Screening And Identification Of Key Proteins For PEDV N Protein-mediated Host Cell S-phase Arrest To Enhance Virus Replication

Porcine epidemic diarrhea virus(PEDV)infection is“the first killer”of piglet death,and the mortality of piglet infected within 7 days of age can reach 100%,which brings serious harm to pig industry in China.At present,although PEDV vaccine is widely used in China,PEDV infection still occurs frequently.Therefore,researches on PEDV infection mechanism and related antiviral strategies based on infection mechanism are very necessary.Previous studies have shown that PEDV N protein enhances viral replication by mediating cell cycle S-phase arrest,but the molecular mechanism of how to enhance viral replication remains unclear.The exploration of this scientific issue will provide a theoretical basis for elucidation of PEDV infection mechanism and antiviral drug target design.In view of this scientific problem,this study utilized TMT labeled quantitative proteomics technology to mine the key proteins of PEDV N protein-mediated S-phase arrest of host cells to enhance viral replication,and further clarified the influence and mechanism of the screened key proteins on PEDV replication.The specific research contents are as follows:1.Screening of key proteins for PEDV N protein-mediated host cells S-phase arrest to enhance viral replication.In order to explore the key proteins of PEDV N protein-mediated host cells S-phase arrest to enhance viral replication,the protein expression profile of Vero E6 cells in S-phase arrest mediated by PEDV N protein and thymidine were analyzed by using TMT labeled quantitative proteomics technology.The differentially expressed proteins were further identified by Western blot.The results showed that PEDV N protein and thymidine groups mediated S-phase arrest in Vero E6 cells in 54%and 42%,respectively,significantly higher than that in control(32%and21%)(p<0.05).The above data indicated that PEDV N protein and thymidine mediated Vero E6cells S-phase arrest samples were successfully prepared in this study.TMT labeled quantitative proteomics showed that a total of 5 709 proteins were identified in PEDV N protein and thymidine groups,among which 58 differentially expressed proteins were found in PEDV N protein group(27 proteins were up-regulated;31 proteins were down-regulated),26differentially expressed proteins were found in thymidine group(15 proteins were up-regulated;11 proteins were down-regulated).The results of GO analysis showed that the biological processes of PEDV N protein group differential proteins were concentrated in chromatin remodeling,nucleosome assembly,chromatin assembly or disassembly,etc.The molecular functions of differential proteins in PEDV N protein group were mainly structural molecule activity,lipid phosphatase activity,oxidoreductase activity and NADH dehydrogenase activity.The cellular components of PEDV N protein group differential proteins were concentrated in intermediate filament,macromolecular complex,intracellular non-membrane-bounded organelle and cytoskeleton,etc.The biological processes of the thymidine group differential proteins were concentrated in d TMP metabolic and biosynthetic processes,pyrimidine deoxyribonucleoside monophosphate metabolic and biosynthetic processes,DNA packaging,etc.The molecular functions of the thymidine group differential proteins were concentrated in thymidylate synthase activity,taurine transmembrane transporter activity,protein transmembrane transporter activity,macromolecule transmembrane transporter activity,etc.The cellular components of the thymidine group differential proteins were concentrated in protein complex,mitochondrial protein complex,nucleosome,DNA packaging complex and so on.KEGG pathway analysis showed that differential proteins in PEDV N protein group were enriched in oxidative phosphorylation,pentose phosphate pathway,neuroactive ligand-receptor interaction,etc.The KEGG pathway of the thymidine group differential proteins were enriched in retinol metabolism,endocytosis and metabolic pathways.Protein-protein interaction network(PPI)analysis showed that PEDV N protein group differential protein axonemal dynein heavy chain 9(DNAH9),60S ribosomal protein L10(RPL10)and 60S ribosomal protein L15(RPL15)could interact with other proteins included in the network database.Western blot results showed that 60S ribosomal protein L18(RPL18)expression