Mechanism Of Porcine Epidemic Diarrhea Virus N Protein Regulating P53 And NF-?B Signal Pathway

Porcine Epidemic Diarrhea Virus(PEDV)is one of the alpha-coronaviruses that cause porcine intestinal diseases.It can cause diarrhea,dehydration and even death of a large number of newborn piglets,which seriously harms the development of the pig industry.The pathogenic mechanism of PEDV is mainly the destruction of intestinal epithelial cells leading to its diarrhea,and the mechanism of PEDV destroying intestinal epithelial cells may include: apoptosis,necrosis and inflammation.p53 has the biological function of regulating cell apoptosis and also participates in the influence on virus replication.The NF-?B signaling pathway is also closely related to cell apoptosis.Therefore,this study analyzed the expression or modification changes of p53 protein and NF-?B signaling pathway related proteins after PEDV infects host cells in order to analyze the molecular mechanism of PEDV.This mechanism aimed at the effect of PEDV on p53 signaling molecules,and obtained the following research results with the help of immunoblotting,indirect immunofluorescence,Co-IP and other technologies: PEDV infects host cells at the later stage(24,48h),it will cause downregulation of p53 protein,and the down-regulation of p53 protein expression caused by PEDV is caused by ubiquitination;In order to analyze the PEDV viral proteins involved in the degradation and ubiquitination of p53,the PEDV-NSP7,PEDV-S2,PEDV-M,and PEDV-N eukaryotic expression plasmids were constructed.The eukaryotic expression screened out that PEDV-N is involved in regulating the total p53 protein.The reduction of protein promotes the increase of p53 modified protein,which is also ubiquitinated;In order to further analyze whether PEDV-N is directly involved in regulating the modification of p53 protein,Co-IP further confirmed that PEDVN can interact with p53,and that p53 is blocked by PEDV-N at the periphery of the nuclear membrane,preventing part of p53 from entering the nucleus,which may affect Its function.This mechanism further explored the expression of molecules related to the NF-?B signaling pathway after PEDV infects host cells,using techniques such as western blotting,indirect immunofluorescence,and laser confocal microscopy imaging,and obtained the following results:24 hours after PEDV infects the cells,the expression of NF-?B family proteins p105/p50 and p65 proteins is significantly down-regulated;The eukaryotic expression of PEDV-N protein can also promote NF-?B family proteins to show the same rules as PEDV virus-infected cells,and as the expression of PEDV-N increases,the expression of p105/p50 and p65 proteins is down-regulated more significantly;IFA and laser confocal microscopy imaging showed that the p105/p50 and p65 proteins of PEDV-N and NF-?B family proteins are all expressed in the cytoplasm,and there is a certain co-localization phenomenon.In summary,after PEDV infects host cells,the down-regulation of p53 protein expression is due to the increased ubiquitination of p53,and PEDV-N participates in this process through colocalization and interaction with p53,and PEDV Infection can also down-regulate the expression of NF-?B-related proteins,and PEDV-N is also involved.Therefore,this study preliminarily analyzes the mechanism by which PEDV infects host cells and affects p53 and NF-?B signaling pathways,which lays a foundation for revealing the molecular mechanism of PEDV in detail A certain foundation.