RESUMEN
Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in infants, the elderly, and immune-compromised patients. It is also a significant contributor to upper respiratory tract infection in the pediatric population. However, its disease mechanisms are still largely unknown. We have recently shown that a tRNA-derived RNA fragment (tRF) from the 5'-end of mature tRNA encoding GluCTC (tRF5-GluCTC), a recently discovered non-coding RNA, is functionally important for RSV replication and host gene regulation at the post-transcriptional level. However, how tRF5-GluCTC carries out the gene regulation is not fully known. In this study, we found that tRF5-GluCTC has impaired gene trans-silencing function in cells deficient of AGO1 or 4, while AGO2 and 3 seem not involved in tRF5-GluCTC-mediated gene regulation. By pulling down individual AGO protein, we discovered that tRF5-GluCTC is detectable only in the AGO4 complex, confirming the essential role of AGO4 in gene regulation and also suggesting that AGO1 contributes to the gene trans-silencing activity of tRF5-GluCTC in an atypical way. We also found that the P protein of RSV is associated with both AGO1 and 4 and AGO4 deficiency leads to reduced infectious viral particles. In summary, this study demonstrates the importance of AGO1 and 4 in mediating the gene trans-silencing function of tRF5-GluCTC.
Asunto(s)
Proteínas Argonautas/genética , Factores Eucarióticos de Iniciación/genética , Silenciador del Gen , ARN de Transferencia/genética , ARN no Traducido/genética , Virus Sincitial Respiratorio Humano/genética , Células A549 , Proteínas Argonautas/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Genes Reporteros , Células HEK293 , Interacciones Huésped-Patógeno/genética , Humanos , Luciferasas/genética , Luciferasas/metabolismo , ARN de Transferencia/metabolismo , ARN no Traducido/metabolismo , Virus Sincitial Respiratorio Humano/metabolismo , Transducción de Señal , Carga Viral , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/metabolismo , Replicación ViralRESUMEN
Respiratory syncytial virus (RSV) is the leading cause of respiratory infection in young children and high-risk adults. However, a specific treatment for this viral infection is not currently available. In this study, we discovered that an exchange protein directly activated by cyclic AMP (EPAC) can serve as a potential therapeutic target for RSV. In both lower and upper epithelial cells, treatment with EPAC inhibitor (ESI-09), but not protein kinase A inhibitor (H89), significantly inhibits RSV replication and proinflammatory cytokine/chemokine induction. In addition, RSV-activated transcriptional factors belonging to the NF-κB and IRF families are also suppressed by ESI-09. Through isoform-specific gene knockdown, we found that EPAC2, but not EPAC1, plays a dominant role in controlling RSV replication and virus-induced host responses. Experiments using both EPAC2 knockout and EPAC2-specific inhibitor support such roles of EPAC2. Therefore, EPAC2 is a promising therapeutic target to regulate RSV replication and associated inflammation.IMPORTANCE RSV is a serious public health problem, as it is associated with bronchiolitis, pneumonia, and asthma exacerbations. Currently no effective treatment or vaccine is available, and many molecular mechanisms regarding RSV-induced lung disease are still significantly unknown. This project aims to elucidate an important and novel function of a protein, called EPAC2, in RSV replication and innate inflammatory responses. Our results should provide an important insight into the development of new pharmacologic strategies against RSV infection, thereby reducing RSV-associated morbidity and mortality.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/genética , Virus Sincitial Respiratorio Humano/crecimiento & desarrollo , Virus Sincitial Respiratorio Humano/genética , Replicación Viral/fisiología , Células A549 , Animales , Línea Celular , Quimiocinas/inmunología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Humanos , Hidrazonas/farmacología , Isoquinolinas/farmacología , Isoxazoles/farmacología , Ratones , FN-kappa B/antagonistas & inhibidores , Interferencia de ARN , ARN Interferente Pequeño/genética , Infecciones por Virus Sincitial Respiratorio/virología , Sulfonamidas/farmacologíaRESUMEN
Recently, linear ubiquitin assembly complex (LUBAC)-mediated linear ubiquitination has come into focus due to its emerging role in activation of NF-κB in different biological contexts. However, the role of LUBAC in LMP1 signaling leading to NF-κB and interferon regulatory factor 7 (IRF7) activation has not been investigated. We show here that RNF31, the key component of LUBAC, interacts with LMP1 and IRF7 in Epstein-Barr virus (EBV)-transformed cells and that LUBAC stimulates linear ubiquitination of NEMO and IRF7. Consequently, LUBAC is required for LMP1 signaling to full activation of NF-κB but inhibits LMP1-stimulated IRF7 transcriptional activity. The protein levels of RNF31 and LMP1 are correlated in EBV-transformed cells. Knockdown of RNF31 in EBV-transformed IB4 cells by RNA interference negatively regulates the expression of the genes downstream of LMP1 signaling and results in a decrease of cell proliferation. These lines of evidence indicate that LUBAC-mediated linear ubiquitination plays crucial roles in regulating LMP1 signaling and functions. IMPORTANCE: We show here that LUBAC-mediated linear ubiquitination is required for LMP1 activation of NF-κB but inhibits LMP1-mediated IRF7 activation. Our findings provide novel mechanisms underlying EBV-mediated oncogenesis and may have a broad impact on IRF7-mediated immune responses.
