responses in the CNS have been known to play an essential role in limiting pathogen infections. Thus, investigating the effects of EV71 infection of neural cells is important for understanding disease pathogenesis. In this study, human neural cells were infected with EV71, and interferonß (IFNß) expression was examined. Our results show that IFNß expression was upregulated in EV71-infected neural cells via pattern recognition receptors (PRRs) sensing of virus RNA. The PRRs Toll-like receptor 3 (TLR3), Toll-like receptor 8 (TLR8), and melanoma differentiation-associated gene-5 (MDA-5), but not retinoic acid-inducible gene-I (RIG-I) and Toll-like receptor 7 (TLR7), were found to be EV71-mediated IFNß induction. Although viral proteins exhibited the ability to cleave mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-ß (TRIF) in neural cells, levels of viral protein expression were low in these cells. Furthermore, neural cells efficiently produced IFNß transcripts upon EV71 vRNA stimulation. Treating infected cells with anti-IFNß antibodies resulted in increased virus replication, indicating that IFNß release may play a role in limiting viral growth. These results indicate that EV71 infection can induce IFNß expression in neural cells through PRR pathways.
Asunto(s)
Enterovirus Humano A/fisiología , Infecciones por Enterovirus/genética , Infecciones por Enterovirus/virología , Expresión Génica , Interferón beta/genética , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/virología , Línea Celular , Infecciones por Enterovirus/metabolismo , Humanos , Interferón beta/metabolismo , Enfermedades del Sistema Nervioso/metabolismo , Neuronas/metabolismo , Fosforilación , ARN Interferente Pequeño/genética , Receptores Toll-Like/metabolismo , Replicación ViralRESUMEN
EV71 is a positive-sense single-stranded RNA virus that belongs to the Picornaviridae family. EV71 infection may cause various symptoms ranging from hand-foot-and-mouth disease to neurological pathological conditions such as aseptic meningitis, ataxia, and acute transverse myelitis. There is currently no effective treatment or vaccine available. Various compounds have been examined for their ability to restrict EV71 replication. However, most experiments have been performed in rhabdomyosarcoma or Vero cells. Since the gastrointestinal tract is the entry site for this pathogen, we anticipated that orally ingested agents may exert beneficial effects by decreasing virus replication in intestinal epithelial cells. In this study, curcumin (diferuloylmethane, C21H20O6), an active ingredient of turmeric (Curcuma longa Linn) with anti-cancer properties, was investigated for its anti-enterovirus activity. We demonstrate that curcumin treatment inhibits viral translation and increases host cell viability. Curcumin does not exert its anti-EV71 effects by modulating virus attachment or virus internal ribosome entry site (IRES) activity. Furthermore, curcumin-mediated regulation of mitogen-activated protein kinase (MAPK) signaling pathways is not involved. We found that protein kinase C delta (PKCδ) plays a role in virus translation in EV71-infected intestinal epithelial cells and that curcumin treatment decreases the phosphorylation of this enzyme. In addition, we show evidence that curcumin also limits viral translation in differentiated human intestinal epithelial cells. In summary, our data demonstrate the anti-EV71 properties of curcumin, suggesting that ingestion of this phytochemical may protect against enteroviral infections.
Asunto(s)
Curcumina/metabolismo , Curcumina/uso terapéutico , Infecciones por Enterovirus/tratamiento farmacológico , Línea Celular , Curcumina/farmacología , Enterovirus/efectos de los fármacos , Enterovirus Humano A/genética , Infecciones por Enterovirus/virología , Células Epiteliales/efectos de los fármacos , Células HT29 , Enfermedad de Boca, Mano y Pie/metabolismo , Interacciones Huésped-Patógeno , Humanos , Sitios Internos de Entrada al Ribosoma , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Intestinos/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , ARN Viral/genética , Proteínas Virales/genética , Replicación Viral/efectos de los fármacosRESUMEN
Neural progenitor cells (NPCs) are stem cells that can differentiate into various neural lineage cells. The damage and loss of NPCs are associated with neurological conditions such as cognitive deficits and memory impairment. In a long-term study of patients with EV71, cognitive disorders were observed. Therefore, we hypothesized that NPCs may be permissive to EV71 infection. We demonstrated that NPCs are prone to EV71 infection and that these stem cells can support the active replication of this virus. Furthermore, EV71 infection triggers apoptosis, resulting in significant cell death in infected NPCs. However, EV71 did not replicate in the differentiated cell types that were tested. Our findings suggest that EV71 can infect NPCs and cause the depletion of these cells.