RESUMEN
Interferon regulatory factor (IRF) 3 and IRF7 are key regulators of type I interferon (IFN) gene expression for the antiviral immune response. In the present study, interferon regulatory factor 3 and 7 from Asian seabass, namely AsIRF3 and AsIRF7 were cloned and characterized. The full-length cDNA sequence of IRF3 and IRF7 consisted of 2965 and 2343 bp respectively. AsIRF3 and AsIRF7 were true orthologes of vertebrate IRF3/7 and showed similar domain organization, with an N-terminal DBD which consisted five tryptophan residues in IRF3 and four in IRF7, a C-terminal IRF3 domain and a serine rich region. Both IRF3 and 7 constitutively expressed during the ontogenesis and in all tissues of healthy fish. The expression of both genes was up-regulated following NNV challenge with obvious transcript abundance in brain heart and kidney. Ectopic expression of AsIRF3 and AsIRF7 displayed activation of ISRE/NF-κB promoters and modulation of interferon, ISGs and pro-inflammatory cytokine gene expression. These observations indicated that IRF3 and IRF7 play an important role in Asian seabass's antiviral defense and the RIG-IRF-IFN axis is conserved in the species.
Asunto(s)
Enfermedades de los Peces/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Perciformes/genética , Perciformes/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Infecciones por Virus ADN/inmunología , Proteínas de Peces/química , Perfilación de la Expresión Génica/veterinaria , Factor 3 Regulador del Interferón/química , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/inmunología , Factor 7 Regulador del Interferón/química , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/inmunología , Nodaviridae/fisiología , Filogenia , Alineación de Secuencia/veterinariaRESUMEN
Interferon regulatory factor 7 (IRF7) plays a crucial role in the interferon (IFN) signaling in mammals, in which it is activated by the TBK1/IKKε complex during host antiviral innate immune response. There are few reports about the relation between IRF7 and IKKε in teleost fishes. In this study, the IRF7 homologue (bcIRF7) of black carp (Mylopharyngodon Piceus) has been cloned and characterized. The transcription of bcIRF7 gene increased in host cells in response to the stimulation of LPS, poly (I:C) and viral infection. bcIRF7 migrated around 56â¯KDa in immunoblot assay and was identified as a predominantly cytosolic protein by immunofluorescent staining. bcIRF7 showed IFN-inducing ability in reporter assay and EPC cells expressing bcIRF7 showed enhanced antiviral ability against both grass carp reovirus (GCRV) and spring viremia of carp virus (SVCV). IKKε of black carp (bcIKKε) was found to be recruited into host innate immune response initiated by SVCV and GCRV in the previous work; in this paper, the kinase dead mutant of bcIKKε, bcIKKε-K39A was constructed and showed no IFN-inducing activity. The data of reporter assay and plaque assay demonstrated that bcIKKε but not bcIKKε-K39A obviously enhanced bcIRF7-mediated IFN production and antiviral activity. Our data support the conclusion that bcIKKε upregulates bcIRF7-mediated antiviral signaling, which most likely depends on its kinase activity.
Asunto(s)
Carpas/genética , Carpas/inmunología , Enfermedades de los Peces/inmunología , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Secuencia de Aminoácidos , Animales , Proteínas de Peces/química , Perfilación de la Expresión Génica/veterinaria , Quinasa I-kappa B/química , Quinasa I-kappa B/genética , Quinasa I-kappa B/inmunología , Factor 7 Regulador del Interferón/química , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/inmunología , Filogenia , Reoviridae/fisiología , Infecciones por Reoviridae/inmunología , Rhabdoviridae/fisiología , Infecciones por Rhabdoviridae/inmunología , Alineación de Secuencia/veterinariaRESUMEN
Transcription factors IRF3, IRF5 and IRF7 (IRF3/5/7) have overlapping, yet distinct, roles in the mammalian response to pathogens. To examine the role that DNA-binding specificity plays in delineating IRF3/5/7-specific gene regulation we used protein-binding microarrays (PBMs) to characterize the DNA binding of IRF3/5/7 homodimers. We identified both common and dimer-specific DNA binding sites, and show that DNA-binding differences can translate into dimer-specific gene regulation. Central to the antiviral response, IRF3/5/7 regulate type I interferon (IFN) genes. We show that IRF3 and IRF7 bind to many interferon-stimulated response element (ISRE)-type sites in the virus-response elements (VREs) of IFN promoters. However, strikingly, IRF5 does not bind the VREs, suggesting evolutionary selection against IRF5 homodimer binding. Mutational analysis reveals a critical specificity-determining residue that inhibits IRF5 binding to the ISRE-variants present in the IFN gene promoters. Integrating PBM and reporter gene data we find that both DNA-binding affinity and affinity-independent mechanisms determine the function of DNA-bound IRF dimers, suggesting that DNA-based allostery plays a role in IRF binding site function. Our results provide new insights into the role and limitations of DNA-binding affinity in delineating IRF3/5/7-specific gene expression.