Asunto(s)
Factor 7 Regulador del Interferón/metabolismo , Complejos Multiproteicos/metabolismo , FN-kappa B/metabolismo , Ubiquitina/metabolismo , Proteínas de la Matriz Viral/metabolismo , Animales , Línea Celular Transformada , Transformación Celular Viral , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Herpesvirus Humano 4/fisiología , Humanos , Quinasa I-kappa B/metabolismo , Ratones , Unión Proteica , Transducción de Señal , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Latencia del VirusRESUMEN
Interferon (IFN) regulatory factor 7 (IRF7) plays a key role in the production of IFN-α in response to viral infection, and phosphorylation at IRF7 C-terminal serine sites is prelude to its function. However, phosphatases that negatively regulate IRF7 phosphorylation and activity have not been reported. In this study, we have identified a conserved protein phosphatase 1 (PP1)-binding motif in human and mouse IRF7 proteins, and shown that PP1 physically interacts with IRF7. Exogenous expression of PP1 subunits (PP1α, ß, or γ) ablates IKKε-stimulated IRF7 phosphorylation and dramatically attenuates IRF7 transcriptional activity. Inhibition of PP1 activity significantly increases IRF7 phosphorylation and IRF7-mediated IFN-α production in response to Newcastle disease virus (NDV) infection or Toll-like receptor 7 (TLR7) challenge, leading to impaired viral replication. In addition, IFN treatment, TLR challenges and viral infection induce PP1 expression. Our findings disclose for the first time a pivotal role for PP1 in impeding IRF7-mediated IFN-α production in host immune responses.
Asunto(s)
Enfermedad de Newcastle/inmunología , Virus de la Enfermedad de Newcastle/inmunología , Proteína Fosfatasa 1/metabolismo , Secuencias de Aminoácidos/genética , Animales , Células HEK293 , Humanos , Inmunidad/genética , Factor 7 Regulador del Interferón/metabolismo , Interferón-alfa/metabolismo , Ratones , Fosforilación/genética , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteína Fosfatasa 1/genética , Células RAW 264.7 , ARN Interferente Pequeño/genética , Activación Transcripcional/genética , Transgenes/genéticaRESUMEN
Human metapneumovirus (hMPV) is a common cause of respiratory tract infection in the paediatrics population. Recently, we and others have shown that retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLRs) are essential for hMPV-induced cellular antiviral signalling. However, the contribution of those receptors to host immunity against pulmonary hMPV infection is largely unexplored. In this study, mice deficient in mitochondrial antiviral-signalling protein (MAVS), an adaptor of RLRs, were used to investigate the role(s) of these receptors in pulmonary immune responses to hMPV infection. MAVS deletion significantly impaired the induction of antiviral and pro-inflammatory cytokines and the recruitment of immune cells to the bronchoalveolar lavage fluid by hMPV. Compared with WT mice, mice lacking MAVS demonstrated decreased abilities to activate pulmonary dendritic cells (DCs) and abnormal primary T-cell responses to hMPV infection. In addition, mice deficient in MAVS had a higher peak of viral load at day 5 post-infection (p.i.) than WT mice, but were able to clear hMPV by day 7 p.i. similarly to WT mice. Taken together, our data indicate a role of MAVS-mediated pathways in the pulmonary immune responses to hMPV infection and the early control of hMPV replication.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/inmunología , Metapneumovirus/inmunología , Infecciones por Paramyxoviridae/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Citocinas/genética , Citocinas/inmunología , Células Dendríticas/inmunología , Femenino , Humanos , Inmunidad Innata , Pulmón/inmunología , Pulmón/virología , Masculino , Metapneumovirus/genética , Ratones , Ratones Noqueados , Infecciones por Paramyxoviridae/genética , Infecciones por Paramyxoviridae/virología , Linfocitos T/inmunologíaRESUMEN
The discovery of small noncoding RNAs (sncRNAs) with regulatory functions is a recent breakthrough in biology. Among sncRNAs, microRNA (miRNA), derived from host or virus, has emerged as elements with high importance in control of viral replication and host responses. However, the expression pattern and functional aspects of other types of sncRNAs, following viral infection, are unexplored. In order to define expression patterns of sncRNAs, as well as to discover novel regulatory sncRNAs in response to viral infection, we applied deep sequencing to cells infected with human respiratory syncytial virus (RSV), the most common cause of bronchiolitis and pneumonia in babies. RSV infection leads to abundant production of transfer RNA (tRNA)-derived RNA Fragments (tRFs) that are ~30 nucleotides (nts) long and correspond to the 5'-half of mature tRNAs. At least one tRF, which is derived from tRNA-Glu-CTC, represses target mRNA in the cytoplasm and promotes RSV replication. This demonstrates that this tRF is not a random by-product of tRNA degradation but a functional molecule. The biogenesis of this tRF is also specific, as it is mediated by the endonuclease angiogenin (ANG), not by other nucleases. In summary, our study presents novel information on the induction of a functional tRF by viral infection.
