RESUMEN
SARS coronavirus (SARS-CoV) papain-like protease (PLpro) has been identified in TGF-ß1 up-regulation in human promonocytes (Proteomics 2012, 12: 3193-205). This study investigates the mechanisms of SARS-CoV PLpro-induced TGF-ß1 promoter activation in human lung epithelial cells and mouse models. SARS-CoV PLpro dose- and time-dependently up-regulates TGF-ß1 and vimentin in A549 cells. Dual luciferase reporter assays with TGF-ß1 promoter plasmids indicated that TGF-ß1 promoter region between -175 to -60, the Egr-1 binding site, was responsible for TGF-ß1 promoter activation induced by SARS-CoV PLpro. Subcellular localization analysis of transcription factors showed PLpro triggering nuclear translocation of Egr-1, but not NF-κB and Sp-1. Meanwhile, Egr-1 silencing by siRNA significantly reduced PLpro-induced up-regulation of TGF-ß1, TSP-1 and pro-fibrotic genes. Furthermore, the inhibitors for ROS (YCG063), p38 MAPK (SB203580), and STAT3 (Stattic) revealed ROS/p38 MAPK/STAT3 pathway involving in Egr-1 dependent activation of TGF-ß1 promoter induced by PLpro. In a mouse model with a direct pulmonary injection, PLpro stimulated macrophage infiltration into lung, up-regulating Egr-1, TSP-1, TGF-ß1 and vimentin expression in lung tissues. The results revealed that SARS-CoV PLpro significantly triggered Egr-1 dependent activation of TGF-ß1 promoter via ROS/p38 MAPK/STAT3 pathway, correlating with up-regulation of pro-fibrotic responses in vitro and in vivo.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Papaína/farmacología , Especies Reactivas de Oxígeno/metabolismo , Factor de Transcripción STAT3/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/enzimología , Factor de Crecimiento Transformador beta1/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Células A549 , Animales , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Fibrosis , Silenciador del Gen/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Humanos , Pulmón/citología , Ratones Endogámicos BALB C , Modelos Biológicos , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Transcripción Sp1/metabolismo , Trombospondina 1/metabolismo , Factor de Crecimiento Transformador beta1/biosíntesis , Factor de Crecimiento Transformador beta1/genética , Vimentina/metabolismoRESUMEN
Japanese encephalitis virus (JEV) nonstructural protein 5 (NS5) exhibits a Type I interferon (IFN) antagonistic function. This study characterizes Type I IFN antagonism mechanism of NS5 protein, using proteomic approach. In human neuroblastoma cells, NS5 expression would suppress IFNß-induced responses, for example, expression of IFN-stimulated genes PKR and OAS as well as STAT1 nuclear translocation and phosphorylation. Proteomic analysis showed JEV NS5 downregulating calreticulin, while upregulating cyclophilin A, HSP 60 and stress-induced-phosphoprotein 1. Gene silence of calreticulin raised intracellular Ca(2+) levels while inhibiting nuclear translocalization of STAT1 and NFAT-1 in response to IFNß, thus, indicating calreticulin downregulation linked with Type I IFN antagonism of JEV NS5 via activation of Ca(2+) /calicineurin. Calcineurin inhibitor cyclosporin A attenuated NS5-mediated inhibition of IFNß-induced responses, for example, IFN-sensitive response element driven luciferase, STAT1-dependent PKR mRNA expression, as well as phosphorylation and nuclear translocation of STAT1. Transfection with calcineurin (vs. control) siRNA enhanced nuclear translocalization of STAT1 and upregulated PKR expression in NS5-expressing cells in response to IFNß. Results prove Ca(2+) , calreticulin, and calcineurin involvement in STAT1-mediated signaling as well as a key role of JEV NS5 in Type I IFN antagonism. This study offers insights into the molecular mechanism of Type I interferon antagonism by JEV NS5.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie)/fisiología , Interferón beta/antagonistas & inhibidores , Proteoma/metabolismo , Proteínas no Estructurales Virales/fisiología , Secuencia de Aminoácidos , Animales , Calcineurina/metabolismo , Inhibidores de la Calcineurina , Línea Celular , Línea Celular Tumoral , Cricetinae , Ciclosporina/farmacología , Regulación de la Expresión Génica/inmunología , Interacciones Huésped-Patógeno , Humanos , Inmunosupresores/farmacología , Interferón beta/metabolismo , Fosforilación , Procesamiento Proteico-Postraduccional , Proteoma/genética , Elementos de Respuesta , Factor de Transcripción STAT1/metabolismo , Espectrometría de Masas en Tándem , Replicación Viral/efectos de los fármacosRESUMEN
