Screening And Identification Of Protein Interacting With RNA-dependent RNA Polymerase And Its Functional Studies In Eimeria Tenella

E.tenella causes coccidiosis in chickens,characterized by bloody stools and diarrhea and resulting in death.It has a significant impact on the poultry industry worldwide.The application of anticoccidial drugs and vaccines is the main strategy used to control coccidiosis in poultry.However,due to limited knowledge of the cellular and molecular biology of the Coccidia parasite and the issue of drug resistance and residues,there are no ideal drugs or vaccines to control coccidiosis.Many protozoa contain double stranded RNA virus,which can affect the virulence or reproduction of the protozoa.Infected with E.tenella with viruses cecum lesion were more serious such as edema,disintegration of intestinal epithelial cell rupture and intestinal mucosa damage fracture compared with infected with E.tenella without viruses.However,the relationship between the E.tenella virus and the E.tenella is unclear.In this study,the yeast two hybrid technique was used to screen the RDRP of E.tenella virus interacting-protein OTU of E.tenella and the deubiquitinase（DUB）activity of OTU was analyzed.It has also been reported that the OTU DUB was a telomere related protein and it associated with activity of telomerase（TERT）.Therefore,the interaction between the E.tenella telomerase reverse transcriptase RNA binding region（TRBD）and E.tenella OTU protein was determined by yeast two hybrid and pull down test,and the effect of OTU on the activity of telomerase reverse transcriptase（TERT）was studied.Screening and Identification of Etv-RDRP interactiing proteins:the recombinant bait plasimd pGBKT7-RDRP was constructed.pGBKT7-RDRP was transformed into yeast competent cells.The results showed that Hypothetical protein,ribosomal protein,Uncharacterized ACR and OTU were obtained.The OTU was found to associate with TRBD based onα-galactosidase assay.The RDRP and OTU were cloned into Eukaryotic expression vector pFast-Bac-HTA and pFast-Bac-dual,respectively.The expression and purification of OTU and RDRP in Sf9 insect cells.SDS-PAGE showed that both recombinant proteins could be expressed in Sf9.The result of pull down and immunoprecipitation verify the interaction in Extracellular and intracellular.Identification of OTU deubiquitinatase activity and the effect of RDRP on the activity of OTU:The selected Pp plasmid was sequenced by Comate Bioscience Co.Ltd.（Jilin,Changchun,China）and analysed using BLASTX from the National Center for Biotechnology Information（NCBI）website.Et-OTU^C239A、Et-OTU^H351A、Et-OTU ^D236A236A was constructed and the protein was expression,and purification in Sf9respectively.Characterisation of the Et-OTU deubiquitination enzyme substrate preference in vitro was analyzed by K63-,K11-,K29-,or K33-linked diubiquitin.Mutations in the catalytic core（C239A and H351A）eliminated the DUB activity of Et-OTU,whereas the D236A mutation did not.The hydrolytic efficiency of GST-OTU towards K48-and K6-linked chains was lower than that of Etv-RDRP combined with Et-OTU.E.tenella OTU interacted with virus RDRP and telomerase.The deubiquitination active site and specificity of Et-OTU were also analysed in vitro.Furthermore,combining Etv-RDRP with Et-OTU enhanced the DUB activity of Et-OTU in vitro.This research provided insights into the symbiosis between E.tenella and Etv.Screening and Identification of Et-TRBD interactiing proteins:the recombinant bait plasimd pGBKT7-TRBD was constructed.pGBKT7-TRBD was transformed into yeast competent cells.The results showed that Uridine phosphorylase,OTU,and disulfide bond isomerase were obtained.The OTU was found to associate with TRBD based onα-galactosidase assay.The TRBD and OTU were cloned into prokaryotic expression vector pET-32a and pGEX-4T-1,respectively.Then expression and purification of E.tenella TRBD and OTU were performed.SDS-PAGE showed that both recombinant proteins could be expressed in the soluble form.GST-OTU and His-TRBD purified were incubated and applied to pull-down assay to verify the interaction.Results showed His-TRBD bound directly to GST-OTU in vitro.The effect of OTU on telomerase activity:Hammerhead ribozyme synthesized for cleavage OTU were cloned into a viral vector derived from the genome of E.tenella virus,and pEtV-GFP-Ham-OTU was obtained.In vitro,the cleavage activity of pEtV-GFP-Ham-OTU transcripts on OTU mRNA was 85%.pEtV-GFP-Ham-OTU transcripts were introduced into E.tenella by electroporation.Western-blot indicated that the expression level of OTU was down-regulated 65%,comparing with WT strain.When the expression of OTU was inhibited,the telomerase activity was down-relugated.The results indicated that OTU may positively regulate telomerase activity.Eimeria tenella OTU interacted with virus RDRP and telomerase.The deubiquitination active site and specificity of Et-OTU were also analysed in vitro.Furthermore,combining Etv-RDRP with Et-OTU enhanced the DUB activity of Et-OTU in vitro.This research provided insights into the symbiosis between E.tenella and Etv.