Asunto(s)
Regulación Viral de la Expresión Génica , ARN Interferente Pequeño/aislamiento & purificación , ARN de Transferencia/aislamiento & purificación , Infecciones por Virus Sincitial Respiratorio/genética , Virus Sincitial Respiratorio Humano/genética , Técnicas Biosensibles , Northern Blotting , Bronquiolitis/genética , Bronquiolitis/virología , Diferenciación Celular , Línea Celular Tumoral , Mapeo Cromosómico , Citoplasma/metabolismo , Células Epiteliales/citología , Células Epiteliales/virología , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Luciferasas/genética , Luciferasas/metabolismo , MicroARNs/genética , MicroARNs/aislamiento & purificación , Plásmidos/genética , Biosíntesis de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/aislamiento & purificación , ARN de Transferencia/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Infecciones por Virus Sincitial Respiratorio/metabolismo , Virus Sincitial Respiratorio Humano/fisiología , Ribonucleasa Pancreática/genética , Ribonucleasa Pancreática/metabolismo , Replicación ViralRESUMEN
Human metapneumovirus (hMPV) is a leading cause of respiratory infections in pediatric populations globally, with no prophylactic or therapeutic measures. Recently, a recombinant hMPV lacking the M2-2 protein (rhMPV-ΔM2-2) demonstrated reduced replication in the respiratory tract of animal models, making it a promising live vaccine candidate. However, the exact nature of the interaction between the M2-2 protein and host cells that regulates viral infection/propagation is largely unknown. By taking advantage of the available reverse genetics system and ectopic expression system for viral protein, we found that M2-2 not only promotes viral gene transcription and replication but subverts host innate immunity, therefore identifying M2-2 as a novel virulence factor, in addition to the previously described hMPV G protein. Since we have shown that the RIG-I/MAVS pathway plays an important role in hMPV-induced signaling in airway epithelial cells, we investigated whether M2-2 antagonizes the host cellular responses by targeting this pathway. Reporter gene assays and coimmunoprecipitation studies indicated that M2-2 targets MAVS, an inhibitory mechanism different from what we previously reported for hMPV G, which affects RIG-I- but not MAVS-dependent gene transcription. In addition, we found that the domains of M2-2 responsible for the regulation of viral gene transcription and antiviral signaling are different. Our findings collectively demonstrate that M2-2 contributes to hMPV immune evasion through the inhibition of MAVS-dependent cellular responses.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Evasión Inmune/genética , Metapneumovirus/metabolismo , Transducción de Señal/fisiología , Transcripción Genética/fisiología , Proteínas Virales/metabolismo , Factores de Virulencia/metabolismo , Análisis de Varianza , Animales , Western Blotting , Chlorocebus aethiops , Cartilla de ADN/genética , Humanos , Inmunoprecipitación , Metapneumovirus/genética , Metapneumovirus/inmunología , Plásmidos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/genética , Células Vero , Proteínas Virales/genética , Factores de Virulencia/genéticaRESUMEN
Human respiratory syncytial virus (RSV), a leading cause of respiratory tract infections in infants, inhibits type I interferon (IFN)-dependent signalling, as well as IFN synthesis. RSV non-structural protein NS1 plays a significant role in this inhibition; however, the mechanism(s) responsible is not fully known. The transcription factor interferon regulatory factor (IRF)-3 is essential for viral-induced IFN-ß synthesis. In this study, we found that NS1 protein inhibits IRF-3-dependent gene transcription in constitutively active IRF-3 overexpressing cells, demonstrating that NS1 directly targets IRF-3. Our data also demonstrate that NS1 associates with IRF-3 and its transcriptional coactivator CBP, leading to disrupted association of IRF-3 to CBP and subsequent reduced binding of IRF-3 to the IFN-ß promoter without blocking viral-induced IRF-3 phosphorylation, nuclear translocation and dimerization, thereby identifying a novel molecular mechanism by which RSV inhibits IFN-ß synthesis.
