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Protein inhibitor of activated signal transducer and activator of transcription (PIAS) family protein involved in gene transcriptional regulation acts as negative regulator in Janus kinase-signal transducer and activator of transcription (JAK/STAT) signaling pathway. But until now, the roles of PIAS in fish are not clear. In this study, we identified the two mammalian PIAS1 orthologs from Ctenopharyngodon idellus, namely CiPIAS1a and CiPIAS1b, respectively. They can respond to the stimulation from Polyribocytidylic acid (Poly I:C), Grass Carp Reovirus (GCRV) and Lipopolysaccharides (LPS) respectively, so we suggested that they could participate in interferon (IFN)-mediated antiviral and antibacterial immune response. The subcellular localization and nuclear cytoplasm extraction showed that CiPIAS1a and CiPIAS1b were mainly distributed in the nucleus. In addition, Co-IP showed that they separately inhibited the phosphorylation of STAT1 via interacting with it, which leads to the reduction of IFN1 expression., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

As a tyrosine phosphatase, Src homology 2-containing protein tyrosine phosphatase 2 (SHP2) serves as an inhibitor in PI3K-Akt pathway. In mammals, SHP2 can phosphorylate GSK3β at Y216 site to control the expression of IFN. So far, the multiple functions of SHP2 have been reported in mammals. However, little is known about fish SHP2. In this study, we cloned and identified a grass carp (Ctenopharyngodon idellus) SHP2 gene (CiSHP2, MT373151). SHP2 is conserved among different vertebrates by amino acid sequences alignment and the phylogenetic tree analysis. CiSHP2 shared the closest homology with Danio rerio SHP2. Simultaneously, SHP2 was also tested in grass carp tissues and CIK (C. idellus kidney) cells. We found that it responded to poly I:C stimulation. CiSHP2 was located in the cytoplasm just as the same as those of mammals. Interestingly, it inhibited the phosphorylation level of GSK3β in a non-contact manner. Meanwhile CiGSK3β interacted with and directly phosphorylated CiTBK1. In addition, we found that CiSHP2 also reduced the phosphorylation level of CiTBK1 by CiGSK3β, and then it depressed the expression of IFN I via GSK3β-TBK1 axis. These results suggested that CiSHP2 was involved in CiGSK3β and CiTBK1 activity but not regulated their transcriptional level. At the same time, we also found that CiSHP2 also influenced the activity of CiIRF3. Therefore, fish SHP2 inhibited IFN I expression through blocking GSK3β-TBK1 signal axis., (Copyright © 2021. Published by Elsevier Ltd.)

As a NAD + -dependent deacetylase, SIRT1 is widely involved in apoptosis and cellular inflammation via multiple pathways such as p53, NF-кB and STAT. More and more studies have shown that p53 is the first non-histone deacetylation target of SIRT1. SIRT1-p53 axis thus plays an important role in mammalian cells. IRF9 is an important member of interferon regulator factor family and performs an important role in innate immunity against foreign virus invasion. More importantly, human IRF9 can suppress the SIRT1-p53 axis. However, the functions and relationship between IRF9 and SIRT1-p53 axis are rarely studied in fish. To this end, we made a preliminary research on the functions of grass carp (Ctenopharyngodon idella) IRF9, SIRT1 and p53 in apoptosis and innate immunity. Firstly, we cloned and identified the ORF of SIRT1 (named CiSIRT1, MN125614) from C. idella and demonstrated that CiIRF9 promoted apoptosis, while CiSIRT1 inhibited apoptosis by flow cytometry and TUNEL experiments. Next, we found the interaction between CiSIRT1 and Cip53 in vivo by co-immunoprecipitation experiments. Moreover, the colocalization analysis also showed CiSIRT1 and Cip53 were mainly distributed in nucleus. Thirdly, we got a conclusion that CiIRF9 can repress the expression of CiSIRT1, implying that CiIRF9 regulates CiSIRT1-p53 axis. Finally, CiSIRT1 mRNA level was significantly up-regulated and the expression reached the highest level at 24 h post poly (I:C) stimulation in CIK cells. So, CiSIRT1 may exert an important function in innate immunity. Furthermore, we found CiSIRT1 down-regulated the expression of CiIFN1. In summary, CiIRF9 promotes apoptosis and innate immunity by inhibiting SIRT1-p53 axis. These findings will provide a new theoretical basis for the research on teleost innate immunity., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