Asunto(s)
Factor 3 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/metabolismo , Factores Reguladores del Interferón/metabolismo , Elementos de Respuesta , Sitios de Unión , ADN/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Humanos , Factor 3 Regulador del Interferón/química , Factor 7 Regulador del Interferón/química , Factores Reguladores del Interferón/química , Interferón Tipo I/genética , Análisis por Matrices de Proteínas , Multimerización de ProteínaRESUMEN
Interferon regulatory factor (IRF) family is a transcription factor family which plays an important role in the regulation of natural immunity and immune cell differentiation. IRF7 is important to regulate the response of type I interferon (IFN) to viral infection. Thus, more researches of the characteristic and functions of IRF7 should be done to get better understanding of the mechanisms underlying immune reactions. Here, the characterization of full-length cDNA of IRF7 was reported from miiuy croaker. Gene characterization analysis of mmiIRF7 showed conservative with other fish and inferred that the difference of tryptophan residues in IRF7 may occurred in the period of fish-specific genome duplication (FSGD) or earlier. Syntenic analysis of IRF7 showed that fish IRF7 had more highly conserved synteny than the higher vertebrates IRF7. Luciferase reporter assays result showed the ability of mmiIRF7 for activation of IFNα, IFNß, IFNγ and ISRE luciferase reporter. In this study, we systematically and comprehensively analyzed evolution and function of mmiIRF7, which will provide the basis for future research on fish IRF family.
Asunto(s)
Enfermedades de los Peces/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/inmunología , Perciformes/genética , Perciformes/inmunología , Secuencia de Aminoácidos , Animales , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Perfilación de la Expresión Génica , Factor 7 Regulador del Interferón/química , Filogenia , Alineación de Secuencia/veterinariaRESUMEN
The interferon regulatory factor 7 (IRF7) is a critical regulator of type-I interferon-dependent immune reaction that defense against virus. To investigate the antiviral function of IRF7 of barbel chub Squaliobarbus curriculus (ScIRF7), the cDNA of ScIRF7 was cloned and characterized. The full length cDNA of ScIRF7 was 1870 bp, consisted of 41 bp 5'-UTR, 560 bp 3'-UTR and a 1269 bp open reading frame (ORF). The ORF encoded 423 amino acids with a molecular weight of 49.426 KDa and a theoretical isoelectric point of 5.71. The putative ScIRF7 protein possesses typical domains of IRF family including a conserved N-terminal DBD-binding domain (DBD), a C-terminal IRF association domain and a serine-rich domain. In the DBD, four tryptophans were found to be highly conserved among all species, whilst in another conserved tryptophan site of mammals, the corresponding amino acids were methionine for fishes. The expression level of ScIRF7 was highest in the spleen and lowest in the liver. The expression level of IFN-ß was highest in the gill and lowest in the liver. After GCRV infection, expression levels changes of ScIRF7 showed an overall tendency of firstly up-regulation and then down-regulation in the spleen and the gill; and expression levels of ScIRF7 in peripheral blood lymphocyte at 24 h post-infection was highest among all time points. In pEGFP-ScIRF7 overexpressing cells, the mRNA level of ScIRF7 was firstly up-regulation and then down-regulation; and the expression of IFN-ß was significantly up-regulated at 12 h post-infection than that of control group (P < 0.05), which was significantly higher than those in pEGFP-N1 overexpressing cells. The results indicated that ScIRF7 may play a key role in immune responses of barbel chub Squaliobarbus curriculus against GCRV and may also functions in the Ctenopharyngodon idellus kidney cells.