Asunto(s)
Factor 3 Regulador del Interferón/antagonistas & inhibidores , Virus Sincitial Respiratorio Humano/inmunología , Proteínas no Estructurales Virales/metabolismo , Factores de Virulencia/metabolismo , Línea Celular , Células Epiteliales/virología , Humanos , Unión ProteicaRESUMEN
The pathogenesis of Japanese encephalitis virus (JEV) is not definitely elucidated as the initial interaction between virus and host cell receptors required for JEV infection is not clearly defined yet. Here, in order to discover those membrane proteins that may be involved in JEV attachment to or entry into virus permissive BHK-21 cells, a chemically mutated cell line (designated 3A10-3F) that became less susceptible to JEV infection was preliminarily established and selected by repeated low moi JEV challenges and RT-PCR detection for viral RNA E gene fragment. The susceptibility to JEV of 3A10-3F cells was significantly weakened compared with parental BHK-21 cells, verified by indirect immunofluorescence assay, virus plague formation assay, and flow cytometry. Finally, two-dimensional electrophoresis (2-DE) coupled with LC-MS/MS was utilized to recognize the most differentially expressed proteins from membrane protein extracts of 3A10-3F and BHK-21 cells respectively. The noted discrepancy of membrane proteins included calcium binding proteins (annexin A1, annexin A2), and voltage-dependent anion channels proteins (VDAC 1, VDAC 2), suggesting that these molecules may affect JEV attachment to and/or entry into BHK-21 cells and worthy of further investigation.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie)/fisiología , Encefalitis Japonesa/genética , Mutación , Animales , Línea Celular , Cricetinae , Virus de la Encefalitis Japonesa (Especie)/inmunología , Encefalitis Japonesa/inmunología , Encefalitis Japonesa/virología , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunologíaRESUMEN
Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infection in young children. It is also a significant contributor to upper respiratory tract infections, therefore, a major cause for visits to the pediatrician. High morbidity and mortality are associated with high-risk populations including premature infants, the elderly, and the immunocompromised. However, no effective and specific treatment is available. Recently, we discovered that an exchange protein directly activated by cyclic AMP 2 (EPAC2) can serve as a potential therapeutic target for RSV. In both lower and upper epithelial cells, EPAC2 promotes RSV replication and pro-inflammatory cytokine/chemokine induction. However, the overall role of EPAC2 in the pulmonary responses to RSV has not been investigated. Herein, we found that EPAC2-deficient mice (KO) or mice treated with an EPAC2-specific inhibitor showed a significant decrease in body weight loss, airway hyperresponsiveness, and pulmonary inflammation, compared with wild-type (WT) or vehicle-treated mice. Overall, this study demonstrates the critical contribution of the EPAC2-mediated pathway to airway diseases in experimental RSV infection, suggesting the possibility to target EPAC2 as a promising treatment modality for RSV.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/fisiología , Infecciones por Virus Sincitial Respiratorio/fisiopatología , Obstrucción de las Vías Aéreas/etiología , Animales , AMP Cíclico/fisiología , Citocinas/biosíntesis , Citocinas/genética , Factor Estimulante de Colonias de Granulocitos/biosíntesis , Factor Estimulante de Colonias de Granulocitos/genética , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Factores de Intercambio de Guanina Nucleótido/deficiencia , Inflamación , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Hipersensibilidad Respiratoria/etiología , Infecciones por Virus Sincitial Respiratorio/complicaciones , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Virus Sincitiales Respiratorios/fisiología , Organismos Libres de Patógenos Específicos , Replicación Viral , Pérdida de PesoRESUMEN