As a dsRNA-dependent and interferon-induced protein kinase, PKR is involved in antiviral immune response and apoptosis mediated by various cytokines. In mammalian cells, PKR can also be activated in the absence of dsRNA. A PKR activator, PACT (PKR activating protein), also referred to as RAX (PKR-associated protein X) plays an important role. In recent years, with the increasing recognition of fish interferon system, PKR and PACT have been gradually revealed in fish. However, the function of fish PACT is unclear. In our previous work, we suggested that grass carp (Ctenopharyngodon idella) PACT must be involved in IRF2 and ATF4-mediated stress response pathways. In the present study, we found that the expression of C. idella PACT (CiPACT) and CiPKR were significantly up-regulated under the stimulation of LPS. It indicated that CiPACT and CiPKR may play an important role in response to LPS stimulation. In addition, the response time of CiPACT to LPS is earlier than that of CiPKR. It has also shown that overexpression of CiPACT in CIK cells can significantly enhance the level of p-eIF2α, induces apoptosis and translocation of Cip65 to nucleus from cytoplasm. To further understand the mechanism, we carried out the co-immunoprecipitation assay. It proved that the interaction of CiPACT and CiPKR made the phosphorylation of CiPKR. Overexpression of CiPACT induced the down-regulation of intracellular expression of bcl-2 and up-regulation of bax. However, in CiPKR knocked-down cells the expression of bcl-2 and bax were just the opposite. Therefore, the mechanism of fish PACT induces apoptosis and activates NF-кB is dependent on PKR., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In vertebrates, IL-10 is an anti-inflammatory factor that serves as a key inhibitory role in a wide range of immune responses. IRAK1 (IL-1 receptor-associated kinase 1), a key molecule in the inflammatory signal of IL-1R/TLR, plays an important pivotal role in regulating the autoimmunity of body. STAT3 (Signal transducer and activator of transcription 3) activated by IRAK1 participates in inflammation, tumorigenesis, metabolic disorders and immune response. Under the stimulation of LPS, IRAK1 enters the nucleus to form a dimer with STAT3 and regulates the expression of IL-10. However, the relationship between fish IRAK1 and STAT3 has not been reported. To explain the anti-inflammation in fish, we amplified and identified the complete open reading frame of grass carp IRAK1 (CiIRAK1) and STAT3 (CiSTAT3) based on the existing sequences. The expression of CiIRAK1 and CiSTAT3 were up-regulated significantly under the stimulation of LPS. This result suggests that both CiIRAK1 and CiSTAT3 may be involved in LPS-induced TLR4 pathway. The subcellular localization experiment revealed that CiIRAK1 is distributed in cytoplasm and enters nucleus after LPS stimulation. CiSTAT3 is distributed in both cytoplasm and nucleus with or without LPS stimulation. Immunoprecipitation assay revealed that CiIRAK1 interacted with CiSTAT3 under LPS stimulation. However in absence of LPS stimulation, CiIRAK1 and CiSTAT3 cannot interact with each other. Subsequently, immunofluorescence colocalization experiment further proved the interaction of CiIRAK1 and CiSTAT3 in nucleus under LPS stimulation. The dual luciferase reporter assays indicated that the binding of CiIRAK1 and CiSTAT3 synergistically enhanced the activity of CiIL-10 promoter., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Bcl-xl, Bax2, and NF-κB are well-known to be involved in anti-apoptosis response. Although Bcl-xl has been reported in fish, the NF-κB-mediated regulatory mechanism and anti-apoptotic function are still unclear. Here, we cloned and characterized the full-length cDNA sequence of grass carp (Ctenopharyngodon idella) Bcl-xl (CiBcl-xl) and its promoter region sequence. The full-length cDNA of CiBcl-xl is 2836 bp with an ORF of 627 bp encoding a polypeptide of 208 amino acids. Phylogenetic tree analysis revealed that CiBcl-xl shared high homology with Dario rerio Bcl-xl (DrBcl-xl). After stimulation with Poly I:C, the expression of CiBcl-xl in CIK cells and various tested tissues of grass carp were significantly upregulated. To further understand the transcriptional control of fish Bcl-xl induced by NF-κB, CiC-rel and Cip65 were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind resin. In vitro, gel mobility shift assays demonstrated the high affinity of CiC-rel and Cip65 with CiBcl-xl promoter. Dual-luciferase reporter assays showed that CiC-rel and Cip65 activated CiBcl-xl promoter. Also, knockdown of CiC-rel and Cip65 reduced the expression of Bcl-xl. Therefore, similar to those of mammals, fish C-rel and p65 can upregulate the transcription of Bcl-xl. In addition, we found that overexpression of CiBcl-xl in CIK cells increased the cell activity and inhibited cell apoptosis, while overexpression of Bax2 promoted cell apoptosis. Meanwhile, co-transfection of CiBcl-xl and CiBax2 into cells can ease up apoptotic rate. To further investigate the molecular basis of synergistic effect of Bcl-xl and Bax2, we showed that Bcl-xl and Bax2 interacted with each other. The results suggested that Bcl-xl executed its anti-apoptotic function by binding to and inhibiting the pro-apoptotic activity of Bax2.