Asunto(s)
Cyprinidae/genética , Cyprinidae/inmunología , Enfermedades de los Peces/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/inmunología , Factor 7 Regulador del Interferón/química , Especificidad de Órganos , Filogenia , Reoviridae/fisiología , Infecciones por Reoviridae/inmunología , Alineación de Secuencia/veterinariaRESUMEN
Interferon regulatory factor 7 (IRF7) plays an important role in regulating the response of type I interferon (IFN) to viral infection. To understand the mechanisms underlying immune reactions in the Pacific cod, Gadus macrocephalus, the gene encoding G. macrocephalus IRF7 was cloned and characterized. The cDNA of G. macrocephalus IRF7 was also cloned and sequenced. A cDNA sequence of 2032 bp was assembled using polymerase chain reaction (PCR) products. It contains an open reading frame of 1323 bp in length, which encoded a 440-amino acid polypeptide that comprised a DNA-binding domain (DBD), an IRF association domain (IAD), and a serine-rich domain (SRD). In the DBD, the tryptophan cluster consisted of only four tryptophans, which is a unique characteristic in fish IRF7. The mRNA of IRF7 was detected in various tissues, including in the spleen, thymus, kidney, intestine, and gills, using relative quantification PCR (R-qPCR). Dynamic expression of IRF7 was observed in larvae throughout post-hatching (ph) development, with the highest level detected at day of ph (dph) 25. Response to immune stimulation was examined by challenging larvae with polyriboinosinic polyribocytidylic acid (pIC) to mimic viral infection and elicit an immune reaction. R-qPCR revealed that the expression of IRF7 significantly increased in pIC-treated groups relative to that in the control groups, in a time-dependent manner, with peak responses at 48 and 72 h after pIC-treatment. These results show that IRF7 is expressed in various tissues of adult fish and larvae and is sensitive to viral infection, suggesting that it plays a role in antiviral immune defense in G. macrocephalus.
Asunto(s)
Proteínas de Peces/genética , Gadiformes/genética , Regulación de la Expresión Génica , Inmunidad Innata , Factor 7 Regulador del Interferón/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Gadiformes/inmunología , Gadiformes/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Inductores de Interferón/farmacología , Factor 7 Regulador del Interferón/química , Factor 7 Regulador del Interferón/metabolismo , Filogenia , Poli I-C/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia/veterinariaRESUMEN
In order to develop novel strategies to protect against increasingly virulent bird-linked pathogens, a better understanding of the avian antiviral response mechanism is essential. Type I interferons (IFNs) are recognized as the first line of defense in a host's antiviral response; and it has been suggested that IRF7, a member of the IFN regulatory factor (IRF) family, plays an important role in modulating the immune response to avian influenza virus infection in chickens. The objective of this study was to identify candidate genes and pathways associated with IRF7 regulation at the transcriptome level as a first step towards elucidating the underlying cellular mechanisms of IRF7 modulation in the chicken antiviral response. IRF7 overexpression and knockdown DF-1 cell lines were established and stimulated by various pathogen-associated molecular patterns. Significant IRF7 and type I IFN expression changes were observed in both the IRF7 overexpression cell line and the IRF7 knockdown cell line upon exposure to the double stranded RNA (dsRNA) analog poly(I:C). Using RNA-seq based transcriptome analysis, we identified potential novel genes that IRF7 may help regulate as part of the host immune response to dsRNA; potential biomarkers and therapeutic targets revealed as a result of this study warrant further investigation. Based on our results, we suggest that IRF7 may have conserved functional activity in the avian antiviral response, and plays a crucial role in type I IFN regulation.
Asunto(s)
Pollos/genética , Técnicas de Silenciamiento del Gen , Factor 7 Regulador del Interferón/genética , Poli I-C/farmacología , ARN Bicatenario/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Ontología de Genes , Factor 7 Regulador del Interferón/química , Factor 7 Regulador del Interferón/metabolismo , Interferón-alfa/genética , Interferón-alfa/metabolismo , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , Moléculas de Patrón Molecular Asociado a Patógenos/metabolismo , ARN Interferente Pequeño/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Análisis de Secuencia de ARNRESUMEN
IFN regulatory factor 7 (IRF7) is a major regulator of type I (αß) IFN secretion. A growing body of evidence shows that IRF7 is involved in a wide variety of pathologic conditions in addition to infections; however, the detailed mechanism of IRF7 transactivation remains elusive. Our current knowledge of IRF7 transactivation is based on studies of IRF3, another major regulator of IFN-ß secretion. IRF3 and IRF7 are closely related homologs with high sequence similarity in their C-terminal regions, and both proteins are activated by phosphorylation of a specific serine cluster (SC). Nevertheless, the functional domains of the two proteins are arranged in an inverted manner. We generated a model structure of the IRF7 C-terminal region using homology modeling and used it to guide subsequent functional domain studies. The model structure led to the identification of a tripod-helix structure containing the SC. Based on the model and experimental data, we hypothesized that phosphorylation-mediated IRF7 transactivation is controlled by a tripod-helix structure. Inducible IκB kinase binds a tripod-helix structure. Serial phosphorylation of the SC by the kinase liberates C-terminal helix from an inhibitory hydrophobic pocket. A histone acetyltransferase P300 binds the liberated helix. The difference in the P300 binding sites explains why the domain arrangement of IRF7 is inverted relative to that of IRF3.
