Cloning, expression and functional analysis of PKR from grass carp (Ctenopharyngodon idellus).

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.

Hu YS ，Li W ，Li DM ，Liu Y ，Fan LH ，Rao ZC ，Lin G ，Hu CY ... -  《-》

Identification and function analysis of the three dsRBMs in the N terminal dsRBD of grass carp (Ctenopharyngodon idella) PKR.

The protein kinase R (PKR) can inhibit protein translation and lead to apoptosis under the circumstances of virus invasion and multiple other stress conditions. PKR is a dsRNA binding protein with a dsRBD and a kinase domain (KD). dsRBD is mostly composed of two (in mammal PKR) or three (in some fish PKR) dsRNA binding motifs (dsRBMs). Multiple sequences alignment and Phylogenetic analysis indicate that the three dsRBMs of fish PKR share analogous structure but show to be divergence origination. In this study, we have identified and analyzed the three dsRBMs from grass carp (Ctenopharyngodon idellus) PKR (CiPKR), which was cloned previously in our laboratory. dsRBMs of CiPKR have two or three conserved regions involved in dsRNA binding. Among the three dsRBMs, dsRBM1 was peculiar to some fish PKRs, while dsRBM2 and dsRBM3 were closely related to the dsRBM1 and dsRBM2 of mammal PKRs respectively. Dimerization assay indicated that dsRBM1 and dsRBM2 formed not only homo-dimer but also homo-multimer; whereas dsRBM3 formed merely homo-dimer. Meanwhile, dsRBM1-2, dsRBM2-3 and dsRBM1-2-3 could homo-dimerize and homo-multimerize also. Poly I:C pull-down assay showed that the binding of dsRBM to Poly I:C needed two or three dsRBMs to cooperate in vitro, meaning one dsRBM from CiPKR could not bind to dsRNA efficiently. To further investigate the effect of dsRBM on the function of CiPKR, we constructed pcDNA3.1/CiPKR-wt and a series of CiPKR mutants recombined plasmids including pcDNA3.1/CiPKR-ΔdsRBM2-3, pcDNA3.1/CiPKR-ΔdsRBM1,3, pcDNA3.1/CiPKR-ΔdsRBM1-2, pcDNA3.1/CiPKR-ΔdsRBM3, pcDNA3.1/CiPKR-ΔdsRBM1. The recombined plasmids respectively were co-transfected with plasmid PGL3 promoter into CIK cells. In comparison with the control group, the luciferase translation inhibitions were 78.7%, 15%, 0, 0.5%, 61.8%, 67.3% respectively. The results indicated that the protein translation inhibition caused by CiPKR mutants with only one dsRBM were very weak, while those with two or three dsRBMs inhibited the protein translation powerfully. Cell viability were 34.2%, 98.2%, 112%, 108%, 50.3%, 47.5% respectively after transfected with pcDNA3.1/CiPKR-wt, pcDNA3.1/CiPKR-ΔdsRBM2-3, pcDNA3.1/CiPKR-ΔdsRBM1,3, pcDNA3.1/CiPKR-ΔdsRBM1-2, pcDNA3.1/CiPKR-ΔdsRBM3, pcDNA3.1/CiPKR-ΔdsRBM1 in order into CIK cells for 48 h. The results from cell counting also indicated that transfection of CiPKR-wt and the mutants CiPKR-ΔdsRBM3, CiPKR-ΔdsRBM1 could inhibit the protein translation and facilitated the decrease of CIK cells number. In conclusion, our observations suggested that two dsRBMs ranking in tandem at N terminal were essential for the function of CiPKR, and the presence of the extra dsRBM1 enhanced its function.

Hu Y ，Fan L ，Wu C ，Wang B ，Sun Z ，Hu C ... -  《-》

Ctenopharyngodon idella IRF2 plays an antagonistic role to IRF1 in transcriptional regulation of IFN and ISG genes.

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.

Gu M ，Lin G ，Lai Q ，Zhong B ，Liu Y ，Mi Y ，Chen H ，Wang B ，Fan L ，Hu C ... -  《-》

IRF-1 acts as a positive regulator in the transcription of grass carp (Ctenopharyngodon idella) IFN gene.

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.

Lai Q ，Lin G ，Ma M ，Huang S ，Li W ，Li D ，Gu M ，Mao H ，Hu C ... -  《-》

Gig1 and Gig2 homologs (CiGig1 and CiGig2) from grass carp (Ctenopharyngodon idella) display good antiviral activities in an IFN-independent pathway.

The virus-induced genes, Gig1 and Gig2, were identified first as IFN-stimulated genes (ISGs) from CAB cells. Previous studies suggested that Gig protein may have some potential antiviral functions. In this study, we cloned and identified the full-length cDNA sequences of Gig1 and Gig2 homologs (designated as CiGig1 and CiGig2, respectively) from grass carp (Ctenopharyngodon idella). The complete cDNA sequences of Gig1 and Gig2 contain 1231 bp and 690 bp, encoding for a 194 amino acid protein and a 158 amino acid protein, respectively. Their structure characteristics of CiGig1 and CiGig2 are highly similar to the corresponding homologues in crucian carp. The tissue-specific expressions of CiGig1 and CiGig2 in liver, spleen, kidney, intestine, gill and heart were significantly up-regulated following GCHV challenge. The results indicated that CiGig1 and CiGig2 may be involved in the antiviral immune responses in cells. To better understand the antiviral functions of CiGig1 and CiGig2 in vivo, CiGig1 or CiGig2 ORF cDNA were inserted into the plasmid pcDNA3.1, respectively. Subsequently, the recombinant plasmids were transfected into C. idellus kidney (CIK) cells. The over-expressions of CiGig1 and CiGig2 were observed in the CIK cells after treatment with GCHV. Cells with pcDNA3.1-CiGig1 or pcDNA-CiGig2 exhibited a relatively higher survival rate of (70.84% or 69.24%) than non-transfection (22.16%) and mock-vehicle controls (24.38%) following the virus infection. Our data showed that both CiGig1 and CiGig2 could exert antiviral effects effectively in vivo. Cycloheximide blocking protein synthesis demonstrated that both CiGig1 and CiGig2 mRNA expression could be induced by GCHV rather than by recombinant grass carp IFN (rCiIFN) directly, suggesting that CiGig1 and CiGig2 may not be IFN-stimulated genes since they display their antivirus activities in an IFN-independent pathway.

Sun C ，Liu Y ，Hu Y ，Fan Q ，Li W ，Yu X ，Mao H ，Hu C ... -  《-》