As a member of NDR protein kinase family and a novel protein kinase of Hippo signal pathway, Serine/threonine kinase 38 (STK38) plays a very significant role in the innate immune. In mammals, STK38 performs its function by combining with GSK3β. Nowadays, there are few reports of STK38 in fish. In order to explain the function of fish STK38 in the innate immunity, we cloned the ORF of grass carp (Ctenopharyngodon idella) STK38 (CiSTK38) and the related kinase GSK3β (CiGSK3β). Phylogenetic trees revealed that CiSTK38 and CiGSK3β evolved closer kinship with sinocyclocheilus grahami STK38 and siniperca chuatsi GSK3β respectively. CiSTK38 and CiGSK3β can respond to the intradermal injection of poly (I:C) in grass carp different tissues and the transfection of poly I:C in CIK cells. Subcellular localization revealed the CiGSK3β were broadly distributed through the cytoplasm, whereas CiSTK38 were observed both in cytoplasm and nucleus. However, when they were co-transferred into cells, the two proteins were found to aggregate in the nucleus. GST-pulldown and co-immunoprecipitation analysis revealed that CiSTK38 can physically interact with CiGSK3β. Phos-tag PAGE illustrated CiSTK38 can decrease the phosphorylation and auto-phosphorylation level of CiGSK3β at Ser9 and at Tyr216. To investigate the functional correlation between CiSTK38 and CiGSK3β, we overexpressed CiSTK38 and CiGSK3β in CIK cells and found that they can up-regulate the expression of IFN I. In short, we demonstrated that CiSTK38 can confer CiGSK3β kinase activity by reducing its phosphorylation level. Result from this study strongly suggested that the anti-viral immune effects elicited by poly (I:C) in part were mediated through activation of CiGSK3β. The findings provided scientific basis for the anti-viral immune mechanism of STK38 and GSK3β in fish., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Recently, studies have shown that IκB kinase β (IKKβ), a critical kinase in the nucleus factor kappa-B (NF-κB) pathway, participates in inflammatory responses associated with unfolded protein response (UPR) and plays an important role in ER stress-induced cell death. The unfolded protein response (UPR), which is a regulatory system to restore cellular homeostasis in the endoplasmic reticulum (ER), such as oxidative stress, bacterial infection, and virus invasion. The UPR pathways have been reported to be involved in immune responses in mammals, including the classical NF-κB pathway. However, the molecular mechanism of their crosstalk remains to be elucidated. Previously, we demonstrated that IKKβ also has some conserved functions between fish and human, as grass carp (Ctenopharyngodon idella) IKKβ (CiIKKβ) can activate NF-κB pathway. In this study, we found that CiIKKβ level in nucleus was elevated under ER stress and CiIKKβ can interact with grass carp X-box-binding protein 1 (CiXBP1S), a key transcription factor in UPR. Consistently, fluorescent histochemical analysis of grass carp kidney (CIK) cells indicated that CiIKKβ and CiXBP1S colocalized under ER stress. Furthermore, overexpression of CiIKKβ in CIK cells enhanced ER stress tolerance by regulating UPR signaling and resulted in the significant increase of cell viability., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In mammals, STAT3 (Signal transducer and activator of transcription 3) plays an absolutely vital role in response to cytokines and growth factors. In mammals, IL-6/JAK/STAT3 pathway is closely linked to immune response and promotes cell proliferation, survival and metastasis. Some recent studies have already demonstrated that STAT3 regulates autophagy. As a downstream target gene of STAT3, Bcl-2 (B-cell lymphoma 2) not only participates in regulating apoptosis, but also responds to autophagy. STAT3 regulates autophagy through Bcl-2. In general, the generation of autophagy is always accompanied by the change of apoptosis, and the occurrence of apoptosis is often accompanied by the decreased of cell viability. In grass carp (Ctenopharyngodon idella), LPS-induced autophagy is involved in the release of pro-inflammatory cytokines. However, only the relationship between autophagy and cytokines was illustrated, in which the signaling pathways were not discussed. In the present study, we found that the autophagy inducer, Tunicamycin (Tm), can induce C.Idella Kidney cells (CIK) autophagy. When the cells were incubated with the recombinant human IL-6 (rIL-6) for a short period of times, the mRNA expression level of C.Idella IL-6R and STAT3 were increased. At the same time, the number of GFP-LC3 puncta and the ratio of LC3-II/LC3-I were both decreased obviously in cells. It indicated that the rIL-6 can significantly alleviate autophagy induced by Tm. We speculated that CiSTAT3 may play a key role in the process. To confirm this hypothesis, we performed a rIL-6 activating CiSTAT3 assay. The result demonstrated that rIL-6 can induce CiSTAT3 to form homologous dimmer. The activated CiSTAT3 regulated the transcription activity of CiBcl-2, finally led to a decrease of autophagy. In addition, when cells were in the state of autophagy, apoptosis was increased and cell viability was decreased. When CiSTAT3 was activated, cell apoptosis weakened and cell viability was increased. The results suggest that CiSTAT3 plays an important role in maintaining the normal physiological process of cells., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