Asunto(s)
Factor 3 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/metabolismo , Serina/metabolismo , Factores de Transcripción p300-CBP/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Línea Celular , Células HEK293 , Humanos , Quinasa I-kappa B/metabolismo , Factor 7 Regulador del Interferón/química , Interferón Tipo I/biosíntesis , Interferón Tipo I/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
Interferon regulatory factor (IRF) 3 and IRF7 have been identified as regulators of type I interferon (IFN) gene expression in mammals. In the present study, the two genes were cloned and characterized in the European eel, Anguilla anguilla. The full-length cDNA sequence of IRF3 and IRF7 in the European eel, named as AaIRF3 and AaIRF7 consists of 2879 and 2419 bp respectively. Multiple alignments showed that the two IRFs have a highly conserved DNA binding domain (DBD) in the N terminus, with the characteristic motif containing five tryptophan residues, which is a feature present in their mammalian homologues. But, IRF7 has only four of the five residues in other species of fish. The expression of AaIRF3 and AaIRF7 both displayed an obvious dose-dependent manner following polyinosinic:polycytidylic acid (PolyI:C) challenge. In vivo expression analysis showed that the mRNA level of AaIRF3 and AaIRF7 was significantly up-regulated in response to PolyI:C stimulation in all examined tissues/organs except in muscle, with a lower level of increase observed in response to lipopolysaccharide (LPS) challenge and Edwardsiella tarda infection, indicating that AaIRF3 and AaIRF7 may be more likely involved in antiviral immune response. In addition, some pattern recognition receptors genes related with the production of type I IFNs and those genes in response to type I IFNs were identified in the European eel genome database, indicating a relatively conserved system in the production of type I IFN and its signalling in the European eel.
Asunto(s)
Anguilla/genética , Infecciones por Enterobacteriaceae/veterinaria , Enfermedades de los Peces/inmunología , Regulación de la Expresión Génica/inmunología , Factor 3 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/genética , Interferones/genética , Secuencia de Aminoácidos , Anguilla/metabolismo , Animales , Secuencia de Bases , Edwardsiella tarda/fisiología , Infecciones por Enterobacteriaceae/inmunología , Infecciones por Enterobacteriaceae/microbiología , Enfermedades de los Peces/microbiología , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Perfilación de la Expresión Génica , Factor 3 Regulador del Interferón/química , Factor 3 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/química , Factor 7 Regulador del Interferón/metabolismo , Lipopolisacáridos/administración & dosificación , Datos de Secuencia Molecular , Filogenia , Poli I-C/administración & dosificación , Alineación de Secuencia/veterinariaRESUMEN
Rotavirus nonstructural protein NSP1 can inhibit expression of interferon (IFN) and IFN-stimulated gene products by inducing proteasome-mediated degradation of IFN-regulatory factors (IRFs), including IRF3, IRF5, and IRF7. All IRF proteins share an N-terminal DNA-binding domain (DBD), and IRF3, IRF5, and IRF7 contain a similar C-proximal IRF association domain (IAD) that mediates IRF dimerization. An autoinhibitory domain (ID) at the extreme C terminus interacts with the IAD, burying residues necessary for IRF dimerization. Phosphorylation of serine/threonine residues in the ID induces charge repulsions that unmask the IAD, enabling IRF dimerization and subsequent nuclear translocation. To define the region of IRF proteins targeted for degradation by NSP1, we generated IRF3 and IRF7 truncation mutants and transiently expressed each with simian SA11-4F NSP1. These assays indicated that the IAD represented a necessary and sufficient target for degradation. Because NSP1 did not mediate degradation of truncated forms of the IAD, NSP1 likely requires a structurally intact IAD for recognition and targeting of IRF proteins. IRF9, which contains an IAD-like region that directs interactions with signal inducer and activator of transcription (STAT) proteins, was also targeted for degradation by NSP1, while IRF1, which lacks an IAD, was not. Analysis of mutant forms of IRF3 unable to undergo dimerization or that were constitutively dimeric showed that both were targeted for degradation by NSP1. These results indicate that SA11-4F NSP1 can induce degradation of inactive and activated forms of IAD-containing IRF proteins (IRF3 to IRF9), allowing a multipronged attack on IFN-based pathways that promote antiviral innate and adaptive immune responses.
