The Molecular Mechanism Of PEDV N Protein-induced Cell Cycle Arrest In S-phase Through P53-DREAM Signaling Pathway

Porcine epidemic diarrhea（PED）is caused by the porcine epidemic diarrhea virus（PEDV）,is an acute and highly contagious intestinal infectious disease,which leads to tremendous financial losses for the swine industry in China.Despite the widespread use of the PEDV vaccine in China,PED still occurs frequently.Therefore,it is necessary to carry out academic researches related to PEDV prevention and control.Viruses can create a cellular microenvironment which is conducive to its replication by mediating host cell cycle arrest during long-term interaction with the host.Thus,the mechanism elucidation of virus-regulated cell cycle arrest can provide a theoretical foundation for the antiviral design.Currently,N protein of coronaviruses such as PEDV can induce cell cycle arrest,but the mechanism has not been clearly explained yet.In this study,the molecular mechanism was investigated to explore PEDV N protein-mediated cell cycle arrest and the antiviral drugs based on this mechanism were further explored.Research contents include the following four parts in detail:1.Confirmation of PEDV N protein-mediated S-phase arrest in Vero E6 cells and the effect on viral replication.To clarify the regulatory effect of PEDV N protein on cell cycle,N protein was overexpressed in non-synchronized Vero E6 cells and synchronized Vero E6 cells which include G0/G1-phase mediated by serum starvation,S-phase mediated by thymidine,as well as G2/M-phase mediated by nocodazole respectively.Then the cell cycle and viral titers were analyzed.The results showed that the percentage of S-phase cells in the non-synchronized cells was 22.91%（1.0μg/m L）,29.66%（2.5μg/m L）,and 36.38%（4.5μg/m L）in the different concentrations of N protein groups,respectively,which were significantly higher than that in the control group（21.68%）.And the concentration of N protein was positively correlated with the number of S-phase cells.Among the G0/G1 phase,S phase and G2/M phase synchronized cells with the overexpressing N protein（4.5μg/m L）,the percentage of S phase cells were 32.07%,58.50%,and 25.50%respectively,which were all obviously higher than those in the control group（14.08%,49.56%,and 8.30%）（P<0.01）.In non-synchronized cells,the virus titers（48 hpi）were 5.45 log₁₀ TCID₅₀/m L,which was significantly higher than that of the control group(4.67 log₁₀ TCID₅₀/m L)（P<0.01）.In synchronized cells,the virus titers（48 hpi）were 5.54 log₁₀ TCID₅₀/m L,which was higher than that in the control group(4.77 log₁₀ TCID₅₀/m L)（P<0.01）.Above all,these results indicate that PEDV N protein mediates S-phase arrest in Vero E6 cells,and S-phase arrest enhances PEDV replication.2.Investigation of the molecular mechanism of PEDV N protein mediating Vero E6 cell cycle arrest in S-phase.To elucidating the mechanism of PEDV N protein-induced cellular S-phase arrest,this study explored the potential role of the p53-DREAM signaling pathway in PEDV N protein-mediated cell cycle S-phase arrest with methods of a living cell imaging system,immunoprecipitation（Co-IP）and construction of truncated mutants of N proteins.The results showed that the expression level of p53 increased obviously in N protein overexpressed Vero E6 cells（P<0.01）.And the percentage of S-phase cells and virus titers were significantly decreased in N protein overexpressed Vero E6 cells after treatment with the p53 inhibitor PFT-α（P<0.05）.In addition,PEDV N protein significantly upregulated the expression of p21,inhibited the phosphorylation of p130,as well as down-regulated the expression of E2F4 and cyclin A（P<0.01）.Meanwhile,PFT-αantagonized the regulatory influences of N protein on p21,p130,E2F4,and cyclin A（P<0.05）.These results suggested that PEDV N protein induces cell cycle arrest in S-phase via activating the p53-DREAM signaling pathway.Further investigation showed that N protein co-localized with p53 in the nucleus,and p53 was stably expressed and