SAMHD1 is an innate immunity restriction factor that inhibits virus infection through IRF3-mediated antiviral and apoptotic responses. Fish SAMHD1 shares some similar properties with those in mammals. In this study, a SAMHD1 orthologue from grass carp (Ctenopharyngodon idellus) was cloned and characterized. The full-length cDNA of CiSAMHD1 is 2792 bp with an ORF of 1884 bp encoding a polypeptide of 627 amino acids. Multiple alignments showed that SAMHD1 is highly conserved among different species. Phylogenetic tree analysis revealed that CiSAMHD1 shared a high degree of homology with Sinocyclocheilus rhinocerous SAMHD1. Expression analysis indicated that CiSAMHD1 was widely expressed in all tissues tested including the brain, eyes, spleen, gill, intestine, liver, heart and kidney. It was significantly up-regulated in spleen, liver and intestines after treatment with poly I:C. Also, CiSAMHD1 can be induced following stimulation with recombinant IFN in CIK cells. The promoter sequence of CiSAMHD1 was identified to explore the mechanism underlying the transcriptional regulation of CiSAMHD1. The promoter sequence of CiSAMHD1 (1370 bp) consists of IRF1, IRF3, IRF9 and p65 binding elements. Gel mobility shift assay also showed that IRF1, IRF3, IRF9 and p65 prokaryotic proteins can separately interact with CiSAMHD1 promoter. Dual luciferase assay and q-PCR suggested that the promoter of CiSAMHD1 can be activated by the overexpression of CiIRF3 and CiIRF9, but cannot be triggered by CiIRF1 and Cip65. In contrast, knockdown of CiIRF3 or CiIRF9 inhibits the transcription of CiSAMHD1. Intriguingly, CCK assay suggested that CiSAMHD1 decreased cell viability. TUNEL apoptosis assay and Hoechst 33258 staining assay indicated that apoptosis is induced by the overexpression of CiSAMHD1. Crystal violet staining, detection of two GCRV genes (vp3 and vp5) and viral titration showed that CiSAMHD1 can suppress the proliferation of grass carp reovirus (GCRV) in CIK cells., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

In mammals, signal transducer and activator of transcription 6 (STAT6) is a broad-spectrum transcriptional regulator involved in cellular immune responses and apoptosis by regulating the immune-related genes and various functional genes. The structure, expression and tyrosine-based phosphorylation of STAT6 are conserved from fish to mammal. However, except the sporadic reports from zebra fish, the function of fish STAT6 has not been well reported. Here, we cloned and characterized the full length cDNA sequence of grass carp (Ctenopharyngodon idella) STAT6 (CiSTAT6). Meanwhile, the activation mechanism and the potential function of CiSTAT6 were studied. The full length cDNA of CiSTAT6 is 2747 bp with an ORF of 2313 bp encoding a polypeptide of 770 amino acids. Phylogenetic tree analysis revealed that CiSTAT6 shares the maximum homology with Cyprinus carpio STAT6. CiSTAT6 was significantly up-regulated and interacted with each other to form the homodimer after treatment with poly I:C. The transfected CiSTAT6 in fish cell lines can activate the promoter activities of CCL20 and Bcl-xl and increase their mRNA levels. In addition, we also found that CiSTAT6 can increase cell viability and inhibit cell apoptosis. Taken together, grass carp STAT6 plays an important part in innate immunity and anti-apoptosis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

IFN regulatory factor (IRF)3 is a central regulator for IFN-β expression in different types of pathogenic infections. Mammals have various pathogenic sensors that are involved in monitoring pathogen intrusions. These sensors can trigger IRF3-mediated antiviral responses through different pathways. Endoplasmic reticulum-associated proteins stimulator of IFN gene (STING) and zinc finger DHHC-type containing 1 (ZDHHC1) are critical mediators of IRF3 activation in response to viral DNA infections. In this study, grass carp STING and ZDHHC1 were found to have some similar molecular features and subcellular localization, and both were upregulated upon stimulation with polyinosinic:polycytidylic acid, B-DNA, or Z-DNA. Based on these results, we suggest that grass carp STING and ZDHHC1 might possess some properties similar to their mammalian counterparts. Overexpression of ZDHHC1 and STING in Ctenopharyngodon idella kidney cells upregulated IFN expression, whereas knockdown of IRF3 inhibited IFN activation. In addition, coimmunoprecipitation and GST pull-down assays demonstrated that STING and ZDHHC1 can interact separately with IRF3 and promote the dimerization and nuclear translocation of IRF3. Furthermore, we also found that small interfering RNA-mediated knockdown of STING could inhibit the expression of IFN and ZDHHC1 in fish cells. Similarly, knockdown of STING resulted in inhibition of the IFN promoter. In contrast, ZDHHC1 knockdown also inhibited IFN expression but had no apparent effect on STING, which indicates that STING is necessary for IFN activation through ZDHHC1. In conclusion, STING and ZDHHC1 in fish can respond to viral DNA or RNA molecules in cytoplasm, as well as activate IRF3 and, eventually, trigger IFN expression., (Copyright © 2017 by The American Association of Immunologists, Inc.)

