RESUMEN

Zika virus (ZIKV), a positive-sense single-stranded RNA virus, causes congenital ZIKV syndrome in children and Guillain-Barré Syndrome (GBS) in adults. ZIKV expresses nonstructural protein 5 (NS5), a large protein that is essential for viral replication. ZIKV NS5 confers the ability to evade interferon (IFN) signalling; however, the exact mechanism remains unclear. In this study, we employed affinity pull-down and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses and found that splicing factor 3b subunit 3 (SF3B3) is associated with the NS5-Flag pull-down complex through interaction with NS5. Functional assays showed that SF3B3 overexpression inhibited ZIKV replication by promoting IFN-stimulated gene (ISG) expression whereas silencing of SF3B3 inhibited expression of ISGs to promote ZIKV replication. GTP cyclohydrolase I (GCH1) is the first and rate-limiting enzyme in tetrahydrobiopterin (BH4) biosynthesis. NS5 upregulates the expression of GCH1 during ZIKV infection. And GCH1 marginally promoted ZIKV replication via the IFN pathway. Additionally, GCH1 expression is related to the regulation of SF3B3. Overexpression of the SF3B3 protein effectively reduced GCH1 protein levels, whereas SF3B3 knockdown increased its levels. These findings indicated that ZIKV NS5 binding protein SF3B3 contributed to the host immune response against ZIKV replication by modulating the expression of GCH1.

Asunto(s)

Infección por el Virus Zika , Virus Zika , Niño , Humanos , Proteínas Portadoras/metabolismo , Proteínas Portadoras/farmacología , Cromatografía Liquida , Unión Proteica , Factores de Empalme de ARN/metabolismo , Espectrometría de Masas en Tándem , Proteínas no Estructurales Virales/genética , GTP Ciclohidrolasa/metabolismo

RESUMEN

Tumour necrosis factor receptor associated factor 3 (TRAF3) is a crucial transducing protein for linking upstream receptor signals and downstream antiviral signalling pathways. Previous studies mostly clarified the functions of TRAF3 in mammals, birds and fish, but little is known about the characterization and function of TRAF3 in amphibians. In this study, the molecular and functional identification of two TRAF3 genes, AdTRAF3A and AdTRAF3B, were investigated in the Chinese giant salamander Andrias davidianus. The complete open reading frames (ORFs) of AdTRAF3A and AdTRAF3B were 1698 bp and 1743 bp in length, encoding 565 and 580 amino acids, respectively. Both AdTRAF3A and AdTRAF3B deduced proteins contained a RING finger, two TRAF-type zinc fingers, a coiled-coil and a MATH domain. Phylogenetic analysis showed that the AdTRAF3 protein clustered together with other known TRAF3 proteins. Gene expression analysis showed that AdTRAF3s were broadly distributed in all examined tissues with similar distribution patterns. AdTRAF3s in the blood or spleen positively responded to Giant salamander iridovirus (GSIV) and poly (I:C) induction but exhibited distinct response patterns. Silencing AdTRAF3A/B remarkably suppressed the expression of IFN signalling pathway-related genes when leukocytes were treated with DNA virus and the viral RNA analogue. Moreover, overexpression of AdTRAF3A may induce the activation of the IFN-ß promoter, and the zinc finger, coiled coil and MATH domains of AdTRAF3A were essential for IFN-ß promoter activation. However, the overexpression of AdTRAF3B significantly suppressed IFN-ß promoter activity, and its inhibitory effect was enhanced when the RING finger or MATH domain was deleted. Furthermore, AdTRAF3A rather than AdTRAF3B significantly induced NF-κB activation, implying that AdTRAF3A may function as an enhancer in both the IFN and NF-κB signalling pathways. Taken together, our results suggest that the two TRAF3 genes play different crucial regulatory roles in innate antiviral immunity in Chinese giant salamanders.

