Proteomic Analysis On PK-15 Cells Infected With Pseudorabies Virus

Pseudorabies virus(PRV)can cause fever,itching and encephalomyelitis in a variety of animals.Pigs are the storage host and source of infection for PRV.PRV infectes pigs causes Pseudorabies,leading to symptoms like fatal encephalitis in newborn piglets,respiratory disorders in finishing pigs,and sow reproductive disorders.PRV belongs to the herpes virus family and is widely distributed throughout the world.In 2012,an unprecedented and large-scale outbreak of PRRSV hit northern and eastern china and resulted in huge economic losses of the pig industry.However,the pathogenesis of PRV is not clear yet.In order to further investigate the changes of gene expressions of host cells caused by PRV infection and to find potential targets for the treatment of PRV,isobaric tags for relative and absolute quantification(iTRAQ)were used to analyze the changes of whole genome protein expression in PRV infected porcine epithelial cells(PK15)in this current study.The expression of differentially expressed protein Bioinformatics analysis found that PRV infection can cause endoplasmic reticulum stress response.This experiment provides the molecular basis for further research on the interaction between PRV and host cells.1.Proteomics study of PRV-infected PK15 cellsThe one-step growth curve showed that the virus titer was highest at 24 h after the infection of PK15 cells by PRV ZJ(Zhejiang)strain.Cell lysates were extracted 24 hours after the infection,and the total protein was extracted.Cell proteomics analysis was performed using iTRAQ combined with liquid chromatography-tandem mass spectrometry(LC-MS/MS).The results showed that there were 4333 identified proteins in total,and 466 of them were the expressed protein(1.5-fold difference,P?0.05)which included 234 up-regulated proteins and232 down-regulated proteins.The Gene Ontology annotation results showed that in the molecular function of genes,the differentially expressed proteins mainly had the binding functions(51.11%),the catelytic activities(26.84%),and the regulator activity(4.56%).Among the cellular components,the differentially expressed proteins mainly in the cell part(18.62%),cell(18.62%)and organelle(17.39%).In the biological processes involved,the differentially expressed proteins were mainly concentrated in the cellular process(12.50%),cellular component organization or biogenesis(6.78%),and multicellular organismmal process accounted(5.43%).The Cluster of Orthologous Groups of proteins annotation results of different functional proteins showed that the differentially expressed proteins mainly focused on the general function prediction only,with 642 proteins.The number of protein in posttranslational modifications,protein turnover and chaperones is 318.The number of protein in translation ribosomal structure and biogenesis is 298.Pathway metabolic function types in KEGG(Kyoto encyclopedia of genes and genomes)annotation are different in all up-regulated and down-regulated proteins.The two Pathway functional annotation results have the same two top ten functions,namely metabolic pathways and microbial metabolism in diverse environments.Compared with annotated results of down-regulated proteins,the proportion of "metabolic pathways" in up-regulation of differential protein function annotation slightly increased.In order to verify the results of proteomics,we further validated the differentially expressed proteins of STAT1,GRB2,PCNA and beta-catenin by Western blot,and the results were basically consistent with those of proteomics.2.Molecular Mechanism of Activated Endoplasmic Reticulum Signaling Pathway in PRV InfectionPathway analysis showed that after PRV infection,the endoplasmic reticulum stress protein pathway in all 21 proteins were up-regulated,indicating that PRV infection activated endoplasmic reticulum stress response.Therefore,the investigation of the endoplasmic reticulum stress response pathways after PRV infection will help to further understand the host response after virus infection.First of all,PRV infection verified the protein pathway.The results showed that PRV infection in the early stage,the expression of GRP78,e IF2? and ATF4 protein were up-regulated,and in the late infection stage the expression of overall were down-regulated.This indicates that PRV infection activates the PERK pathway and ultimately inhibits protein synthesis.These genes can serve as potential molecular markers for PRV infected cells and provide new insights into the regulatory mechanisms of PRV infection in host cells...