IRF9 is a key factor in the JAK-STAT pathway. Under the stimulation of type I IFN, IRF9 interacts with STAT1 and STAT2 to form the IFN-I-stimulated gene factor 3 (ISGF3) which activates the transcription of ISG. However, many studies also showed that the dimmer IRF9/STAT2 rather than the tripolymer IRF9/STAT1/STAT2 acts as the ISGF3 in cells in response to IFN signals. In the present study, the full-length cDNA sequence of IRF9 (termed CiIRF9, KT601055) and STAT2 (term CiSTAT2, KT781914) from grass carp were cloned and identified. A low level of constitutive expression of CiIRF9 was detected by RT-PCR in grass carp tissues, but it was significantly up-regulated by LPS and poly I:C stimulation. In vitro, a high-affinity interaction between CiIRF9 and the promoter of CiIFN or CiPKR was demonstrated by gel mobility shift assay. In vivo, the promoter activities of CiIFN and CiPKR were not only increased by transient transfection of CiIRF9, but also prominently increased by co-transfection of CiIRF9 and CiSTAT2. Moreover, the interaction of CiIRF9 and CiSTAT2 was further investigated by in vivo and in vitro protein interaction assays. Recombinant CiIRF9 and CiSTAT2, both tagged with FLAG (or HA), were expressed in HEK 293T cells by transient transfection experiment. Co-immunoprecipitation assays showed that CiIRF9 can interact with CiSTAT2 in vivo. Soluble GST-ST2-936 (containing the N-terminal and coiled-coil domain of CiSTAT2) was expressed and purified from E. coli. A GST pull-down assay suggested that GST-tagged ST2-936 efficiently bound to FLAG-tagged IRF9. The data indicated that interaction of IRF9 and STAT2 synergistically up-regulated the transcriptional level of IFN and ISG genes., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Grass carp (Ctenopharyngodon idella) was one of the economically important freshwater fish in China. However, hemorrhagic disease caused by grass carp reovirus (GCRV) results in a tremendous loss in the process of grass carp cultivation. Transcriptome analysis could provide a comprehensive understanding of the molecular mechanisms involved in specific biological processes and diseases for the resistance to reovirus infection of grass carp. In this study, the raw data from NCBI (accession number: SRA099702) were analyzed, in which, 50 significant differentially expressed genes by routine transcriptome analysis and 84 notably differentially expressed genes by co-expression network method. KEGG analysis revealed that the pathway in hemorrhagic diseases in grass carp was similar to the influenza A induced pathway. The interferon-stimulated gene ISG15 and sacsin-like gene, which were up-regulated in data (SRA099702), were also up-regulated in data (SRP049081) from a similar assay. QPCR experiment was performed to validate these up-regulated genes. The ISG15 gene was shown to be the core gene in the co-expression network. The results would enhance our understanding of the antivirus system of grass carp infected by reovirus., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

As one member of ADAR family, ADAR1 (adenosine deaminase acting on RNA 1) can convert adenosine to inosine within dsRNA. There are many ADAR1 splicing isoforms in mammals, including an interferon (IFN) inducible ∼150 kD protein (ADAR1-p150) and a constitutively expressed ∼110 kD protein (ADAR1-p110). The structural diversity of ADAR1 splicing isoforms may reflect their multiple functions. ADAR1 splicing isoforms were also found in fish. In our previous study, we have cloned and identified two different grass carp ADAR1 splicing isoforms, i.e. CiADAR1 and CiADAR1-like, both of them are IFN-inducible proteins. In this paper, we identified a novel CiADAR1 splicing isoform gene (named CiADAR1a). CiADAR1a gene contains 15 exons and 14 introns. Its full-length cDNA is comprised of a 5' UTR (359 bp), a 3' UTR (229 bp) and a 2952 bp ORF encoding a polypeptide of 983 amino acids with one Z-DNA binding domain, three dsRNA binding motifs and a highly conserved hydrolytic deamination domain. CiADAR1a was constitutively expressed in Ctenopharyngodon idella kidney (CIK) cells regardless of Poly I:C stimulation by Western blot assay. In normal condition, CiADAR1a was found to be present mainly in the nucleus. After treatment with Poly I:C, it gradually shifted to cytoplasm. To further investigate the mechanism of transcriptional regulation of CiADAR1a, we cloned and identified its promoter sequence. The transcriptional start site of CiADAR1a is mapped within the truncated exon 2. CiADAR1a promoter is 1303 bp in length containing 4 IRF-Es. In the present study, we constructed pcDNA3.1 eukaryotic expression vectors with IRF1 and IRF3 and co-transfected them with pGL3-CiADAR1a promoter into CIK cells. The results showed that neither the over-expression of IRF1 or IRF3 nor Poly I:C stimulation significantly impacted CiADAR1a promoter activity in CIK cells. Together, according to the molecular and expression characteristics, subcellular localization and transcriptional regulatory mechanism, we deduced that CiADAR1a shared a high degree of homology with mammalian ADAR1-p110., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Protein kinase R (PKR), the double-stranded RNA-activated protein kinase, exists in mammalian and fish. PKZ, a PKR-like protein kinase containing Z-DNA binding domains, just exists in fish. PKR and PKZ work synergistically in the antiviral defense by inhibiting intracellular protein translation. The transcriptional factor IRF3 (interferon regulatory factor 3) acts as a key regulator of type I IFN (Interferon) and ISG (interferon stimulated gene). On the basis of the cloned CiIRF3 previously, CiIRF3 with His-tag was over-expressed in BL21 Escherichia coli, and the expressed protein was purified by affinity chromatography with Ni-NTA His-Bind Resin. In this study, we have demonstrated that grass carp (Ctenopharyngodon idellus) PKR (CiPKR) and PKZ (CiPKZ) genes were inducible by Poly I:C in C. idella kidney (CIK) cells. So, they might be implicated in the intracellular antiviral activity. To understand the up regulatory mechanism of CiPKR and CiPKZ genes upon virus induction, we constructed wild type (pGL3-CiPKR-luc and pGL3-CiPKZ-luc) and the mutant (pGL3-CiPKR-nISRE-luc and pGL3-CiPKZ-nISRE-luc) reporter gene vectors according to the promoter sequences of CiPKR (KJ704845) and CiPKZ (KJ704844). In vitro, gel mobility shift assays demonstrated that CiIRF3 can combine CiPKR and CiPKZ promoters with high affinity. However, CiIRF3 bound to the mutants CiPKR-nISRE and CiPKZ-nISRE faintly. Whereafter, the recombinant plasmids of pGL3-CiPKR-luc, pGL3-CiPKZ-luc were transiently co-transfected with pcDNA3.1-CiIRF3, pcDNA3.1-CiIRF7 respectively into CIK cells. Cell transfection assays indicated that CiIRF3 and CiIRF7 up-regulated the transcriptional level of CiPKR and CiPKZ. The results also revealed that the consensus sequence of ISRE (interferon stimulated response element) is an important regulatory element for the transcriptional initiation of CiPKR and CiPKZ., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Interferon Regulatory Factors (IRFs) belong to a family of transcription factor involved in transcriptional regulation of type I IFN and IFN-stimulated genes (ISG) in cells. In the present study, an IRF3 full-length cDNA (termed CiIRF3, JX999055) and its promoter sequence were cloned by homology cloning strategy and genome walking from grass carp (Ctenopharyngodon idella). The full-length cDNA sequence of CiIRF3 is comprised of a 5'UTR (195 bp), a 3'UTR (269 bp) and a largest open reading frame (ORF) of 1377 bp encoding a polypeptide of 458 amino acids. CiIRF3 has a conservative DNA-binding domain (DBD) at N-terminal and a relatively conserved interferon regulatory factors association domain (IAD). Phylogenetic tree analysis indicated that CiIRF3 gathers together with other IRF-3 from higher vertebrates in the same branch. The promoter sequence of CiIRF3 (596 bp) consists of three IRF-E, a C/EBP beta, a NF-kappa B and a TATA-BOX. CiIRF3 was constitutively expressed at low level in different grass carp tissues but was rapidly up-regulated with Poly I:C stimulation. To study the molecular mechanism of CiIRF3 regulating the transcription of IRFs, CiIRF3 was expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind Resin. Gel mobility shift assays revealed the affinity of CiIRF3 protein with promoters of CiIRF1, CiIRF2, CiIRF3 and CiIRF7 respectively. Then, CIK cells were co-transfected with pcDNA3.1-CiIRF3, pGL3-promotor (pGL3-CiIRF1, pGL3-CiIRF2, pGL3-CiIRF3, pGL3-CiIRF7) and luciferase reporter vector respectively. The cotransfection experiment showed that CiIRF3 increased the promoter activity of CiIRF1, CiIRF2, CiIRF3 and CiIRF7. Furthermore, overexpression of CiIRF3 in CIK cells also up-regulated the expressions of CiIRF1, CiIRF2, CiIRF3 and CiIRF7. So, CiIRF3 can improve the transcriptional level of CiIRF1, CiIRF2, CiIRF3 and CiIRF7., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Interferon Regulatory Factors (IRFs) make up a family of transcription factors involved in transcriptional regulation of type I IFN and IFN-stimulated genes (ISG) in cells. In the present study, an IRF2 gene (termed CiIRF2, JX628585) was cloned and characterized from grass carp (Ctenopharyngodon idella). The full-length cDNA of CiIRF2 is 1809 bp in length, with the largest open reading frame (ORF) of 981 bp encoding a putative protein of 326 amino acids. CiIRF2 contains a conserved DNA-binding domain (DBD) in N-terminal and a non-conserved C-terminal region. Protein sequence analysis revealed that CiIRF2 shares significant homology to the known IRF2 counterparts. Phylogenetic reconstruction confirmed its closer evolutionary relationship with other fish counterparts, especially with zebra fish IRF2. CiIRF2 was ubiquitously expressed at low level in all tested grass carp tissues and significantly up-regulated except in brain following poly I:C 6-12 h post stimulation. In order to understand fish innate immune and resistance to virus diseases, recombinant CiIRF2 with His-tag was over-expressed in BL21 Escherichia coli, and the expressed protein was purified by affinity chromatography with Ni-NTA His-Bind Resin. Promoter sequences of grass carp type I IFN gene (CiIFN) and two ISG genes (CiPKR and CiPKZ) were amplified and cloned. In vitro, gel mobility shift assays were employed to analyze the interaction of CiIRF2 protein with promoters of CiIFN, CiPKR and CiPKZ respectively. The results showed that CiIRF2 bound to these promoters with high affinity by means of its DBD. Afterwards, recombinant plasmids of pGL3-CiIFN, pGL3-CiPKR and pGL3-CiPKZ were constructed and transiently co-transfected with pcDNA3.1-CiIRF2 or pcDNA3.1-CiIRF1 respectively into C. idella kidney (CIK) cells. Dual-luciferase reporter assays demonstrated that CiIRF2 down-regulates the transcription activity of CiIFN, CiPKR and CiPKZ genes in CIK cells. To further understand the function of fish IRF2, expression plasmids (pcDNA3.1-IRF2 and pcDNA3.1-IRF1) were transiently co-transfected with pGL3-IFN or pGL3-CiPKZ into CIK cells, respectively. The results