Severe acute respiratory syndrome coronavirus (SARS-CoV) papain-like protease (PLpro), a deubiquitinating enzyme, reportedly blocks poly Iâ:âC-induced activation of interferon regulatory factor 3 and nuclear factor kappa B, reducing interferon (IFN) induction. This study investigated type I IFN antagonist mechanism of PLpro in human promonocytes. PLpro antagonized IFN-α-induced responses such as interferon-stimulated response element- and AP-1-driven promoter activation, protein kinase R, 2'-5'-oligoadenylate synthetase (OAS), interleukin (IL)-6 and IL-8 expression, and signal transducers and activators of transcription (STAT) 1 (Tyr701), STAT1 (Ser727) and c-Jun phosphorylation. A proteomics approach demonstrated downregulation of extracellular signal-regulated kinase (ERK) 1 and upregulation of ubiquitin-conjugating enzyme (UBC) E2-25k as inhibitory mechanism of PLpro on IFN-α-induced responses. IFN-α treatment significantly induced mRNA expression of UBC E2-25k, but not ERK1, causing time-dependent decrease of ERK1, but not ERK2, in PLpro-expressing cells. Poly-ubiquitination of ERK1 showed a relationship between ERK1 and ubiquitin proteasome signalling pathways associated with IFN antagonism by PLpro. Combination treatment of IFN-α and the proteasome inhibitor MG-132 showed a time-dependent restoration of ERK1 protein levels and significant increase of ERK1, STAT1 and c-Jun phosphorylation in PLpro-expressing cells. Importantly, PD098059 (an ERK1/2 inhibitor) treatment significantly reduced IFN-α-induced ERK1 and STAT1 phosphorylation, inhibiting IFN-α-induced expression of 2'-5'-OAS in vector control cells and PLpro-expressing cells. Overall results proved downregulation of ERK1 by ubiquitin proteasomes and suppression of interaction between ERK1 and STAT1 as type I IFN antagonist function of SARS-CoV PLpro.
Asunto(s)
Cisteína Endopeptidasas/metabolismo , Regulación hacia Abajo , Interferón-alfa/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Transducción de Señal , Proteínas Virales/metabolismo , Línea Celular , Proteasas 3C de Coronavirus , Humanos , Interferón-alfa/inmunología , Monocitos/virología , Proteoma/análisis , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidadRESUMEN
In this study, aloe-emodin was identified as a potential interferon (IFN)-inducer by screening compounds from Chinese herbal medicine. Aloe-emodin showed low cytotoxicity to human HL-CZ promonocyte cells and TE-671 medulloblastoma cells and significantly activated interferon-stimulated response element (ISRE) and gamma-activated sequence (GAS)-driven cis-reporting systems. Moreover, aloe-emodin upregulated expression of IFN-stimulated genes such as dsRNA-activated protein kinase and 2',5'-oligoisoadenylate synthase. Aloe-emodin resulted in significant activation of nitric oxide production. The antiviral activity of aloe-emodin against Japanese encephalitis virus (JEV) and enterovirus 71 (EV71) was evaluated using dose- and time-dependent plaque reduction assays in HL-CZ cells and TE-671 cells. The 50% inhibitory concentration (IC(50)) of aloe-emodin ranged from 0.50microg/mL to 1.51microg/mL for JEV and from 0.14microg/mL to 0.52microg/mL for EV71. Aloe-emodin showed clearly potent virus inhibitory abilities and achieved high therapeutic indices, in particular for HL-CZ cells. Therefore, the study demonstrated dose- and time-dependent actions of aloe-emodin on the inhibition of JEV and EV71 replication via IFN signalling responses.