Chronic exposure to environmental heavy metals is a worldwide health concern. It is acknowledged to be an important cause of lower respiratory tract damage in children. However, the molecular mechanisms underlying the heavy metal-induced cellular stress/toxicity are not completely understood. Small non-coding RNAs (sncRNAs), such as microRNAs (miRNA) and more recently identified tRNA-derived RNA fragments (tRFs), are critical to the posttranscriptional control of genes. We used deep sequencing to investigate whether cellular sncRNA profiles are changed by environmental heavy metals. We found that the treatment of arsenite, an important groundwater heavy metal, leads to abundant production of tRFs, that are ~30 nucleotides (nts) long and most of which correspond to the 5'-end of mature tRNAs. It is unlikely for these tRFs to be random degradation by-products, as the type of induced tRFs is heavy metal-dependent. Three most inducible tRFs and their roles in arsenite-induced cellular responses were then investigated. We identified that p65, an important transcription factor belonging to NF-κB family and also a key factor controlling inflammatory gene expression, is a regulated target of a tRF derived from 5'-end of mature tRNA encoding AlaCGC (tRF5-AlaCGC). tRF5-AlaCGC activates p65, subsequently leading to enhanced secretion of IL-8 in arsenite response. In this study, we also identified that endonuclease Dicer and angiogenin temporally control the induction of tRF5-AlaCGC, providing an insight into the control of tRF biogenesis and subsequently the prevention of cellular damage.
Asunto(s)
Arsenitos/farmacología , ARN de Transferencia/metabolismo , Secuencia de Bases , Línea Celular , Humanos , Inflamación/patología , Metales Pesados/toxicidad , Virus Sincitiales Respiratorios/efectos de los fármacos , Virus Sincitiales Respiratorios/fisiología , Ribonucleasa III/metabolismo , Ribonucleasa Pancreática/metabolismoRESUMEN
Japanese encephalitis virus (JEV) is a member of mosquito-borne Flaviviridae. To date, the mechanisms of the early events of JEV infection remain poorly understood, and the cellular receptors are unidentified. There are evidences that the structure of the virus attachment proteins (VAP), envelope glycoprotein of mosquito-borne flaviviruses is very similar, and the vector-virus interaction of mosquito-borne flaviviruses is also very similar. Based on the studies previously demonstrated that the similar molecules present on the mosquito cells involved in the uptake process of JEV, West Nile virus (WNV) and Dengue virus (DV), it is proposed that the same receptor molecules for mosquito-borne flaviviruses (JEV, WNV and DV) may present on the surface of C6/36 mosquito cells. By co-immunoprecipitation assay, we investigated a 74-KDa protein on the C6/36 cells binds JEV, and the mass spectrometry results indicated it may be heat shock cognate protein 70(HSC70) from Aedes aegypti. Based upon some other viruses use of heat shock protein 70 (HSP70) family proteins as cell receptors, its possible HSC70's involvement in the fusion of the JEV E protein with the C6/36 cells membrane, and known form of cation channels in the interaction of HSC70 with the lipid bilayer, it will further be proposed that HSC70 as a penetration receptor mediates JEV entry into C6/36 cells.
