RESUMEN
Interferon (IFN)-induced Janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathway is important in controlling immune responses and is negatively response-regulated by the suppressor of cytokine signaling (SOCS) proteins. However, several viruses have developed various strategies to inhibit this pathway to circumvent the anti-viral immunity of the host. The infectious spleen and kidney necrosis virus (ISKNV) is the type species of the genus Megalocytivirus in the family Iridoviridae and a causative agent of epizootics in fish. ISKNV ORF103R encodes a predicted viral SOCS (vSOCS) with high homology to the vertebrate SOCS1, but lacks a SOCS-box domain. Interestingly, vSOCS only exists in the genus Megalocytivirus. ISKNV-vSOCS can block the IFN-α-induced Jak/Stat pathway in HepG2 cells. Over-expression of ISKNV-vSOCS inhibited the activities of IFN-stimulated response element (ISRE) promoter; however, the inhibitions by ISKNV-vSOCS were dose-dependent. ISKNV-vSOCS interacted with Jak1 protein and inhibited its tyrosine kinase activity in vitro. ISKNV-vSOCS also impaired the phosphorylation of Stat1 and Stat3 proteins and suppressed their activations. The point mutations (F18D, S66A, S85A, and R64K) of ISKNV-vSOCS significantly impaired the inhibition of IFN-α-induced ISRE-promoter activation. In conclusion, vSOCS inhibits IFN-α-induced Stat1/Stat3 signaling, suggesting that Megalocytivirus has developed a novel strategy to evade IFN anti-viral immunity via vSOCS protein.
Asunto(s)
Interferón-alfa/farmacología , Janus Quinasa 1/metabolismo , Riñón/virología , Factores de Transcripción STAT/metabolismo , Proteínas Supresoras de la Señalización de Citocinas/metabolismo , Virus de la Necrosis Esplénica del Pato de Trager , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Animales , Biología Computacional , Genes Reporteros/genética , Células Hep G2 , Humanos , Ratones , Datos de Secuencia Molecular , Fosforilación/efectos de los fármacos , Mutación Puntual , Unión Proteica , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Proteínas Supresoras de la Señalización de Citocinas/química , Proteínas Supresoras de la Señalización de Citocinas/genética , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
The IκB kinase ß (IKKß) plays crucial roles in regulating activation of nuclear factor-kappa B (NF-κB) in response to proinflammatory factors and microbial and viral infections. Here, we report the cloning of an IKKß cDNA (named SicIKKß) from the mandarin fish Siniperca chuatsi. The full-length cDNA is 4052bp and contains an ORF that encodes a predicted protein of 743-amino acid residues. The deduced amino acid sequence of SicIKKß has the same domain organization as human IKKß, which consists of a serine/threonine kinase domain, a leucine zipper motif and a putative helix-loop-helix motif. Quantitative RT-PCR showed that SicIKKß was ubiquitously expressed in tissues of mandarin fish, and its expression in mandarin fish fry (MFF-1) cells was up-regulated during the course of ISKNV infection.
Asunto(s)
Infecciones por Virus ADN/veterinaria , Enfermedades de los Peces/enzimología , Quinasa I-kappa B/genética , Perciformes/inmunología , Animales , Clonación Molecular , Infecciones por Virus ADN/enzimología , Infecciones por Virus ADN/genética , Infecciones por Virus ADN/inmunología , Enfermedades de los Peces/genética , Enfermedades de los Peces/inmunología , Iridoviridae/inmunología , FN-kappa B/metabolismo , Perciformes/genética , Perciformes/metabolismo , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Análisis de Secuencia de ADN/veterinaria , Regulación hacia ArribaRESUMEN
The JAK/STAT signal transduction pathway plays a critical role in host defence against viral and bacterial infections. In the present study, we report cDNA cloning and characterization of the JAK family (mJAK1-3 and mTYK2) and STAT family members (mSTAT1, mSTAT3-6) from the mandarin fish Siniperca chuatsi. To our knowledge, JAK2, TYK2 and STAT6 genes were cloned from fish for the first time. The mJAK family proteins consist of 1112-1177 residues with a FERM domain, an SH2 domain, a pseudokinase domain, and a tyrosine kinase domain. The mSTAT family members contain 716-786 residues with similar architecture, including an N-terminal domain, a coiled coil domain, a DNA binding domain, a linker domain, an SH2 domain, and a transcription activation domain. Multiple sequence alignments of mJAKs/mSTATs and phylogenetic analysis showed that mJAK1 was closed to mTYK2, and mJAK2 was closed to mJAK3. Quantitative real-time PCR results revealed that mJAK/mSTAT family members were expressed in most tissues examined except muscle. In mandarin fish fry cells, the expressions of IRF-1, Mx, SOCS1 and SOCS3 genes were significantly induced by poly(I:C) stimulation, indicating that the mJAK/mSTAT signal pathway is activated by poly(I:C). Furthermore, expressions of all four mJAKs and four mSTATs were all up-regulated after poly(I:C) stimulation, but expression of mSTAT5 was inhibited by poly(I:C). These results suggest that mandarin fish has the JAK/STAT signal transduction pathways similar to those in mammals, and these signalling pathways may play an important role in regulation of antiviral responses in fish.