Asunto(s)
Factores Reguladores del Interferón/metabolismo , Rotavirus/inmunología , Proteínas no Estructurales Virales/inmunología , Proteínas no Estructurales Virales/metabolismo , Inmunidad Adaptativa , Células HEK293 , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata , Factor 3 Regulador del Interferón/química , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/química , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/metabolismo , Factores Reguladores del Interferón/química , Factores Reguladores del Interferón/genética , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/química , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/genética , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/metabolismo , Modelos Moleculares , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Rotavirus/patogenicidad , Rotavirus/fisiologíaRESUMEN
Open reading frame 45 (ORF45) of Kaposi's sarcoma-associated herpesvirus (KSHV) is an immediate-early and tegument protein that plays critical roles in antagonizing host antiviral responses. We have previously shown (Zhu et al, Proc. Natl. Acad. Sci. U. S. A., 99:5573-5578, 2002) that ORF45 suppresses activation of interferon regulatory factor 7 (IRF7), a crucial regulator of type I interferon gene expression, by blocking its virus-induced phosphorylation and nuclear accumulation. We report here further characterization of the mechanisms by which ORF45 inhibits IRF7 phosphorylation. In most cell types, IRF7 is phosphorylated and activated by IKKε and TBK1 after viral infection. We found that phosphorylation of IRF7 on Ser477 and Ser479 by IKKε or TBK1 is inhibited by ORF45. The inhibition is specific to IRF7 because phosphorylation of its close relative IRF3 is not affected by ORF45, implying that ORF45 does not inactivate the kinases directly. In fact, we found that ORF45 is phosphorylated efficiently on Ser41 and Ser162 by IKKε and TBK1. We demonstrated that ORF45 competes with the associated IRF7 and inhibits its phosphorylation by IKKε or TBK1 by acting as an alternative substrate.
Asunto(s)
Herpesvirus Humano 8/genética , Herpesvirus Humano 8/metabolismo , Quinasa I-kappa B/metabolismo , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Factor 7 Regulador del Interferón/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Unión Competitiva , ADN Viral/genética , Células HEK293 , Interacciones Huésped-Patógeno/fisiología , Humanos , Quinasa I-kappa B/antagonistas & inhibidores , Quinasa I-kappa B/genética , Factor 7 Regulador del Interferón/química , Modelos Biológicos , Datos de Secuencia Molecular , Fosforilación , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , ARN Interferente Pequeño/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina/química , Especificidad por SustratoRESUMEN
The interferon regulatory factor (IRF)3 and IRF7 are considered to play essential roles in innate immune system's antiviral responses. In this report, the full-length cDNA and genomic structure and immune response characterizations of IRF3 and IRF7 were investigated in large yellow croaker, Larimichthys crocea. The full-length cDNA of L. crocea (Lc)IRF3 was of 2204 bp, including a 5'-terminal untranslated region (UTR) of 41 bp, a 3'-terminal UTR of 774 bp and an open reading frame (ORF) of 1389 bp encoding a polypeptide of 462 amino acids residues. The full-length cDNA of LcIRF7 was of 1979 bp, including a 5'-terminal UTR of 47 bp, a 3'-terminal UTR of 636 bp and an ORF of 1296 bp encoding a polypeptide of 431 amino acids. The putative amino acid sequence of both LcIRF3 and LcIRF7 contained a typical IRF domain at the N-terminal and an IRF3 domain at the C-terminal. Furthermore, we obtained 4517 nucleotides (nt) LcIRF3 genome sequence based on the full-length cDNA, which contained 11 exons and 10 introns. The full-length genome sequence of LcIRF7 was of 3991 nucleotides, including 9 exons and 8 introns. Quantitative real-time reverse transcription PCR analysis revealed a broad expression of LcIRF3 and LcIRF7 with the most predominant expression of LcIRF3 and LcIRF7 in the liver and in the gill, respectively. The expression levels of LcIRF3 and LcIRF7 after challenged with LPS, poly I:C and Vibrio parahaemolyticus were tested in blood, spleen and liver. The results showed that the highest relative expression of LcIRF3 was in the liver at 24 h after poly I:C injection with 90 times greater than that of the non-injection group (p < 0.05). Moreover, LcIRF3 transcription increased significantly at most time point in blood and spleen tissue after poly I:C stimulation compared with that of the control group. After LPS injection, the peak value of LcIRF7 was in the liver with 207 times (at 3 h) as much as that in the control group (p < 0.05). In addition, LcIRF7 expression was significantly induced by poly I:C injection in spleen. Both LcIRF3 and LcIRF7 transcripts did not show significant change after V. parahaemolyticus stimulation. These results indicated that IRF3 and IRF7 might play an important role in large yellow croaker's defense against viral and bacterial infection.