accumulated in the nucleus.What’s more,N protein-mediated S-phase arrest was depended on the nuclear co-localization of N protein and p53and the nuclear localization signal of N protein(S⁷¹NWHFYYLGTGPHADLRYRT⁹⁰).Further research confirmed that N protein and p53 have direct interaction.The key domain of PEDV N protein interacting with p53 was S¹⁷¹RGNSQNRGNNQGRGASQNRGGNN¹⁹⁴(N^S171-N194)in which G¹⁸³RG¹⁸⁵ are core sites.The interaction of the N protein mutant N^mut(R¹⁸³RR¹⁸⁵)with p53was attenuated,which further decreases the N protein-mediated S-phase arrest significant（32.79%）（P<0.01）.Moreover,the virus titers in N^mut group were significantly lower(5.38 log₁₀ TCID₅₀/m L)than that in control group(5.67 log₁₀ TCID₅₀/m L)（P<0.05）.Above results indicate that,after entering in the nucleus the N^S171-N194 domain of PEDV N protein interacts with p53 to maintain consistently high-level expression of p53,which further activating the p53-DREAM signaling pathway and mediating the S-phase arrest in Vero E6 cells.3.Design of antiviral drugs based on the mechanism of PEDV N protein-mediated S-phase arrest.In order to obtain small molecule compounds that can interfere with PEDV N protein-mediated cell cycle S-phase arrest,small molecular drugs targeting the N^S171-N194 domain of PEDV N protein were screened through molecular docking and the antiviral effect of the selected small molecule compounds was further evaluated in this study.Molecular docking analysis showed that Hyperoside had a higher affinity with PEDV N protein with-CDOCKER ENERGY（24.4758）and-CDOCKER INTERACTION ENERGY（49.5773）.Biolayer interferometry（BLI）assay revealed that Hyperoside exhibited a strong affinity to N protein of PEDV in a dose-dependent manner.In addition,Hyperoside weakened the interaction between PEDV N protein and p53（P<0.05）,and then interfered with the cell cycle S-phase arrest（P<0.05）.The virus titers of PEDV in the Hyperoside-treated cells was significantly decreased(5.13 log₁₀ TCID₅₀/m L)compared with that in control group(5.63 log₁₀ TCID₅₀/m L)（P<0.05）.All together,these results demonstrated that Hyperoside could antagonize the interaction between N protein and p53 to relieve N protein-induced S-phase arrest,and thereby inhibit PEDV replication.4.Validation of the molecular mechanism that PEDV N protein induces S-phase arrest in IPEC-J2cells.Due to Vero E6 is not a natural host cell for PEDV,the mechanism of PEDV N protein-mediated cell cycle arrest was further validated in porcine intestinal cells IPEC-J2.The results showed that compared with the control group（37.57%）,the proportion of S-phase cells（40.94%）was significantly higher in the IPEC-J2 cells overexpressing N protein（P<0.01）.And PEDV replication was enhanced obviously（P<0.05）.Further studies confirmed that the interaction of PEDV N protein and pig p53 was presented in the IPEC-J2 cells.Compared with control group（40.94%）,the proportion of cells in the S-phase was significantly decreased in N^mut group（37.57%）（P<0.05）.Hyperoside could antagonize the N protein-induced IPEC-J2 cell cycle S-phase arrest（P<0.05）,and inhibit virus replication（P<0.05）.The above results indicated that the mechanism that PEDV N protein promotes viral replication through inducing cell cycle S-phase arrest was validated in the IPEC-J2 cells,which was consistent with that in Vero E6 cells.Above all,after PEDV N protein entering into the nucleus,PEDV N protein interacts with p53 through its N^S171-N194 region and activates the p53-DREAM signaling pathway,which then induces host cell cycle S-phase arrest and creates a favorable microenvironment for PEDV replication.Hyperoside,a small molecule drug designed based on the mechanism of PEDV N protein-mediated cell cycle arrest,could antagonize the PEDV N protein-induced S-phase arrest to inhibit virus replication by impacting on the binding of N protein to p53.This study reveals a new mechanism of PEDV N protein regulating the host cell cycle,which would provide a novel antiviral strategy of PEDV and other coronaviruses.