Endoplasmic Reticulum Transmembrane Proteins ZDHHC1 and STING Both Act as Direct Adaptors for IRF3 Activation in Teleost.

IFN regulatory factor (IRF)3 is a central regulator for IFN-ß 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.

Ctenopharyngodon idella IKKß interacts with PKR and IκBα.

Inhibitor of nuclear factor kappa-B kinase ß (IKKß) is a subunit of the IKK complex. It can activate the NF-κB pathway through phosphorylating IκB in response to a wide range of stimuli. In the present study, an IKKß gene from grass carp (Ctenopharyngodon idella; KT282114) was cloned and identified by homologous cloning and rapid-amplification of cDNA ends (RACE) technique. The complete CiIKKß cDNA is 3428 bp in length, with the longest open reading frame (ORF) of 2337 bp encoding a polypeptide of 778 amino acids. The deduced amino acid sequence of CiIKKß has similar domain distribution to those of mammalian. For example, CiIKKß consists of a serine/threonine kinase domain at the N-terminal, a basic region leucin zipper (BRLZ) domain in the middle, a homeobox associated leucin zipper (HALZ) domain and an IKKß NEMO (NF-κB essential modulator) binding domain at the C-terminal. Phylogenetic tree analysis also showed that CiIKKß is highly homologous to zebrafish IKKß (DrIKKß) and clearly distinct from the mammalian and amphibian counterparts. The expression of CiIKKß was ubiquitously found in the liver, intestine, kidney, gill, spleen, heart, and brain tissues of grass carp and significantly up-regulated in CIK cells under the stimulation with Poly I:C and UV-inactivated grass carp hemorrhagic virus. To investigate the activation mechanism of NF-κB pathway in fish and the role of CiIKKß in the pathway, we explored the protein interactions of protein kinase R (PKR) with IKKß and IKKß with IκBα by co-immunoprecipitation and GST-pull down assays. The interaction between each pair was confirmed. The results suggest that CiIKKß may be a primary member in the activation of NF-κB pathway in fish.

Transcriptome data analysis of grass carp (Ctenopharyngodon idella) infected by reovirus provides insights into two immune-related genes.

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.

Poly I:C facilitates the phosphorylation of Ctenopharyngodon idellus type I IFN receptor subunits and JAK kinase.

Members of the Janus kinase (JAK) family, JAK1 and TYK2 take part in JAK-STAT signaling pathway mediated by interferon in mammalian cells. Similar to the mammalian counterparts, fish JAK1 and TYK2 also perform their potential biological activities by phosphorylating cytokine receptors and STAT. In the present study, Ctenopharyngodon idellus JAK1 (CiJAK1) and TYK2 (CiTYK2) were cloned and identified. The full-length cDNA of CiJAK1 (KT724352.1) is 3829 bp, with an Open Reading Frame (ORF) of 3465 bp encoding a putative protein of 1154 amino acids. The full-length cDNA of CiTYK2 (KT724353.1) is 4337 bp, including an ORF of 3168 bp encoding 1055 amino acids. Structurally, both of them have B41, SH2, TyrKc and TyrKc common domains. CiJAK1 and CiTYK2 share a high degree of homology with their respective counterparts from Danio rerio and Cyprinus carpio by phylogenetic tree analysis. Polyinosinic-polycytidylic acid (Poly I:C), a synthetic dsRNA analogue, can launch the JAK-STAT antiviral signaling pathway. To elucidate the molecular mechanism of Poly I:C initiating the antiviral signaling pathway in fish, C. idellus kidney (CIK) cells were stimulated with Poly I:C and then the cell lysates were separated on 10% SDS-PAGE. The results showed that not only Poly I:C drastically increased the expression level of CiJAK1 and CiTYK2, but also it induced the phosphorylation of CiJAK1 and CiTYK2, as well as C. idellus type I IFN receptor subunits, CiCRFB1 and CiCRFB5. In detail, the levels of p-CiJAK1 and p-CiTYK2 were evidently up-regulated at 3 h post stimulation; however the phosphorylation levels of CiCRFB1 and CiCRFB5 displayed a sharp up-regulation at 12 h post stimulation of Poly I:C. As a basic mechnism of feedback regulation of JAK-STAT signaling pathway, overexpression of CiCRFB1 and CiCRFB5 in CIK cells facilitated the phosphorylation of CiJAK1 and CiTYK2.

Ctenopharyngodon idella NF-κB subunit p65 modulates the transcription of IκBα in CIK cells.

NF-κB is an important transcription factor for regulating the multiple inflammatory and immune related gene transcription. It can bind with the nuclear factor κB site within the promoter of target genes to regulate their transcriptions. p65, the all-important subunit of NF-κB, is ubiquitously expressed in cells. In the present study, we cloned and identified the p65 subunit from grass carp (Ctenopharyngodon idella) (named Cip65) by homologous cloning and RACE technique. The full length of Cip65 cDNA is 2481 bp along with 9 bp 5' UTR, 639 bp 3' UTR and the largest open reading frame (1833 bp) encoding a polypeptide of 610 amino acids with a well conserved Rel-homology domain (RHD) in N-terminal and a putative transcription activation domain (TAD) in C-terminal. Cip65 gathers with other teleost p65 proteins to form a fish-specific clade clearly distinct from those of mammalian and amphibian counterparts on the phylogenetic tree. In CIK (C. idellus kidney) cells, the expression of Cip65 was significantly up-regulated under the stimulation with Poly I:C. As one member of the NF-κB inhibitor protein (IκB) family, IκBα can dominate the activity of NF-κB by interacting with it. To study the molecular mechanisms of negative feedback loop of NF-κB signaling in fish, we cloned grass carp IκBα (CiIκBα) promoter sequence. CiIκBα promoter is 414 bp in length containing two RelA binding sites and a putative atypical TATA-box. Meanwhile, Cip65 and its mutant proteins including C-terminus deletion mutant of Cip65 (Cip65-ΔC) and N-terminus deletion mutant of Cip65 (Cip65-ΔN) were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind resin. In vitro, Cip65 rather than Cip65-ΔC and Cip65-ΔN showed high affinity with CiIκBα promoter sequence by gel mobility shift assays. In vivo, the cotransfection of pcDNA3.1-Cip65 (or pcDNA3.1-Cip65-ΔC, pcDNA3.1-Cip65-ΔN respectively) with pGL3-CiIκBα and pRL-TK renilla luciferase plasmid into CIK cells showed that pcDNA3.1-Cip65 rather than pcDNA3.1-Cip65-ΔC and pcDNA3.1-Cip65-ΔN, can increase the luciferase activity. Taken together, these results suggested that Cip65 can regulate the expression of CiIκBα and works as a negative feedback loop in NF-κB pathway.