revealed that CiIRF2 plays an antagonistic role to CiIRF1 in transcriptional regulation of IFN and ISG genes., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Grass carp reovirus (GCRV)-induced gene 2 (Gig2) is recognized as a new antiviral factor involved in response to viral infection. However, little is known about the mechanisms behind the transcriptional regulation of Gig2 when infected by virus. In this study, the upstream promoter region of grass carp (Ctenopharyngodon idella) Gig2 gene (CiGig2) was identified by homology cloning strategy. CiGig2 promoter sequence was found to be 859 bp in length and contained three scattered IFN-stimulated response elements (ISRE). In addition, some grass carp IRFs (CiIRF1, CiIRF2 and CiIRF3) ORF sequences were subcloned into the expression plasmids pET-32a and expressed in Escherichia coli BL21, then the expressed proteins were purified by affinity chromatography with the Ni-NTA His-Bind Resin. Gel mobility shift assay was employed to screen the transcriptional regulatory factor for CiGig2. The results revealed that the recombinant polypeptides of CiIRF1, CiIRF2 and CiIRF3 bound to CiGig2 promoter with high affinity; indicating that IRF1, IRF2 and IRF3 could be the potential transcriptional regulatory factors for Gig2. Subsequently, CiGig2 promoter sequence was cloned into pGL3-Basic vector and the ORFs of CiIRF1, CiIRF2 and CiIRF3 were cloned into the expression plasmids pcDNA3.1 (+). Then, pGL3-CiGig2 promoter sequence and pcDNA3.1-CiIRFs were co-transfected into C. idella kidney (CIK) cells. The in vivo effects of CiIRFs on CiGig2 promoter were measured by dual-luciferase assays in the transfected CIK cells. Our results showed that the roles of CiIRFs were diversified in regulating CiGig2 transcription, e.g., CiIRF3 played a positive role in during this process; on the contrary CiIRF1 worked as a suppressor; however the effect of CiIRF2 on CiGig2 transcription was not obvious. For further study the roles of the three ISREs in CiGig2 transcription, we cloned three mutant CiGig2 promoters called ISRE1mut-luc (deleted ISRE1), ISRE2mut-luc (deleted ISRE2) and ISRE3mut-luc (deleted ISRE3), respectively. In vitro, gel mobility shift assays showed that all three mutant promoters also were combined with CiIRFs. CIK cells were co-transfected with CiGig2 promoter mutants (ISRE1mut-luc, ISRE2mut-luc or ISRE3mut-luc, respectively) and pcDNA3.1-IRFs. The results suggested that different ISRE played the diverse roles. ISRE2 is more important than ISRE1 and ISRE3 to the transcription of CiGig2 induced by CiIRF1. ISRE1 and ISRE3 are important to the transcription of CiGig2 induced by CiIRF2 and CiIRF3., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The interferon-induced, dsRNA-activated protein kinase (PKR) is considered as an important component of innate immune system and as a representative effector protein of interferon system. In the present study, PKR gene (CiPKR, JX511974) from grass carp (Ctenopharyngodon idellus) was isolated and identified using homology-based PCR. CiPKR shares high sequence identity with the counterparts of goldfish (Crucian carp) and zebrafish (Danio rerio). The full-length cDNA of CiPKR was found to be 2436 bp, with an ORF of 2067 bp that encodes a polypeptide of 688 amino acids. The deduced polypeptide CiPKR contains three tandem dsRNA-binding motifs (dsRBMs) at the N-terminus and a conserved Ser/Thr kinase domain at the C-terminus. CiPKR was expressed ubiquitously at a low-level under normal conditions, but it could be up-regulated after intraperitoneal (ip) injection with grass carp haemorrhagic virus (GCHV). CiPKR was dramatically up-regulated at 6 h post-injection and then recovered rapidly to normal levels within 24 h; however, it was obviously up-regulated once again at 48 h or 72 h post-injection. It seemed that CiPKR could respond to GCHV infection in an IFN-independent as well as an IFN-dependent pathway. To further investigate its mechanism of biological actions, we constructed a series of recombinant plasmids including pcDNA3.1/PKR-wt, pcDNA3.1/PKR-K430R, pcDNA3.1/PKR-C (deletion of dsRBD sequence) and pcDNA3.1/PKR-C-K430R, and then each recombinant plasmid was transfected into CIK cells. In comparison with those of controls, a 79% and a 64% decrease of luciferase activities were detected in the tested cells transfected with CiPKR and CiPKR-C, respectively; however, luciferase activities were increased in those cells transfected with PKR-K430R and PKR-C-K430R, with a 160% and 115% up-regulation, respectively. Similarly, MTT colorimetric assay indicated that cell viabilities of CIK cells transfected with pcDNA3.1/PKR-wt, pcDNA3.1/PKR-K430R, pcDNA3.1/PKR-C and pcDNA3.1/PKR-C-K430R were 49%, 90%, 54% and 100%, respectively. Our observations suggested that the expression of CiPKR could be up-regulated following viral infection, and then resulted in the inhibition of protein synthesis and the induction of potential apoptosis., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Interferon regulatory factors (IRFs) are well-known to be crucial for modulating the innate immune responses to viral infections. In the present study, the IRF-1 gene of grass carp (Ctenopharyngodon idella) (termed CiIRF-1) was cloned and characterized. The complete genomic sequence of CiIRF-1 was 3150 bp in length and comprised 9 exons and 8 introns. The CiIRF-1 promoter sequence was 558 bp in length. The largest open reading frame (ORF) of the full CiIRF-1 cDNA sequence was 870 bp, and encoded a polypeptide of 289 amino acids. The putative CiIRF-1 was