Asunto(s)
Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/inmunología , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/inmunología , Perciformes/genética , Perciformes/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Factor 3 Regulador del Interferón/química , Factor 7 Regulador del Interferón/química , Datos de Secuencia Molecular , Poli I-C/farmacología , Alineación de Secuencia , Vibrio/fisiologíaRESUMEN
Signal transducers and activators of transcription (STATs) and interferon regulatory factors (IRFs) share common target genes. Here we show that the Irf7 gene is regulated by transcription factors STAT1 and IRF9 in response to the type II interferon (IFN) IFN-γ. IRF7 cooperated with STAT1 and IRF1 to stimulate the expression of a subset of IFN-γ-induced STAT1 target genes. IRF7-mediated control of the Gbp2 gene required the presence and basal activity of the S/T kinase TANK-binding kinase 1 (TBK1), whereas the binding of IRF7 to the Gbp2 promoter did not. Analysis of RNA polymerase II (Pol II) recruitment to the Gbp2 promoter revealed a role for IRF7 at later stages of the IFN-γ response. In support of the role of IRF7 in establishing an effective antibacterial response, IFN-γ-pretreated Irf7(-/-) macrophages showed an increased bacterial burden after infection with Listeria monocytogenes. Our data thus describe a biologically relevant basal activity of TBK1 and identify IRF7 as a novel player in the IFN-γ response.
Asunto(s)
Regulación de la Expresión Génica , Factor 7 Regulador del Interferón/genética , Interferón gamma/inmunología , Proteínas Serina-Treonina Quinasas/inmunología , Factor de Transcripción STAT1/inmunología , Secuencia de Aminoácidos , Animales , Células Cultivadas , Proteínas de Unión al GTP/genética , Factor 1 Regulador del Interferón/inmunología , Factor 7 Regulador del Interferón/química , Macrófagos/inmunología , Macrófagos/microbiología , Ratones , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Transducción de SeñalRESUMEN
Interferon regulatory factors IRF-3 and IRF-7 are transcription factors essential in the activation of interferon-ß (IFN-ß) gene in response to viral infections. Although, both proteins recognize the same consensus IRF binding site AANNGAAA, they have distinct DNA binding preferences for sites in vivo. The X-ray structures of IRF-3 and IRF-7 DNA binding domains (DBDs) bound to IFN-ß promoter elements revealed flexibility in the loops (L1-L3) and the residues that make contacts with the target sequence. To characterize the conformational changes that occur on DNA binding and how they differ between IRF family members, we have solved the X-ray structures of IRF-3 and IRF-7 DBDs in the absence of DNA. We found that loop L1, carrying the conserved histidine that interacts with the DNA minor groove, is disordered in apo IRF-3 but is ordered in apo IRF-7. This is reflected in differences in DNA binding affinities when the conserved histidine in loop L1 is mutated to alanine in the two proteins. The stability of loop L1 in IRF-7 derives from a unique combination of hydrophobic residues that pack against the protein core. Together, our data show that differences in flexibility of loop L1 are an important determinant of differential IRF-DNA binding.