Asunto(s)

Inmunidad Innata/inmunología , Iridovirus/inmunología , Factor 3 Asociado a Receptor de TNF/inmunología , Urodelos/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Interacciones Huésped-Patógeno/inmunología , Inmunidad Innata/genética , Iridovirus/fisiología , FN-kappa B/inmunología , FN-kappa B/metabolismo , Filogenia , Isoformas de Proteínas/genética , Isoformas de Proteínas/inmunología , Isoformas de Proteínas/metabolismo , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transducción de Señal/genética , Transducción de Señal/inmunología , Factor 3 Asociado a Receptor de TNF/clasificación , Factor 3 Asociado a Receptor de TNF/genética , Urodelos/genética , Urodelos/virología

RESUMEN

Inhaled corticosteroids (ICS) are recommended treatments for all degrees of asthma severity and in combination with bronchodilators are indicated for COPD patients with a history of frequent exacerbations. However, the long-term side effects of glucocorticoids (GCs) may include increased risk of respiratory infections, including viral triggered exacerbations. Rhinovirus (RV) infection is the main trigger of asthma and COPD exacerbations. Thus, we sought to explore the influence of GCs on viral replication. We demonstrate the ICS fluticasone propionate (FP) and two selective non-steroidal (GRT7) and steroidal (GRT10) glucocorticoid receptor (GR) agonists significantly suppress pro-inflammatory (IL-6 and IL-8) and antiviral (IFN-λ1) cytokine production and the expression of the interferon-stimulated genes (ISGs) OAS and viperin in RV-infected bronchial epithelial cells, with a consequent increase of viral replication. We also show that FP, GRT7 and GRT10 inhibit STAT1 Y701 and/or STAT2 Y690 phosphorylation and ISG mRNA induction following cell stimulation with recombinant IFN-ß. In addition, we investigated the effects of the ICS budesonide (BD) and the long-acting ß2 agonist (LABA) formoterol, alone or as an ICS/LABA combination, on RV-induced ISG expression and viral replication. Combination of BD/formoterol increases the suppression of OAS and viperin mRNA observed with both BD and formoterol alone, but an increase in viral RNA was only observed with BD treatment and not with formoterol. Overall, we provide evidence of an impairment of the innate antiviral immune response by GC therapy and the potential for GCs to enhance viral replication. These findings could have important clinical implications.

Asunto(s)

Bronquios/efectos de los fármacos , Células Epiteliales/efectos de los fármacos , Glucocorticoides/toxicidad , Mediadores de Inflamación/metabolismo , Interferón Tipo I/metabolismo , Rhinovirus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , 2',5'-Oligoadenilato Sintetasa/genética , 2',5'-Oligoadenilato Sintetasa/metabolismo , Agonistas de Receptores Adrenérgicos beta 2/toxicidad , Bronquios/inmunología , Bronquios/metabolismo , Bronquios/virología , Quimioterapia Combinada , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Células Epiteliales/virología , Fumarato de Formoterol/toxicidad , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Inmunidad Innata/efectos de los fármacos , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Proteínas/genética , Proteínas/metabolismo , Rhinovirus/crecimiento & desarrollo , Rhinovirus/inmunología , Transducción de Señal