level was significantly increased in PEDV N protein and thymidine mediated S-phase arrest in Vero E6 cells and porcine intestinal epithelial cells(IPEC J2)(p<0.05),which was consistent with TMT quantitative proteomics identification.The expression level of ribosomal protein RPL18 in Vero E6 and IPEC J2 cells were significantly increased after infected by PEDV CV777 classical strain and HM2017 variant strain(p<0.05).These results showed that the cellular ribosomal protein RPL18 was highly correlated with PEDV N protein-mediated host cells S-phase arrest to enhance viral replication.2.Effect of ribosomal protein RPL18 on replication of PEDV virus.In order to clarify the influence of ribosomal protein RPL18 on replication of PEDV in vitro,this study detected the replication level of PEDV in target cells by overexpression and silencing of ribosomal protein RPL18 in Vero E6 and IPEC J2 cells.The interaction between ribosomal protein RPL18 and PEDV N protein was further detected by Bio-Layer Interferometry(BLI).The results showed that in IPEC J2 cells,after the PEDV CV777 classical strain infected the target cells at 48 h,the TCID₅₀was 4.625 log₁₀TCID₅₀/m L with RPL18 overexpressed group,which was significantly higher than that in the control group(4.167 log₁₀TCID₅₀/m L)(p<0.05);after the PEDV HM2017 variant strain infected the target cells at 48 h,the TCID₅₀was 5.417log₁₀TCID₅₀/m L with RPL18 overexpressed group,which was significantly higher than that in the control group(4.542 log₁₀TCID₅₀/m L)(p<0.001).Western blot results showed that the expression of PEDV S protein was significantly increased after PEDV CV777 classical strain and HM2017 variant strain was infected in IPEC J2 cells overexpressing RPL18(p<0.05).The results showed that overexpression of ribosomal protein RPL18 in IPEC J2 cells could significantly enhance PEDV replication.When the ribosomal protein RPL18 was silenced in Vero E6 cells,after the PEDV CV777 classical strain infected the target cells at 48 h,the TCID₅₀was 4.208 log₁₀TCID₅₀/m L with RPL18 silenced group,which was significantly lower than that in the control group(4.833 log₁₀TCID₅₀/m L)(p<0.05);after the PEDV HM2017 variant strain infected the target cells at 48 h,the TCID₅₀was 5.417 log₁₀TCID₅₀/m L with RPL18 silenced group,which was significantly lower than that in the control group(6.125 log₁₀TCID₅₀/m L)(p<0.01).Western blot results showed that the expression of PEDV S protein was significantly decreased after PEDV CV777 classical strain and HM2017 variant strain was infected in Vero E6 cells with si RNA-RPL18(p<0.05).When the ribosomal protein RPL18 was silenced in IPEC J2 cells,after the PEDV CV777 classical strain infected the target cells at 48 h,the TCID₅₀was5.125 log₁₀TCID₅₀/m L with RPL18 silenced group,which was significantly lower than that in the control group(5.583 log₁₀TCID₅₀/m L)(p<0.05);after the PEDV HM2017 variant strain infected the target cells at 48 h,the TCID₅₀was 6.208 log₁₀TCID₅₀/m L with RPL18 silenced group,which was significantly lower than that in the control group(6.958 log₁₀TCID₅₀/m L)(p<0.05).Western blot results showed that the expression of PEDV S protein was significantly decreased after PEDV CV777 classical strain and HM2017 variant strain was infected in IPEC J2 cells with si RNA-RPL18(p<0.05).The results showed that ribosomal protein RPL18 could significantly inhibit PEDV replication after silencing in Vero E6 cells and IPEC J2 cells.These results suggest that ribosomal protein RPL18 can enhance PEDV replication in Vero E6 cells and IPEC J2 cells.In this study,Bio-Layer Interferometry(BLI)technology was further used to analyze the interaction between ribosomal protein RPL18 and PEDV N protein.The results showed that there was no direct interaction between ribosomal protein RPL18 and PEDV N protein.In summary,TMT labeled quantitative proteomics was used to systematically identify the differentially expressed protein profile of PEDV N protein-mediated S-phase arrest in Vero E6cells,revealled the key proteins information and important biological processes of PEDV N protein-mediated S-phase arrest that enhanced virus replication,confirmed that ribosomal protein RPL18 enhanced replication of PEDV classical and variant strains.This study provided a theoretical basis for elucidating the molecular mechanism of PEDV-mediated host cell cycle arrest promoting virus replication and designing new antiviral drug targets.