The Effect Of DsRBM On CiPKR Controlling The Protein Translation

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 ds RNA binding protein with a ds RBD and a kinase domain(KD). Ds RBD is mostly composed of two(in mammal PKR) or three(in some fish PKR) ds RNA binding motifs(ds RBMs). Multiple sequences alignment and Phylogenetic analysis indicate that the three ds RBMs of fish PKR share analogous structure but show to be divergence origination. In this study, we have identified and analyzed the three ds RBMs from grass carp(Ctenopharyngodon idellus) PKR(Ci PKR). ds RBMs of Ci PKR have two or three conserved regions involved in ds RNA binding. Among the three ds RBMs, ds RBM1 was peculiar to some fish PKRs, while ds RBM2 and ds RBM3 were closely related to the ds RBM1 and ds RBM2 of mammal PKRs respectively. Dimerization assay indicated that ds RBM1 and ds RBM2 formed not only homo-dimer but also homo-multimer; whereas ds RBM3 formed merely homo-dimer. Meanwhile, ds RBM1-2, ds RBM2-3 and ds RBM1-2-3 could homo-dimerize and homo-multimerize also. Poly I:C pull-down assay showed that the binding of ds RBM to Poly I:C needed two or three ds RBMs to cooperate in vitro, meaning one ds RBM from Ci PKR could not bind to ds RNA efficiently. To further investigate the effect of ds RBM on the function of Ci PKR, we constructed pc DNA3.1/Ci PKR-wt and a series of Ci PKR mutants recombined plasmids including, Ci PKR-Δds RBM2-3, Ci PKR-Δds RBM1,3, Ci PKR-Δds RBM1-2, Ci PKR-Δds RBM3, Ci PKR-Δds RBM1. 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 Ci PKR mutants with only one ds RBM were very weak, while those with two or three ds RBMs inhibited the protein translation powerfully. Cell viability were 34.2 %, VII 98.2 %, 112 %, 108 %, 50.3 %, 47.5 % respectively after transfected with Ci PKR-wt, Ci PKR-Δds RBM2-3, Ci PKR-Δds RBM1,3, Ci PKR-Δds RBM1-2, Ci PKR-Δds RBM3, Ci PKR-Δds RBM1 in order into CIK cells for 48 h. The results from cell counting also indicated that transfection of Ci PKR-wt and the mutants Ci PKR-Δds RBM3, Ci PKR-Δds RBM1 could inhibit the protein translation and facilitated the decrease of CIK cells number. In conclusion, our observations suggested that two ds RBMs ranking in tandem at N terminal were essential for the function of Ci PKR, and the presence of the extra ds RBM1 enhanced its function.In order to study the relationship between the protein translation inhibition by Ci PKR and the translation of the immune antiviral cytokines in the antiviral response of the cells, we had cloned the most important targeted protein of Ci PKR, namely eukaryotic initiation factor 2α from grass carp(Cie IF2α). Interesting, we discovered two e IF2α genes in the grass carp cell for the first time, and it were named as Cie IF2α(Accession number: KJ126860.1, GI: 597914306) and Cie IF2α-like(Accession number: KJ126861.1, GI: 597914308) respectively. Moreover, we have also cloned the possibly existing eukaryotic translation initiation factor 2A(Cie IF2 A, Accession number: KJ126862.1, GI: 597914310) in the CIK cells, which was considered as the factor to bypass the e IF2α-dependent translation pathway in an e IF2α-independent manner, and it was closely related the translation of the cytokine proteins in the host antiviral response. It were showed by the quasi-quantitative assay and Q-PCR analysis of their m RNA level that Cie IF2α was downregulated and Cie IF2 A was upregulated by GCHV in the antiviral response of CIK cells. The varying transcipted expression patern implied that both of the two genes might display various function in the antivial response of CIK cells. Furthermore, there possibly existed other protein translation pathways to bypass the control of Ci PKR, it might be Cie IF2 A or Cie IF2α-like-dependent translation pathway. Then we constructed plasmids to express the fusion protein of Ci PKR、Cie IF2α and Cie IF2α-like in E. Coli., and the proteins were applied for the preparation of polyclonal antibodies by immune stimulus rabits. The efficiency of the polyclonal antibodies were indicated by ELISA and WB assay.By designing and synthesis Ci PKR specific si RNA oligoes, we have knockdown Ci PKR by using the method of RNAi after the induction of GCHV to the CIK cells. Then the expression levels of Ci PKR and Ci IFN were determined by Q-PCR and WB approaches. The results indicated that the levels of m RNA and protein of Ci PKR were significantly downregulated by the specific si RNA knocking down of Ci PKR; whereas the levels of the m RNA and protein of Ci IFN were upregulated. By using the PKR inhibitor 2-AP, the m RNA level of Ci PKR could not induced by GCHV, but the m RNA level of Ci IFN was upregulated too. The Ci PKR RNAi or PKR inhibitor could shut down the PKR-NF-κB pathway to induce the expression of Ci IFN and other ISGs, so the upregulation of Ci IFN might through other way to bypass the PKR/ NF-κB pathway. The TLR inhibitor chloroquine was also applied to check whether the antivral response induced by GCHV was transduced by TLR, and the results indicated that the induction by GCHV was controlled by TLR which was similar to the mammalian cells.