Asunto(s)
ADN/química , Factor 3 Regulador del Interferón/química , Factor 7 Regulador del Interferón/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Humanos , Factor 3 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/genética , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
We describe a procedure for detection and comparison of protein-DNA interactions using DNA-BIND plate and horseradish peroxidase (HRP)-based colorimetric assay. Amino-modified oligonucleotide was covalently immobilized on the surface of DNA-BIND plate. After the complementary oligonucleotide was annealed, the plate was incubated with protein to allow sequence-specific DNA binding. Primary antibody and HRP-labeled secondary antibody were then employed, and colorimetric assay was conducted before the absorbance was read. This is a sensitive, specific, and high-throughput method that has been applied not only in the detection of protein-DNA interaction but also in the quantitative comparison of DNA-binding capabilities among wild-type and mutant proteins.
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Colorimetría/métodos , Proteínas de Unión al ADN/química , ADN/química , Peroxidasa de Rábano Silvestre/química , Anticuerpos/inmunología , Factor 7 Regulador del Interferón/química , Oligonucleótidos/química , Unión ProteicaRESUMEN
IRF-7 is the master regulator of type I interferon-dependent immune responses controlling both innate and adaptive immunity. Given the significance of IRF-7 in the induction of immune responses, many viruses have developed strategies to inhibit its activity to evade or antagonize host antiviral responses. We previously demonstrated that ORF45, a KSHV immediate-early protein as well as a tegument protein of virions, interacts with IRF-7 and inhibits virus-mediated type I interferon induction by blocking IRF-7 phosphorylation and nuclear translocation (Zhu, F. X., King, S. M., Smith, E. J., Levy, D. E., and Yuan, Y. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 5573-5578). In this report, we sought to reveal the mechanism underlying the ORF45-mediated inactivation of IRF-7. We found that ORF45 interacts with the inhibitory domain of IRF-7. The most striking feature in the IRF-7 inhibitory domain is two α-helices H3 and H4 that contain many hydrophobic residues and two ß-sheets located between the helices that are also very hydrophobic. These hydrophobic subdomains mediate intramolecular interactions that keep the molecule in a closed (inactive) form. Mutagenesis studies confirm the contribution of the hydrophobic helices and sheets to the autoinhibition of IRF-7 in the absence of viral signal. The binding of ORF45 to the critical domain of IRF-7 leads to a hypothesis that ORF45 may maintain the IRF-7 molecule in the closed form and prevent it from being activated in response to viral infection.
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Herpesvirus Humano 8 , Proteínas Inmediatas-Precoces/metabolismo , Factor 7 Regulador del Interferón/antagonistas & inhibidores , Factor 7 Regulador del Interferón/metabolismo , Modelos Moleculares , Secuencia de Aminoácidos , Células HEK293 , Herpesvirus Humano 8/fisiología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Factor 7 Regulador del Interferón/química , Factor 7 Regulador del Interferón/genética , Datos de Secuencia Molecular , Mutagénesis , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Eliminación de Secuencia , Especificidad por Sustrato , Activación TranscripcionalRESUMEN
BACKGROUND: FUS1/TUSC2 is a novel tumor suppressor located in the critical 3p21.3 chromosomal region frequently deleted in multiple cancers. We previously showed that Tusc2-deficient mice display a complex immuno-inflammatory phenotype with a predisposition to cancer. The goal of this study was to analyze possible involvement of TUSC2 in malignant pleural mesothelioma (MPM) - an aggressive inflammatory cancer associated with exposure to asbestos. METHODS: TUSC2 insufficiency in clinical specimens of MPM was assessed via RT-PCR (mRNA level), Representational Oligonucleotide Microarray Analysis (DNA level), and immunohistochemical evaluation (protein level). A possible link between TUSC2 expression and exposure to asbestos was studied using asbestos-treated mesothelial cells and ROS (reactive oxygen species) scavengers. Transcripional effects of TUSC2 in MPM were assessed through expression array analysis of TUSC2-transfected MPM cells. RESULTS: Expression of TUSC2 was downregulated in approximately 84% of MM specimens while loss of TUSC2-containing 3p21.3 region observed in approximately 36% of MPMs including stage 1 tumors. Exposure to asbestos led to a transcriptional suppression of TUSC2, which we found to be ROS-dependent. Expression array studies showed that TUSC2 activates transcription of multiple genes with tumor suppressor properties and down-regulates pro-tumorigenic genes, thus supporting its role as a tumor suppressor. In agreement with our knockout model, TUSC2 up-regulated IL-15 and also modulated more than 40 other genes (approximately 20% of total TUSC2-affected genes) associated with immune system. Among these genes, we identified CD24 and CD274, key immunoreceptors that regulate immunogenic T and B cells and play important roles in systemic autoimmune diseases. Finally, clinical significance of TUSC2 transcriptional effects was validated on the expression array data produced previously on clinical specimens of MPM. In this analysis, 42 TUSC2 targets proved to be concordantly modulated in MM serving as disease discriminators. CONCLUSION: Our data support immuno-therapeutic potential of TUSC2, define its targets, and underscore its importance as a transcriptional stimulator of anti-tumorigenic pathways.