Asunto(s)
Culicidae/virología , Virus del Dengue/fisiología , Virus de la Encefalitis Japonesa (Especie)/fisiología , Proteínas del Choque Térmico HSC70/aislamiento & purificación , Receptores Virales/aislamiento & purificación , Acoplamiento Viral , Virus del Nilo Occidental/fisiología , Animales , Línea Celular , Proteínas del Choque Térmico HSC70/química , Proteínas del Choque Térmico HSC70/metabolismo , Inmunoprecipitación , Espectrometría de Masas , Unión Proteica , Receptores Virales/química , Receptores Virales/metabolismo , Internalización del VirusRESUMEN
Human metapneumovirus (hMPV) is a leading cause of lower respiratory infection in young children, the elderly and immunocompromised patients. Repeated hMPV infections occur throughout life. However, immune evasion mechanisms of hMPV infection are largely unknown. Recently, our group has demonstrated that hMPV M2-2 protein, an important virulence factor, contributes to immune evasion in airway epithelial cells by targeting the mitochondrial antiviral-signaling protein (MAVS). Whether M2-2 regulates the innate immunity in human dendritic cells (DC), an important family of immune cells controlling antigen presenting, is currently unknown. We found that human DC infected with a virus lacking M2-2 protein expression (rhMPV-ΔM2-2) produced higher levels of cytokines, chemokines and IFNs, compared to cells infected with wild-type virus (rhMPV-WT), suggesting that M2-2 protein inhibits innate immunity in human DC. In parallel, we found that myeloid differentiation primary response gene 88 (MyD88), an essential adaptor for Toll-like receptors (TLRs), plays a critical role in inducing immune response of human DC, as downregulation of MyD88 by siRNA blocked the induction of immune regulatory molecules by hMPV. Since M2-2 is a cytoplasmic protein, we investigated whether M2-2 interferes with MyD88-mediated antiviral signaling. We found that indeed M2-2 protein associated with MyD88 and inhibited MyD88-dependent gene transcription. In this study, we also identified the domains of M2-2 responsible for its immune inhibitory function in human DC. In summary, our results demonstrate that M2-2 contributes to hMPV immune evasion by inhibiting MyD88-dependent cellular responses in human DC.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Inmunidad Innata , Metapneumovirus/genética , Metapneumovirus/inmunología , Proteínas Virales/genética , Animales , Línea Celular , Quimiocinas/biosíntesis , Cricetinae , Citocinas/biosíntesis , Expresión Génica , Humanos , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Unión Proteica , Transducción de Señal , Proteínas Virales/metabolismoRESUMEN
Human metapneumovirus (hMPV) is a recently identified RNA virus belonging to the Paramyxoviridae family. It is a common cause of respiratory tract infections in children, adults, and immunocompromised patients, for which no specific treatment or vaccine is available. Recent investigations in our lab identified hMPV glycoprotein G as an important virulence factor, as a recombinant virus lacking the G protein (rhMPV-ΔG) exhibited enhanced production of important immune and antiviral mediators, such as cytokines, chemokines and type I interferon (IFN) in airway epithelial cells, and expression of G protein alone inhibits cellular signaling dependent on retinoic induced gene (RIG)-I, a RNA helicase with a fundamental role in initiating hMPV-induced cellular responses. In this study, we have further investigated the mechanism underlying the inhibitory role of hMPV G protein on RIG-I-dependent signaling. We found that the interaction of hMPV G with RIG-I occurs primarily through the CARD domains of RIG-I N-terminus, preventing RIG-I association with the adaptor protein MAVS (mitochondrial antiviral signaling protein), recruitment of RIG-I to mitochondria, as well as the interaction between mitochondria and mitochondria-associated membrane (MAM) component of the endoplasmic reticulum (ER), which contains STINGS, an important part of the viral-induced RIG-I/MAVS signaling pathway, leading in the end to the inhibition of cytokine, chemokine and type I IFN expression. Mutagenesis analysis showed that hMPV G protein cytoplasmic domain played a major role in the observed inhibitory activity, and recombinant viruses expressing a G protein with amino acid substitution in position 2 and 3 recapitulated most of the phenotype observed with rhMPV-ΔG mutant upon infection of airway epithelial cells.
Asunto(s)
Glicoproteínas/metabolismo , Metapneumovirus/metabolismo , Mitocondrias/metabolismo , Mucosa Respiratoria/metabolismo , Transducción de Señal , Proteínas Virales/metabolismo , Línea Celular , Retículo Endoplásmico/metabolismo , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Metapneumovirus/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Receptores de Ácido Retinoico/metabolismo , Mucosa Respiratoria/virología , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Human metapneumovirus (hMPV) is a common cause of lung and airway infections in infants and young children. Recently, we and others have shown that hMPV infection induces Toll-like receptor (TLR)-dependent cellular signaling. However, the contribution of TLR-mediated signaling in host defenses against pulmonary hMPV infection and associated disease pathogenesis has not been elucidated. In this study, mice deficient in MyD88, a common adaptor of TLRs, was used to investigate the contribution of TLRs to in vivo pulmonary response to hMPV infection. MyD88(-/-) mice have significantly reduced pulmonary inflammation and associated disease compared with wild-type (WT) C57BL/6 mice after intranasal infection with hMPV. hMPV-induced cytokines and chemokines in bronchoalveolar lavage fluid (BALF) and isolated lung conventional dendritic cells (cDC) are also significantly impaired by MyD88 deletion. In addition, we found that MyD88 is required for the recruitment of DC, T cells, and other immune cells to the lungs, and for the functional regulation of DC and T cells in response to hMPV infection. Taken together, our data indicate that MyD88-mediated pathways are essential for the pulmonary immune and pathogenic responses to this viral pathogen.
