Transcriptome And Proteome Analysis Of Porcine Epidemic Diarrhea Virus-infected Marc-145 Cells

Porcine epidemic diarrhea(PED)is an acute and highly contagious viral enteric epizootic caused by porcine epidemic diarrhea virus(PEDV),with the transmission via fecal-oral route and typical symptoms such as watery diarrhea,fever,anorexia,and lethargy.Although adult pigs only show mild illness when infected with PEDV and scarcely die of it,the virus is able to reach a morbidity of 100% and a mortality of 90% to 100% when infecting newborn piglets.PEDV was first identified in UK,1978,and has spread to many countries around the world,including China,undermining the development of swine industry.Therefore,greater demands are placed on the PEDV fundamental researches.Given modern sequencing technology based data can provide certain guides and supports,this study investigated the transcriptome and proteome of Marc-145 cells,infected or not with PEDV,via RNA sequencing(RNA-seq)as well as isobaric Tags for Relative and Absolute Quantification(iTRAQ)based LC-MS/MS.Both transcriptomic and proteomic data were obtained and submitted to bioinformatic analyses and associate analysis subsequently.The results were listed as following:PEDV-and mock-infected Marc-145 cells at 24 hpi were submitted to RNA-seq.The sequencing reads were acquired and then mapped to the reference genome sequence of Chlorocebus sabaeus.After the functional annotation and exprssion calculation,the differentially expressed genes(DEGs)were screened and submitted to the GO and KEGG Pathway enrichment analysis.The results showed as follows: A total of 22198 genes were identified and 832 DEGs were screened,with 497 up-regulated and 335 down-regulated.440,291 and 2700 GO terms were enriched in molecular function,cellular component and biological process ontologies respectively in GO analysis.27 GO terms were significantly enriched in biological process with most of them involved in signal transduction,stress response,and apoptosis,while the most enriched GO terms in molecular function and cellular component were mainly concerned with DNA binding and cytoskeleton components respectively.167 pathways,including ones associated with immunoreaction,apoptosis,andcell cycle,were enriched in KEGG analysis,with MAPK signaling pathway and Jak-STAT signaling pathway significantly enriched.Genes related to innate immune and inflammatory responses showed significant alterations.IFNB1,CXCL10,TRIM56,IL11 were observed being up-regulated,suggesting that host cells' innate immune and inflammatory responses were activated during the infection.However,genes like IFNA and IL23 A were significantly down-regulated,indicating a part of innate immune and inflammatory responses might be inhibited by PEDV through certain mechanisms.Significant alterations were also observed in genes associated with apoptosis,of which TRAILR,DFF40,NGF were up-regulated while TRAIL and BIRC3 were down-regulated.This interplay of both pro-and anti-apoptotic elements might contribute to the maximum viral replication before cell death.Cell cycle-related genes were significantly altered.The up-regulations of GADD45,p300,Mdm2 as well as the down-regulations of HSP105,HSP70,etc suggest that cell cycle arrest might be induced by PEDV.Proteomic sequencing was performed via LC-MS/MS coupled to iTRAQ labeling.Differentially expressed proteins(DEPs)during PEDV infection were screened and submitted to GO and KEGG analyses,and the following results were obtained: A total of3346 proteins were identified and 478 DEPs were screened,with 423 up-regulated and 55down-regulated.201,211 and 1342 GO terms were enriched in molecular function,cellular component and biological process ontologies respectively in GO analysis.And in the above ontologies,there were 45,66 and 255 GO term showing significant enrichment,with most of them concerning RNA binding and translation regulation,cytoskeleton and channel complex,cell cycle and transmembrane transportation respectively.A total of 67 pathways including p53,mTOR,MAPK signaling pathways were enriched in KEGG analysis,with 29 of them significantly enriched,including ErbB,HIF-1,Wnt and cell cycle signaling pathways.Various ribosomal proteins showed significant up-regulation,suggesting the infected cells' protein biosynthesis function might be hijacked by PEDV to contribute to its replication.And the up-regulations of ribosomal proteins such as RPL11,RPL23,RPL26,RPS25,and RPS27 might also indicate that ribosomal stress was activated.The expressions of cytoskeleton related proteins were found altered.The the up-regulations of DCTN2,TUBA4 A,MAP4,etc as well as the down-regulation of HSP27 suggest the cytoskeleton lesions was involved during the infection.In addition,the down-regulated HSP27 also indicates the cells were undergoing apoptosis.Proteins associated with cell cycle such as Cdc2,Mad1 and Bub3 were significantly up-regulated,also indicating cell cycle arrest might be induced.Associate analysis was performed on the identified genes and proteins obtained from transcriptomic and proteomic sequencing.A total of 103 proteins were found altered both at translation and transcription levels,and there were 52 of them whose translation shared the same pattern with the transcription,with 35 up-regulated and 17 down-regulated.These proteins were mainly involved cytoskeleton,cell cycle,signal transduction,etc.With significant alterations at translation and transcription levels,TRIM56 might take part in the immunoreaction during the infection.However,to understand the exact functions of TRIM56 in PEDV-infected cells,further researches are required.This is the first report on TRIM56's involvement in PEDV infection.