Study On Subcellular Localization Signal Of Porcine Epide Mic Diarrhea Virus ORF3 Protein

Porcine epidemic diarrhea(PED)is a contagious intestinal disease caused by porcine epidemic diarrhea virus(PEDV)in swine with symptoms including vomiting,diarrhea and dehydration.Pigs of different ages can be infected,b ut usually suckling piglets suffer the most serious harm,resulting in serious economic losses in pig industry.The ORF3 is the only accessary gene of PEDV ORF3 gene encodes ORF3 protein,which is related with the pathogenecity of the vir us.The study on sub-cellular localization signal of PEDV ORF3 protein was carried o ut to understand the trafficking and pathogenecity mechanism of the protein.The contents and conclusions of this study are as follows:⑴ At the beginning of the experiment,the ORF3 protein was cloned into eukaryotic expression vector pEGFP-C1 to construct recombinant plasmid and transfected into Vero cells.After 16 h.p.t,ORF3 protein could be found in the cytoplasm of p EGFP-PEDV-ORF3 transfected cells,while in pEGFP-C1 transfected cells green fluorescence could be found both in the cytoplasm and nucleus.This suggests that the presence of a certain structure in the ORF3 protein or the additiona l ORF3 protein itself that influences cellular distribution of the fusion protein.⑵ To analysis whether there was a domain in the ORF3 protein that could influence trafficking of the protein,we divided the protein into two regions based on amino acid sequence feature of DR13 at t ORF3 and the predicted transmembrane region of CV777 wt.The first part is ORF3(1-173aa)which contains fo ur predicted transmembrane domains and the second part is ORF3(174-225aa)which contains non-transmembrane domains.Recombinant p lasmids were transfected into Vero cells.LCM analysis was made at 16 h.p.t.It was found that fusion protein was dstributed both in cytoplasm and nucleus of the cells transfected with p EGFP-PEDV-ORF3(174-225aa),while fusion protein was mainly distributed in the cytoplasm of the cells transfected with p EGFP-PEDV-ORF3(1-173aa),indicating that there was a domain in the part of the ORF3(1-173aa)that limited trafficking of the chimeric protein to the nucleus.⑶ To further analysis which part of ORF3(1-173aa)limited the ORF3 protein trafficking to the nucleus,the ORF3(1-173aa)was fragmented into two parts: the first part is O RF3(1-91aa)which contains TM1 and TM2.The second part is ORF3(92-173aa)which contains TM3 and TM4.Recombinant plasmids pEGFP-PEDV-ORF3(1-91aa),pEGFP-PEDV-ORF3(92-173aa)were transfected into Vero cells.LCM analysis was made at 16 h.p.t.It was shown that the the fusion protein was distributed both in cytoplasm and nucleus in the p EGFP-PEDV-ORF3(92-173aa)transfected cell.However,fusion protein was mainly distributed in the cytoplasm in the pEGFP-PEDV-ORF3(1-91aa)transfected cell,indicating that O RF3(1-91aa)is the key part of ORF3 that interfere the fusion protein to traffic to nucleus.⑷ In order to analysis which part of ORF3(1-91aa)had the cytoplasm location function,it was fragmented into ORF3(1-39aa)which contains no transmembrane domain and ORF3(40-91aa)which contains TM1 and TM2.Recombinant p lasmids pEGFP-PEDV-ORF3(1-39aa)and p EGFP-PEDV-ORF3(40-91aa)were transfected into Vero cells.LCM analysis was made at 16 h.p.t.It was shown that fusion protein existed both in the cytoplasm and nucleus in the p EGFP-PEDV-ORF3(1-39aa)transfected cells,but in the pEGFP-PEDV-ORF3(40-91aa)transfected cells fusion protein only existed in the cytoplasm.To verify that ORF3(40-91aa)is the key domain that b locked the fused protein trafficking to nucleus,the gene of 40-91 aa fragment was knocked o ut from the entire ORF3 gene.The construction was designated O RF3(1-39+92-225aa)and cloned into pEGFP-C1 to obtain the vector pEGFP-PEDV-ORF3(1-39+92-225aa).After transfecting the vector to Vero cells,LCM analysis was made at 16 h.p.t.It was to find o ut the fusion protein was distributed both in the cytoplasm and nucleus.The result further proved ORF3(40-91aa)is the key part of ORF3 that interfere the fusion protein trafficking to nucleus.⑸ There is a possibility that a part of ORF3(40-91aa)served as domain to block the fusion protein trafficking to nucleus.Therefore the ORF3(40-91aa)was fragmented into three parts:ORF3(40-63aa)which contains TM1,O RF3(64-74aa)which is the polypeptide between TM1 and TM2,ORF3(75-91aa)which contains TM2.Construction of recombinant p lasmids pEGFP-PEDV-ORF3(40-63aa)、pEGFP-PEDV-ORF3(64-74aa)、pEGFP-PEDV-ORF3(75-91aa)were transfected into Vero cells.LCM analysis was made at 16 h.p.t.It was found each vector had fusion protein expression both in the cytoplasm and nucleus,indicating that the three amino acid segments(40-63 aa,64-74 aa,75-91aa)could not block the fusion protein trafficking to nucleus separetly.⑹ There was a possibility that any two of three parts interact with each other to have the function.For this purpose we constructed vectors p EGFP-PEDV-ORF3(40-74aa),pEGFP-PEDV-ORF3(64-91aa)and pEGFP-PEDV-ORF3(40-63+75-91aa).The vector was transfected into Vero cells and checked the expression of the fusion protein by LCM.The results showed all the three vectors had fusion protein expression which distrib uted both in cytoplasm and nucleus.Therefore we could make a conclusion the 40-91 aa sequence was the smallest unit carrying the cytoplasm trafficking signal.