RESUMEN

BACKGROUND: A multitude of recent studies has observed common epigenetic changes develop in tumour cells of multiple lineages following exposure to stresses such as hypoxia, chemotherapeutics, immunotherapy or targeted therapies. A significant increase in the transcriptionally repressive mark trimethylated H3K9 (H3K9me3) is becoming associated with treatment-resistant phenotypes suggesting upstream mechanisms may be a good target for therapy. We have reported that the increase in H3K9me3 is derived from the methyltransferases SETDB1 and SETDB2 following treatment in melanoma, lung, breast and colorectal cancer cell lines, as well as melanoma patient data. Other groups have observed a number of characteristics such as epigenetic remodelling, increased interferon signalling, cell cycle inhibition and apoptotic resistance that have also been reported by us suggesting these independent studies are investigating similar or identical phenomena. MAIN BODY: Firstly, this review introduces reports of therapy-induced reprogramming in cancer populations with highly similar slow-cycling phenotypes that suggest a role for both IFN signalling and epigenetic remodelling in the acquisition of drug tolerance. We then describe plausible connections between the type 1 IFN pathway, slow-cycling phenotypes and these epigenetic mechanisms before reviewing recent evidence on the roles of SETDB1 and SETDB2, alongside their product H3K9me3, in treatment-induced reprogramming and promotion of drug resistance. The potential mechanisms for the activation of SETDB1 and SETDB2 and how they might arise in treatment is also discussed mechanistically, with a focus on their putative induction by inflammatory signalling. Moreover, we theorise their timely role in attenuating inflammation after their activation in order to promote a more resilient phenotype through homeostatic coordination of H3K9me3. We also examine the relatively uncharacterized functions of SETDB2 with some comparison to the more well-known qualities of SETDB1. Finally, an emerging overall mechanism for the epigenetic maintenance of this transient phenotype is outlined by summarising the collective literature herein. CONCLUSION: A number of converging phenotypes outline a stress-responsive mechanism for SETDB1 and SETDB2 activation and subsequent increased survival, providing novel insights into epigenetic biology. A clearer understanding of how SETDB1/2-mediated transcriptional reprogramming can subvert treatment responses will be invaluable in improving length and efficacy of modern therapies.

Asunto(s)

Resistencia a Antineoplásicos , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Neoplasias/metabolismo , Animales , Reprogramación Celular , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Humanos , Interferón Tipo I/metabolismo , Neoplasias/tratamiento farmacológico , Fenotipo , Transducción de Señal

RESUMEN

OBJECTIVE: ß-catenin is one of the most critical oncogenes associated with many kinds of human cancers, especially in the human CRC. Innate immunity recognizes tumour derived damage-associated molecular patterns (DAMPs) and primes the anti-tumour adaptive responses. While the function of ß-catenin in CRC tumourigenesis is well established, its impact on innate immune evasion is largely unknown. The aim of this study is to characterize the role of ß-catenin in inhibiting RIG-I-like receptor (RLR)-mediated IFN-ß signalling in colorectal cancer. MATERIALS AND METHODS: Immunohistochemical staining and western blotting were conducted to study the expression of ß-catenin, IRF3 and phospho-IRF3 (p-IRF3) in CRC samples and cell lines. Plaque assay determining virus replication was performed to assess the regulation of ß-catenin on IFN-ß signalling. The inhibition of ß-catenin on RLR-mediated IFN-ß signalling was further studied by real-time analyses and reporter assays in the context of lentiviral-mediated ß-catenin stably knocking down. Lastly, co-immunoprecipitation and nuclear fractionation assay were conducted to monitor the interaction between ß-catenin and IRF3. RESULTS: We found that high expression of ß-catenin positively correlated with the expression of IRF3 in CRC cells. Overexpression of ß-catenin increased the viral replication. Conversely knocking down of ß-catenin inhibited viral replication. Furthermore, our data demonstrated that ß-catenin could inhibit the expression of IFN-ß and interferon-stimulated gene 56 (ISG56). Mechanistically, we found that ß-catenin interacted with IRF3 and blocked its nuclear translocation. CONCLUSION: Our study reveals an unprecedented role of ß-catenin in enabling innate immune evasion in CRC.

