Study On Proteomic Of MA-104 Cells Infected With GPRV Strain CH-1

MA-104 cells(rhesus monkey kidney cells)are suitable for the proliferation of rotavirus(RV)in vitro and are also important model cells for molecular biology research of RV.GPRV(rotavirus derived from giant panda)CH-1 strain was isolated from first strains of giant panda rotavirus diarrhea cases,can proliferate and cause typical cytopathic effects in MA-104 cells(CPE).To study the pathogenic mechanism of the virus,a quantitative proteomic approach using isobaric tags for relative and absolute quantitation(i TRAQ)-coupled liquid chromatography-tandem mass spectrometry(LC-MS/MS)identification was performed to identify the differentially expressed proteins(DEPs)in MA-104 cells infected with GPRV strain CH-1.To identify an optimal time point after GPRV infection for the proteomic analysis,MA-104 cells were infected with GPRV strain CH-1 at a MOI of 2 and microscopically monitored for CPE and relative expression of VP4 m RNA at 6,12,18,24,30,36 and 42 hpi.Minimal CPEs were visible at 12 hpi and obvious CPE could be observed at 30 hpi;at 36 hpi,almost all cells fell off.The VP4 m RNA levels of GPRV strain CH-1 increased gradually over time,and reached a peak at 30 hpi.Following this time point,the viral m RNA levels appear to decrease.To make sure higher proportion of the infected cells and to avoid excessive CPE,we thus chose 24 hpi as the time point under our infection conditions for further proteomic analysis.We idetified all DEPs in MA-104 cells infected with GPRV strain CH-1 at 24 hpi by quantitative proteomic analysis.Totally,272 DEPs were identified,including142 up-regulated proteins and 130 downregulated proteins,by GO functional enrichment analysis,they involved in 26 biological processes,12 cell components,and 16 molecular function.Moreover,among these DEPs,64,42 and 57 proteins were involved in cell death process,viral replication,and the immune response process,respectively.However,KEGG pathway enrichment analysis,we found that the these DEPs are significant enriched in pathways which related to protein processing in endoplasmic reticulum,regulation of actin cytoskeleton and MAPK signaling pathway.Network and KEGG pathway analyses revealed that the DEPs mainly involved in MAPK,p53 and PI3K-Akt signaling pathways.By real-time RT-PCR,we analyzed the expression of KRT8,HSP27,14-3-3 ?/? and Annexin2(ANXA2)in the control group and 24 hpi,respectively,which accorded with the results of the proteomics analysis.The present study started with comparsion of the changes in cellular proteome upon GPRV strain CH-1 infection in MA-104 cells to analyze the virus-host cell interactions,selected the DEPs linking to virus replication,cell apoptosis and immune response.Our results contributed to understanding of the molecular mechanism GPRV strain CH-1 replication,host cell apoptosis,the pathogenic mechanism involved in innate immune response against GPRV strain CH-1infection.