Asunto(s)
Antraquinonas/farmacología , Antivirales/farmacología , Virus de la Encefalitis Japonesa (Especie)/efectos de los fármacos , Enterovirus Humano A/efectos de los fármacos , Interferón-alfa/metabolismo , Medicina Tradicional China , Aloe/química , Animales , Línea Celular , Cricetinae , Humanos , Interferón-alfa/efectos de los fármacos , Transducción de Señal , Ensayo de Placa Viral , Replicación Viral/efectos de los fármacosRESUMEN
The interferon (IFN) antagonists of Japanese encephalitis virus (JEV) proteins contribute to the JE pathogenesis. Most flavivirus non-structural (NS) proteins correlate with virus-induced inflammation and immune escape. NS4A proteins of West Nile virus and dengue type 2 virus have been demonstrated to inhibit IFN signaling. In this study, JEV NS4A without the C-terminal 2K domain has been demonstrated to partially block activation of an IFN-stimulated response element (ISRE)-based cis-reporter by IFN-alpha/beta. In addition, JEV NS4A significantly inhibited the phosphorylation levels of STAT1 and STAT2, but not TYK2 in the IFN-treated cells. Moreover, the N-terminus of a RNA helicase DDX42 protein identified using a phage display human brain cDNA library have been demonstrated to specifically bind to JEV NS4A in vitro using a co-immunoprecipitation assay. The interaction between JEV NS4A and RNA helicase DDX42 showed partial co-localization in human medulloblastoma TE-671 cells by confocal microscopy. Importantly, the expression of N-terminal DDX42 is able to overcome JEV-induced antagonism of IFN responses. Therefore, these results show that JEV NS4A without the C-terminal 2K domain is associated with modulation of the IFN response and the interaction of JEV NS4A with RNA helicase DDX42 could be important for JE pathogenesis.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Virus de la Encefalitis Japonesa (Especie)/metabolismo , Encefalitis Japonesa/virología , Interferón Tipo I/antagonistas & inhibidores , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Proteínas Virales/metabolismo , Animales , Línea Celular Tumoral , Chlorocebus aethiops , Cricetinae , Virus de la Encefalitis Japonesa (Especie)/genética , Regulación Viral de la Expresión Génica , Biblioteca de Genes , Proteínas Fluorescentes Verdes/genética , Humanos , Interferón Tipo I/metabolismo , Quinasas Janus/metabolismo , Proteínas Luminiscentes/genética , Microscopía Confocal , Fosforilación , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción STAT/metabolismo , Transducción de Señal , Células Vero , Proteínas Virales/genética , Proteína Fluorescente RojaRESUMEN
The pathogenesis of severe acute respiratory syndrome coronavirus (SARS CoV) is an important issue for treatment and prevention of SARS. Previously, SARS CoV 3C-like protease (3CLpro) has been demonstrated to induce apoptosis via the activation of caspase-3 and caspase-9 (Lin, C. W., Lin, K. H., Hsieh, T. H., Shiu, S. Y. et al., FEMS Immunol. Med. Microbiol. 2006, 46, 375-380). In this study, proteome analysis of the human promonocyte HL-CZ cells expressing SARS CoV 3CLpro was performed using 2-DE and nanoscale capillary LC/ESI quadrupole-TOF MS. Functional classification of identified up-regulated proteins indicated that protein metabolism and modification, particularly in the ubiquitin proteasome pathway, was the main biological process occurring in SARS CoV 3CLpro-expressing cells. Thirty-six percent of identified up-regulated proteins were located in the mitochondria, including apoptosis-inducing factor, ATP synthase beta chain and cytochrome c oxidase. Interestingly, heat shock cognate 71-kDa protein (HSP70), which antagonizes apoptosis-inducing factor was shown to down-regulate and had a 5.29-fold decrease. In addition, confocal image analysis has shown release of mitochondrial apoptogenic apoptosis-inducing factor and cytochrome c into the cytosol. Our results revealed that SARS CoV 3CLpro could be considered to induce mitochondrial-mediated apoptosis. The study provides system-level insights into the interaction of SARS CoV 3CLpro with host cells, which will be helpful in elucidating the molecular basis of SARS CoV pathogenesis.