Affection On Cell Cycle By Porcine Epidemic Diarrhea Virus And Its Nucleoli Localization Signal Peptides Of Nucleocapsid Protein

Porcine epidemic diarrhea virus (PEDV) is a member of coronavirus genus and the causative agentfor porcine epidemic diarrhea (PED) characterized by watery diarrhea and high mortality rate. The Nprotein is the most abundant virus-derived protein and closely related with the pathogenicity. The studyof interaction PEDV and its N protein with cell will provide information for the pathogenic mechanismof PEDV and antiviral strategy.In this study, the first concerns focus on the interaction PEDV with cells. Vero E6cells infectedwith PEDV, the flow cytometer was used to analysis the cell cycle in4h、8h、12h、24h、36h、48h、72h post-infection. The results showed that the Vero E6cells exhibited cell growth retardation throughG2/M arrest in12h post-infection. These indicated that PEDV may interact with cells to influence thecells in the G2/M phaseThe flow cytometer was used to analysis the Vero E6cell cycle with the chemical drug nocodazoletreated.It showed that cell cycle was blocked in the G2/M phase. Serum-free cultured Vero E6cells canbe observed in G0/G1arrested after detected by flow cytometer. With established quantitative PCRmethod, PEDV viral load was analyzed in treated and normal cells. The results suggested that viral loadin normal cells were significantly lower than that in cell of G2/M phase, but higher than that in cell ofG0/G1phase. These results indicate that Vero E6cell of G2/M phase can facilitate virus propagation.To further investigate the affection on the cell cycle by PEDV, relative quantitative PCR was used todetect the changes of p21, p53, cyclin D and cyclin A and cyclin B in normal cells and infected cells.Taking GAPDH as the reference gene, RNA in Vero E6cells was extracted in post infected48h andnormal cells, The results revealed that compared with normal cells, the level of p21, p53and cyclinDwere rosed, cyclin A and cyclin B declined in infected cells, which suggests that PEDV can further alterthe level of cyclin and cell-cycle regulated molecule.The nucleoli signal sequence of N protein have been identified early, that is71SNWHFYYLGTGPHGDLRYRT90, the PEDV N gene was amplified with the deletion of nucleolilocalization signal sequence by EOS-PCR and connected to the eukaryotic expression vectorpAc-GFP-C1,so the recombinant plasmid pAc-GFP-dN was constructed. Vero E6cells were transfectedwith the pAc-GFP-dN and the subcellular localization of the expressed dN protein was analyzed byconfocal microscopy. The affection on cell cycle of the transfected cell was detected by flow cytometry.The results showed that the dN protein was unable to localize in the nucleus compared to the N proteinas expected; However, the dN was also unable to induce the arresting on cell cycle G2/M caused by Nprotein. These results revealed that the nucleoli localization signal in PEDV N protein has importantfunction on host cell.