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Mesotelioma/genética , Neoplasias Pleurales/genética , Transcripción Genética , Proteínas Supresoras de Tumor/deficiencia , Secuencia de Aminoácidos , Amianto/toxicidad , Línea Celular Tumoral , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Depuradores de Radicales Libres/metabolismo , Eliminación de Gen , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunohistoquímica , Factor 7 Regulador del Interferón/química , Mesotelioma/patología , Modelos Biológicos , Datos de Secuencia Molecular , Neoplasias Pleurales/patología , Estructura Terciaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reproducibilidad de los Resultados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Three interferon regulatory factor (IRF) genes, CaIRF-1, CaIRF-2 and CaIRF-7, and their promoters of snakehead (Channa argus) were cloned and characterized. The CaIRF-1 gene consists of ten exons, spans 4.3 kb and encodes a putative peptide of 299 aa. The CaIRF-2 gene consists of nine exons, spans 8 kb and encodes a putative peptide of 328 aa. The gene organizations of CaIRF-1 and CaIRF-2 are very similar to that of human IRF-1 and IRF-2 except more compact. Comparison of exon-intron organization of the two genes indicated a common evolutionary structure, notably within the exons encoding the DNA binding domain (DBD) of the two factors. The CaIRF-7 gene spans 4.1 kb and encodes a putative peptide of 437 aa. However, the gene organization of CaIRF-7 consisting of ten exons is different to human IRF-7a gene which has an intron in 5' UTR. Three CaIRFs share homology in N-terminal encompassing the DBD that contains a characteristic repeat of tryptophan residues. The promoters of CaIRF-1 and CaIRF-2 genes contain the conserved sites for NF-kappaB and Sp1. The gamma-IFN activation sites (GAS) were found in the promoters of CaIRF-1 and CaIRF-7. The promoter of CaIRF-7 contains conserved interferon stimulating response element (ISRE) which is characteristic of IFN-induced gene promoter, and suggests that there also exist intracellular amplifier circuit in fish IFN signal pathway. Moreover, the element GAAANN oriented in both directions is repeated in CaIRF promoter regions, which confers to further inducibility by IFN. The constitutive expression of CaIRF genes were found to increase obviously in response to induction by the known IFN-inducer poly I:C.
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Peces/genética , Factores Reguladores del Interferón/genética , Regiones Promotoras Genéticas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , ADN Complementario/aislamiento & purificación , Exones/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Factor 1 Regulador del Interferón/química , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/metabolismo , Factor 2 Regulador del Interferón/química , Factor 2 Regulador del Interferón/genética , Factor 2 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/química , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/metabolismo , Factores Reguladores del Interferón/química , Factores Reguladores del Interferón/metabolismo , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ProteínaRESUMEN
Upon viral infection, the major defense mounted by the host immune system is activation of the interferon (IFN)-mediated antiviral pathway that is mediated by IFN regulatory factors (IRFs). In order to complete their life cycle, viruses must modulate the host IFN-mediated immune response. Kaposi's sarcoma-associated herpesvirus (KSHV), a human tumor-inducing herpesvirus, has developed a unique mechanism for antagonizing cellular IFN-mediated antiviral activity by incorporating viral homologs of the cellular IRFs, called vIRFs. Here, we report a novel immune evasion mechanism of KSHV vIRF3 to block cellular IRF7-mediated innate immunity in response to viral infection. KSHV vIRF3 specifically interacts with either the DNA binding domain or the central IRF association domain of IRF7, and this interaction leads to the inhibition of IRF7 DNA binding activity and, therefore, suppression of alpha interferon (IFN-alpha) production and IFN-mediated immunity. Remarkably, the central 40 amino acids of vIRF3, containing the double alpha helix motifs, are sufficient not only for binding to IRF7, but also for inhibiting IRF7 DNA binding activity. Consequently, the expression of the double alpha helix motif-containing peptide effectively suppresses IRF7-mediated IFN-alpha production. This demonstrates a remarkably efficient means of viral avoidance of host antiviral activity.