Asunto(s)

Neoplasias Colorrectales/genética , Inmunidad Innata/genética , Factor 3 Regulador del Interferón/genética , Transducción de Señal/genética , beta Catenina/genética , Adulto , Anciano , Anciano de 80 o más Años , Animales , Línea Celular , Chlorocebus aethiops , Femenino , Células HCT116 , Células HEK293 , Células HT29 , Humanos , Inmunoprecipitación/métodos , Interferón beta/genética , Masculino , Persona de Mediana Edad , Factores de Transcripción/genética , Células Vero , Replicación Viral/genética , Adulto Joven

RESUMEN

Systemic lupus erythematosus (SLE) is a complex disease targeting multiple organs as a result of overactivation of the type I interferon (IFN) system, a feature currently being targeted by multiple biologic therapies against IFN-α. We have identified an estrogen-regulated microRNA, miR-302d, whose expression is decreased in SLE patient monocytes and identify its target as interferon regulatory factor (IRF)-9, a critical component of the transcriptional complex that regulates expression of interferon-stimulated genes (ISGs). In keeping with the reduced expression of miR-302d in SLE patient monocytes, IRF9 levels were increased, as was expression of a number of ISGs including MX1 and OAS1. In vivo evaluation revealed that miR-302d protects against pristane-induced inflammation in mice by targeting IRF9 and hence ISG expression. Importantly, patients with enhanced disease activity have markedly reduced expression of miR-302d and enhanced IRF9 and ISG expression, with miR-302d negatively correlating with IFN score. Together these findings identify miR-302d as a key regulator of type I IFN driven gene expression via its ability to target IRF9 and regulate ISG expression, underscoring the importance of non-coding RNA in regulating the IFN pathway in SLE.

Asunto(s)

Regulación de la Expresión Génica , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/genética , Lupus Eritematoso Sistémico/genética , MicroARNs/genética , Interferencia de ARN , Animales , Análisis por Conglomerados , Modelos Animales de Enfermedad , Estrógenos/farmacología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Interferón Tipo I/metabolismo , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Ratones , Monocitos/efectos de los fármacos , Monocitos/inmunología , Monocitos/metabolismo , Transducción de Señal/efectos de los fármacos

RESUMEN

AIMS/HYPOTHESIS: Three hallmarks of the pancreatic islets in early human type 1 diabetes are overexpression of HLA class I, endoplasmic reticulum (ER) stress and beta cell apoptosis. The mediators of these phenomena remain to be defined. The type I interferon IFNα is expressed in human islets from type 1 diabetes patients and mediates HLA class I overexpression. We presently evaluated the mechanisms involved in IFNα-induced HLA class I expression in human beta cells and determined whether this cytokine contributes to ER stress and apoptosis. METHODS: IFNα-induced inflammation, ER stress and apoptosis were evaluated by RT-PCR, western blot, immunofluorescence and nuclear dyes, and proteins involved in type I interferon signalling were inhibited by small interfering RNAs. All experiments were performed in human islets or human EndoC-ßH1 cells. RESULTS: IFNα upregulates HLA class I, inflammation and ER stress markers in human beta cells via activation of the candidate gene TYK2, and the transcription factors signal transducer and activator of transcription 2 and IFN regulatory factor 9. Furthermore, it acts synergistically with IL-1ß to induce beta cell apoptosis. CONCLUSIONS/INTERPRETATION: The innate immune effects induced by IFNα may induce and amplify the adaptive immune response against human beta cells, indicating that IFNα has a central role in the early phases of diabetes.

Asunto(s)

Apoptosis/fisiología , Diabetes Mellitus Tipo 1/metabolismo , Estrés del Retículo Endoplásmico/fisiología , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Secretoras de Insulina/metabolismo , Interferón-alfa/farmacología , Apoptosis/efectos de los fármacos , Apoptosis/genética , Western Blotting , Línea Celular , Supervivencia Celular/efectos de los fármacos , Diabetes Mellitus Tipo 1/genética , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Células Secretoras de Insulina/efectos de los fármacos , Interleucina-1beta/farmacología , Interferencia de ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

Loss of DNA methylation can activate endogenous retroviral expression and dsRNA in cancer cells. This leads to induction of toll-like receptor signaling stimulating an antiviral interferon response. Recent findings provide a therapeutic rationale for combining DNA methylation inhibitors with blockage of immune checkpoint proteins to fight cancer.