Identification and characterization of a constitutively expressed Ctenopharyngodon idella ADAR1 splicing isoform (CiADAR1a).

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.

The transcription regulation analysis of Ctenopharyngodon idellus PKR and PKZ genes.

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.

Fish IRF3 up-regulates the transcriptional level of IRF1, IRF2, IRF3 and IRF7 in CIK cells.

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.

A splicing isoform of Ctenopharyngodon idella ADAR1 (CiADAR1-like): Genome organization, tissue specific expression and transcriptional regulation.

Catalyzing the deamination of adenosine to inosine in RNA, ADAR1 (adenosine deaminase that act on RNA 1) belongs to ADAR family. In our previous work, we have cloned the complete genomic sequence of ADAR1 from grass carp (Ctenopharyngodon idella), named CiADAR1. In the process, we found a splicing isoform of CiADAR1 (CiADAR1-like). CiADAR1 and CiADAR1-like are possessed by different promoters but share a common exon 2. The complete genomic CiADAR1-like has 9 exons and 8 introns. Its full-length cDNA is comprised of a 5' UTR (417 bp), a 3' UTR (118 bp) and a 3324 bp-long ORF encoding a polypeptide of 1107 amino acids. The deduced amino acid sequence of CiADAR1-like contains two Z-DNA binding domains, three dsRNA binding motifs and a truncate catalytic domain. CiADAR1-like shared higher homology with Danio rerio ADAR1 and lower homology with HsADAR1-like in phylogenetic tree. qRT-PCR showed that CiADAR1-like were ubiquitously expressed and significantly up-regulated after stimulation with Poly I:C. Its mRNA reached the peak at 12 h post-stimulation in all tested tissues. Western-blotting experiment proved CiADAR1-like was factually expressed in C. idella kidney (CIK) cells. To further study the transcriptional regulatory mechanism of CiADAR1-like, we cloned its promoter sequence. CiADAR1-like promoter is 1173 bp in length containing 3 ISRE and 8 IRF-E. Subsequently, grass carp IRF1 (CiIRF1) and IRF3 (CiIRF3) were expressed in Escherichia coli BL21 and purified by affinity chromatography with the Ni-NTA His-Bind Resin. In vitro, CiIRF1 and CiIRF3 were able to bind to CiADAR1-like promoter with high affinity in gel mobility shift assays, revealing that IRF1 and IRF3 could be the potential transcriptional regulatory factors for CiADAR1-like. In vivo, Co-transfection of pcDNA3.1-IRF1 (or pcDNA3.1-IRF3) with pGL3-CiADAR1-like promoter into CIK cells showed that both IRF1 and IRF3 significantly increased the luciferase activity, suggesting that they play a positive role in CiADAR1-like transcription.

Cloning, identification of the two cytokine receptor family B subunits CRFB1 and CRFB5 from grass carp (Ctenopharyngodon idella).

Similar to the mammalian counterparts, fish type I interferon (IFN) performs its potential biological activities via binding to the corresponding receptor on target cell membrane. Fish type I IFN receptor, a kind of enzyme-linked receptor, consists of two subunits and belongs to the class II cytokine receptor family B (CRFB). In the present study, we cloned and identified two putative grass carp (Ctenopharyngodon idella) type I interferon receptor subunits (termed CiCRFB1 and CiCRFB5) by homology cloning techniques. Phylogenetic tree analysis suggested that CiCRFB1 and CiCRFB5 shared highly homology to Danio rerio CRFB1 and CRFB5 respectively. CiCRFB1 and CiCRFB5 were up-regulated after the stimulation with Grass Carp Hemorrhagic Virus (GCHV) and Polyinosinic-polycytidylic acid (Poly I:C), indicating that they are related to the intracellular antiviral activity. In order to know more about the roles of CiCRFB1 and CiCRFB5 in the process, the extracellular domains of CiCRFB1 (CiCRFB1-EC) and CiCRFB5 (CiCRFB5-EC), as well as grass carp type I IFN (CiIFN) were expressed in Escherichia coli BL21, and purified by affinity chromatography with the Ni-NTA His-Bind resin. Cross-linking reactions were employed to analyze the affinity of the ligand (CiIFN) with the two putative receptor subunits (CiCRFB1-EC and CiCRFB5-EC). The result suggested the formation of (CiCRFB5)2 homodimer was more easily than that of (CiCRFB1)2 under the induction of CiIFN in vitro. However, CiIFN was inclined to bind to (CiCRFB1)2 homodimer. Interestingly, although CiIFN seemed unable to facilitate the formation of (CiCRFB1 + CiCRFB5) heterodimer in the absence of DSS cross linker, however it can bind to the heterodimer in the presence of DSS. This indicated that the homodimer and the heterodimer were the potential receptor for CiIFN.