Screening And Validation Of Host Proteins Interacting With HEV ORF3 Of Different Genotypes

Hepatitis E is an acute self-limiting hepatitis caused by infection with Hepatitis E virus.The mortality rate of pregnant women infected with HEV can reach 20%.In recent years,hepatitis E has become one of the important diseases that endanger human health.In addition,it has important public health significance as a zoonotic disease.HEV is a member of the family Hepeviridae family and consists of non-eveloped,single-straneded and positive sense RNA viruses.The HEV isolated from mammalian can be divided into 8genotypes,of which genetypes 1 and 2 only infect humans,and genetypes 3,4 and 8 have human and animal infectivity.In China,the main epidemic genotypes in human are type 1and type 4,epidemic in swine is genotype 4 and epidemic in rabbit is genotype 3.HEV ORF3 encodes a small phosphorylated protein with strong antigenicity(p ORF3),which is about 113 amino acids in length.In recent years,with the in-depth study of HEV ORF3 protein,it was found that this protein is closely related to the release of virus and pathogenic damage to the host.However,the host proteins involved in this process have no relevant systematic research reports,of which hinders the study of the pathogenesis of HEV.In this study,we used a proteomic system to analyze the interaction between cellular proteins and ORF3 proteins from four different genotypes: SAR-55 for genotype 1,Kernow-C1 and CHN-SX-r HEV for genotype 3,genotype 4 for CHN-SD-s HEV.The bioinformatics was further used to analyze the physiological functions and their distribution in tissues of the host proteins interacting with ORF3 proteins.The research contents and results are briefly described as follows.1.Expression of HEV ORF3 protein carrying Fc tag in HEK293 T cellsPCR successfully amplified the gene fragment of the different genotype HEV ORF3 expected at 350 bp and the Ig G Fc fragment expected at 670 bp.Then,the recombinant eukaryotic expression plasmids of different genotype HEV ORF3 and Fc fusion proteins were successfully constructed by restriction enzyme digestion and ligation.The plasmids named p CAGEN-SAR-55-ORF3-Fc,p CAGEN-Kernow-C1-ORF3-Fc,p CAGEN-s HEV-ORF3-Fc,p CAGEN-r HEV-ORF3-Fc and p CAGEN-Fc.After transfecting the recombinant expression vector into HEK293 T,the results of immunofluorescence and Western blot indicated that different genotypes of HEV ORF3 and Fc fusion protein were successfully expressed in HEK293 T cells.2.Screening and validation of host proteins interacting with different genotypes of HEV ORF3 proteinsUsing the binding properties of Fc protein with Protein G,cell mixture of different genotypes of HEV ORF3 protein interacting with HEK293 T cell proteins were obtained by Co-IP.Silver staining analysis after SDS-PAGE revealed that there are multiple specific host proteins interacting with HEV ORF3 protein.Then,the interaction mixture was identified by LS-MS/MS and the results showed that there were 140 proteins interacting with Kernow-C1 ORF3 and 117 proteins interacting with SAR-55 ORF3,134 proteins interacting with the CHN-SD-s HEV ORF3 and 91 proteins interacting with the CHN-SX-r HEV ORF3.Using Gene Ontology(GO)to analyze protein function and signaling pathways,the cellular components(CC)of these proteins are mainly extracellular exosome,myelin sheath,mitochondrial,mainly involved in biological process is viral process,positive regulation of gene expression and cell-cell ashesion.From the interaction of 22 host proteins,MYPT1,MYO1 D,DBN1 and TXNDC5 host proteins were selected according to the results of mass spectrometry and protein function.Their interaction with HEV ORF3 protein was further confirmed by Western blot using specific antibodies against the above proteins.The endoplasmic reticulum protein TXNDC5 was identified as a research object by LS-MS/MS and experimental purposes.3.Identification of critical regions between host protein TXNDC5 and ORF3 protein interactionThe c DNA encoding the host protein TXNDC5 was amplified by RT-PCR;then,the eukaryotic expression vector p CAGEN-TXNDC5-Myc of TXNDC5 was successfully constructed by restriction enzyme digestion and ligation.HEK293 T was co-transfected with the eukaryotic expression vector p CAGEN-ORF3-Fc-His and p CAGEN-TXNDC5-Myc.Co-immunoprecipitation and Western blot further confirmed the interaction of TXNDC5 with ORF3 of different genotypes.The functional regions of different genotypes of HEV ORF3 protein were analyzed by bioinformatics software.The ORF3 protein fragment of different functional regions was amplified by PCR.The eukaryotic expression vector was constructed and co-transfected with p CAGEN-TXNDC5-Myc to HEK293 T.Co-IP and Western blot confirmed that the interaction region between ORF3 protein and TXNDC5 protein is in the N-terminal(amino acid 5-23)region of ORF3,which is highly conserved in ORF3 of different genotypes and is rich in cysteine.