characterized by a conserved N-terminal DBD (113 aa), and included a signature of five conserved tryptophan residues. Phylogenetic relationship analysis revealed that CiIRF-1 was highly homologous to the counterparts of other teleosts and mammalians. CiIRF-1 was expressed at a low constitutive level but was significantly up-regulated following stimulation with either Poly I:C or recombinant grass carp (C. idella) IFN (rCiIFN) in all 6 tested tissues, especially in spleen and gill. The recombinant CiIRF-1 was expressed in BL21 Escherichia coli, and the expressed protein was purified by affinity chromatography with the Ni-NTA His-Bind Resin. Three different fragments of promoter sequences from grass carp type I IFN (CiIFN) gene (GU139255) were amplified. These fragments included the proximal region (CiIFNP2), the distal region (CiIFNP6), and the full length of CiIFN promoter sequences (CiIFNP7). Gel mobility shift assays were employed to analyze the interaction between CiIRF-1 and CiIFN promoter sequences. The results revealed that CiIRF-1 could bind to CiIFN promoter with high affinity in vitro. Subsequently, the recombinant plasmid of pGL3-CiIFNPs and pcDNA3.1-CiIRF-1 were constructed and transiently co-transfected into C. idella kidney (CIK) cells. The impact of CiIRF-1 on CiIFN promoter sequences were measured by luciferase assays. These results demonstrated that CiIRF-1 acts as a positive regulator in the transcription of grass carp IFN gene., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Type I interferons and interferon regulatory factor 7 (IRF7), which are crucial for innate immunity against viral infection, have been identified in many teleost fishes in recent years. In this study, the complete genomic sequence of grass carp (Ctenopharyngodon idella) type I interferon (termed CiIFN) (GU139255) and the full-length IRF7 cDNA sequence of grass carp (termed CiIRF7) (GQ141741) were cloned and characterized. CiIFN consists of 3368 bp, retaining the characteristic 5-exon/4-intron gene organization in fish type I IFNs. The CiIFN spans 5 exons and encodes a polypeptide of 180 amino acids, with the first 22 amino acids representing a putative signal peptide. The CiIFN promoter sequence was found to be 760 bp, which can be divided into a proximal region (from -1 to -140 bp) and a distal region (from -400 to -700 bp). The cDNA of CiIRF7 was found to be 1808 bp in full length, with an ORF of 1293 bp that encodes a putative protein of 430 amino acids. The putative amino acid sequence of CiIRF7 possesses a DNA-binding domain (DBD) in the N-terminal region. Real-time PCR analysis revealed that CiIFN displayed a low constitutive expression in all the tissues tested. After stimulation by polyinosinic:polycytidylic acid (Poly I:C), the expression of CiIFN was significantly up-regulated in most tissues of grass carp, with a relatively strong expression in spleen, kidney and intestine. The recombinant polypeptides of CiIRF7 and CiIRF7-nDBD were analyzed in gel mobility shift assays, along with the PCR amplification products of the proximal region (CiIFNP2), the distal region (CiIFNP6) and the full-length (CiIFNP7) of CiIFN promoter sequence. The results revealed that CiIRF7 could bind to the distal region as well as to the proximal region of CiIFN promoter sequence in vitro. Subsequently, the CiIFNPs (CiIFNP7/2/6) were cloned into pGL3-Basic vectors and CiIRF7 was subcloned into pcDNA3.1 vectors, then pGL3-CiIFNPs were separately transiently transfected or co-transfected with pcDNA3.1-CiIRF7 into the mouse myeloma cell lines (MMCL) SP2/0 and the grass carp kidney cell lines (CIK), and the impact of CiIRF7 on CiIFN promoter activity was measured by luciferase assays in the transfected cells. These results demonstrated that CiIRF7 acted as a positive regulator on the transcription of CiIFN., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The new teleost fish PKZ (PKR-like) full-length cDNA (GU299765) had been cloned and identified from grass carp (Ctenopharyngodon idellus). The cDNA of grass carp PKZ (CiPKZ) has 2185 bp in length with a largest open reading frame (ORF) encoding 513aa. CiPKZ possesses a conserved C-terminal catalytic domain of eIF2α kinase family. Within its N-terminal there are two binding domain (Zα) named Zα1 (1-67aa) and Zα2 (81-152aa). BLAST homologous search reveals that CiPKZ has a high-level homology with other fish PKZs and PKRs. Like other fish PKZs and PKRs, CiPKZ is a ubiquitous tissue expression gene that had a very low level of constitutive expression but up-regulated in response to Poly I:C or hot stress (34 °C). For the purpose of searching for the potential function of CiPKZ, we obtained CiPKZ polypeptide via Escherichia coli Rosetta prokaryotic expression and purified with Ni-NTA His-Bind Resin affinity chromatography. CiPKZ polypeptide was used for the test of phosphorylating eIF2αin vitro. The results demonstrated that CiPKZ could be activated by Z-DNA but not by Poly I:C, and with subsequent could phosphorylate eIF2α. Meanwhile, four pcDNA3.1/PKZ recombinant plasmids, including pcDNA3.1/PKZ-wet, pcDNA3.1/PKZ-wet-K198R, pcDNA3.1/PKZ-wet-C, pcDNA3.1/PKZ-wet-C-K198R had been constructed, respectively. Mouse Myeloma cells (Sp2/0) and Human Umbilical Vein Endothelial Cells (HUVEC) were transiently cotransfected with pcDNA3.1/PKZ recombinant plasmid and PGL-3-promoter plasmid. The results revealed that CiPKZ could greatly decrease luciferase level in these cells. Zα and the K198 amino acid residue may play a key role in its function., (Copyright © 2011. Published by Elsevier Ltd.)