Asunto(s)
Interacciones Huésped-Patógeno , Pulmón/patología , Metapneumovirus/fisiología , Factor 88 de Diferenciación Mieloide/metabolismo , Infecciones por Paramyxoviridae/patología , Animales , Líquido del Lavado Bronquioalveolar/química , Movimiento Celular , Citocinas/análisis , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Pulmón/inmunología , Pulmón/virología , Metapneumovirus/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/deficiencia , Infecciones por Paramyxoviridae/inmunología , Infecciones por Paramyxoviridae/virologíaRESUMEN
Human metapneumovirus (hMPV), a leading cause of respiratory tract infections in infants, inhibits type I interferon (IFN) signaling by an unidentified mechanism. In this study, we showed that infection of airway epithelial cells with hMPV decreased cellular level of Janus tyrosine kinase (Jak1) and tyrosine kinase 2 (Tyk2), due to enhanced proteosomal degradation and reduced gene transcription. In addition, hMPV infection also reduced the surface expression of type I IFN receptor (IFNAR). These inhibitory mechanisms are different from the ones employed by respiratory syncytial virus (RSV), which does not affect Jak1, Tyk2 or IFNAR expression, but degrades downstream signal transducer and activator of transcription proteins 2 (STAT2), although both viruses are pneumoviruses belonging to the Paramyxoviridae family. Our study identifies a novel mechanism by which hMPV inhibits STAT1 and 2 activation, ultimately leading to viral evasion of host IFN responses.
Asunto(s)
Regulación hacia Abajo , Células Epiteliales/virología , Interferón beta/metabolismo , Janus Quinasa 1/metabolismo , Metapneumovirus/fisiología , Transducción de Señal , TYK2 Quinasa/metabolismo , Células Epiteliales/citología , Células Epiteliales/enzimología , Células Epiteliales/metabolismo , Humanos , Interferón beta/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Subunidades de Proteína/metabolismo , Proteolisis , Receptor de Interferón alfa y beta/metabolismo , Factor de Transcripción STAT2/metabolismo , Transcripción Genética , Replicación ViralRESUMEN
Punta Toro virus (PTV; family Bunyaviridae, genus Phlebovirus) causes severe hepatic damage through brisk apoptosis of hepatocytes. In the present study, two viral proteins encoded by the S segment of the viral genome, non-structural (NSs) and nucleocapsid protein (N), were examined for their roles in apoptosis. Expression of NSs in HepG2 cells led to apoptosis in 45% of transfected cells, and with N, 28%, on average. These levels represent a four- to an eightfold increase over cells transfected with the mutated protein vectors. Caspase-3, -8 and -9 activities were increased by N protein when compared with the control NC (P < 0.05), and by NSsA and NSsB, as compared to control NSsC (P < 0.01). Treatment of the transfected cells with caspase-8 or -9 inhibitors markedly decreased apoptosis. Neutralization of TNF-alpha or Fas ligand had no effect on apoptosis. These results indicate that both NSs and N are responsible for causing hepatocyte apoptosis by triggering the extrinsic caspase-8 and intrinsic caspase-9 pathways.
Asunto(s)
Apoptosis/fisiología , Hepatocitos/citología , Proteínas de la Nucleocápside/fisiología , Phlebovirus/metabolismo , Proteínas no Estructurales Virales/fisiología , Animales , Apoptosis/genética , Western Blotting , Caspasa 8/metabolismo , Caspasa 9/metabolismo , Chlorocebus aethiops , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Etiquetado Corte-Fin in Situ , Proteínas de la Nucleocápside/genética , Phlebovirus/genética , Reacción en Cadena de la Polimerasa , Transducción de Señal/genética , Transducción de Señal/fisiología , Células Vero , Proteínas no Estructurales Virales/genéticaRESUMEN
West Nile virus (WNV) can cause encephalitis or meningitis that affects brain tissue, which can also lead to permanent neurological damage that can be fatal. To our knowledge, no consistent double immunohistochemical staining of neurons, neuroglia cells, and WNV has yet been reported. To establish a method for performing double-label immunohistochemical detection of neurons, neuroglia cells and WNV, examining the pathological characteristics of WNV-infected neurons, neuroglia cells, and investigating distribution of WNV in monkey brain, paraffin-embedded monkey brain tissue were retrospectively studied by immunohistochemical staining of neurons, neuroglia cells and WNV. Antibodies against neuron-specific enolase (NSE), glial fibrillary acidic protein (GFAP) and WNV were used to develop the method of double-label immunohistochemical staining, which allowed independent assessment of neuron status and WNV distribution. A range of immunohistochemical WNV infection in monkey brain was observed in both neurons and neuroglia cells in terms of the thickness of lesion staining, and the WNV staining was slightly higher in neuroglia cells than in neurons. All these findings suggest that WNV invasion in the brain plays a crucial role in neurological damage by inducing central nervous system (CNS) cell dysfunction or cell death directly.
Asunto(s)
Antígenos Virales/inmunología , Encéfalo/inmunología , Haplorrinos/inmunología , Neuroglía/inmunología , Neuronas/inmunología , Virus del Nilo Occidental/inmunología , Animales , Encéfalo/virología , InmunohistoquímicaRESUMEN
Hemorrhagic fever with renal syndrome (HFRS) is a fulminant infectious disease characterized by fever, hemorrhage, renal impairment, and thrombocytopenia. Hantaviruses associated with this belong to different serotypes: Hantaan (HTN), Seoul (SEO), Dobrava/Belgrade (DOB), and Puumala (PUU). The first two, HTN and SEO, are endemic in China. To investigate the epidemiology of HFRS and virus transmission in China, we constructed prokaryotic plasmids encoding truncated recombinant HTN and SEO nucleocapsid proteins (NPs), which lacked 154 amino acid (aa), 99 aa, or 49 aa in the N-terminal region, respectively. After expression, the truncated rNPs were tested as serotyping antigens, particularly for use in the enzyme-linked immunosorbent assay (ELISA). In addition, 68 acute and 52 convalescent sera were collected from HFRS patients from Harbin, Lantian, and Kaifeng regions in China in 2004, which had hantavirus specific antibodies by IFA. A neutralization test was used to differentiate these, which showed that 73 were due to HTN infection, 33 to SEO infection, and 14 undetermined. By ELISA, the truncated rNPs, that lacked 99 (rNP100) or 49 (rNP50) N-terminal amino acids of the NPs of HTN and SEO, were able to differentiate HTNV and SEOV-specific immune sera, but the rNP155 could not. Particularly, the ELISAs based on the rNP50s had a result comparable to PRNT. Thus, the rNP50 is recommended as efficient serotyping antigen for hantavirus infection diagnosis by ELISA.
Asunto(s)
Fiebre Hemorrágica con Síndrome Renal/virología , Proteínas de la Nucleocápside/inmunología , Orthohantavirus/clasificación , Orthohantavirus/genética , Serotipificación/métodos , Animales , Anticuerpos Antivirales , China/epidemiología , Ensayo de Inmunoadsorción Enzimática , Femenino , Virus Hantaan/clasificación , Virus Hantaan/genética , Virus Hantaan/inmunología , Orthohantavirus/inmunología , Fiebre Hemorrágica con Síndrome Renal/diagnóstico , Fiebre Hemorrágica con Síndrome Renal/epidemiología , Humanos , Ratones , Pruebas de Neutralización , Proteínas de la Nucleocápside/genética , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Virus Seoul/clasificación , Virus Seoul/genética , Virus Seoul/inmunologíaRESUMEN
AIM: To prepare monoclonal antibody (mAb) against human mu chain with high titer and establish a capture ELISA for early serological diagnosis of infectious diseases. METHODS: BALB/c mice were immunized with human IgM. Hybridoma cell line which could stably secret the mAb to human IgM was established by routine cell fusion technique. mAb's characteristics (titer, Ig subclass, specificity and relative affinity) were identified by indirect ELISA and Western blot, respectively. A capture ELISA was established by using purified mAb to capture specific IgM for early diagnosis of Japanese encephalitis. RESULTS: One hybridoma cell line 2E5 stably secreting mAb against human IgMmu chain was obtained. The titer of ascites of the mAb was 1 x 10(-6) and the Ig subclass was IgG1(kappa). Relative affinity of 2E5 was 1 x 10 (-5). Western blot analysis showed that mAb 2E5 reacted specifically to mu chain. Both sensitivity and specificity of the capture ELISA in detecting specific IgM in Japanese encephalitis patients sera were high. CONCLUSION: mAb 2E5 against human mu chain was prepared successfully, and a capture ELISA for early serological diagnosis of Japanese encephalitis was set up.