Coronavirus Porcine Epidemic Diarrhea Virus Nucleocapsid Protein Interacts with p53 To Induce Cell Cycle Arrest in S-Phase and Promotes Viral Replication

• Published in: Journal of virology 2021-07, Vol.95 (16), p.e0018721-e0018721
• Main Authors: Su, Mingjun, Shi, Da, Xing, Xiaoxu, Qi, Shanshan, Yang, Dan, Zhang, Jiyu, Han, Yuru, Zhu, Qinghe, Sun, Haibo, Wang, Xiaoran, Wu, Haoyang, Wang, Meijiao, Wei, Shan, Li, Chunqiu, Guo, Donghua, Feng, Li, Sun, Dongbo
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Antiviral Agents - chemistry; Antiviral Agents - pharmacology; Binding Sites; Cell Line; Chlorocebus aethiops; Coronavirus Infections - drug therapy; Coronavirus Infections - genetics; Coronavirus Infections - metabolism; Coronavirus Infections - virology; Coronavirus Nucleocapsid Proteins - antagonists & inhibitors; Coronavirus Nucleocapsid Proteins - chemistry; Coronavirus Nucleocapsid Proteins - genetics; Coronavirus Nucleocapsid Proteins - metabolism; Epithelial Cells - drug effects; Epithelial Cells - virology; Gene Expression Regulation; High-Throughput Screening Assays; Host-Pathogen Interactions - drug effects; Host-Pathogen Interactions - genetics; Molecular Docking Simulation; Nuclear Localization Signals; Porcine epidemic diarrhea virus - drug effects; Porcine epidemic diarrhea virus - genetics; Porcine epidemic diarrhea virus - metabolism; Protein Binding; Protein Conformation; Protein Interaction Domains and Motifs; Quercetin - analogs & derivatives; Quercetin - chemistry; Quercetin - pharmacology; S Phase Cell Cycle Checkpoints - drug effects; S Phase Cell Cycle Checkpoints - genetics; Signal Transduction; Swine; Swine Diseases - drug therapy; Swine Diseases - genetics; Swine Diseases - metabolism; Swine Diseases - virology; Tumor Suppressor Protein p53 - antagonists & inhibitors; Tumor Suppressor Protein p53 - chemistry; Tumor Suppressor Protein p53 - genetics; Tumor Suppressor Protein p53 - metabolism; Vero Cells; Virus Replication - drug effects; Virus-Cell Interactions
• ISSN: 0022-538X; 1098-5514
• DOI: 10.1128/JVI.00187-21

Subversion of the host cell cycle to facilitate viral replication is a common feature of coronavirus infections. Coronavirus nucleocapsid (N) protein can modulate the host cell cycle, but the mechanistic details remain largely unknown. Here, we investigated the effects of manipulation of porcine epidemic diarrhea virus (PEDV) N protein on the cell cycle and the influence on viral replication. Results indicated that PEDV N induced Vero E6 cell cycle arrest at S-phase, which promoted viral replication ( 0.05). S-phase arrest was dependent on the N protein nuclear localization signal S NWHFYYLGTGPHADLRYRT and the interaction between N protein and p53. In the nucleus, the binding of N protein to p53 maintained consistently high-level expression of p53, which activated the p53-DREAM pathway. The key domain of the N protein interacting with p53 was revealed to be S RGNSQNRGNNQGRGASQNRGGNN (N ), in which G RG are core residues. N and G RG were essential for N-induced S-phase arrest. Moreover, small molecular drugs targeting the N domain of the PEDV N protein were screened through molecular docking. Hyperoside could antagonize N protein-induced S-phase arrest by interfering with interaction between N protein and p53 and inhibit viral replication ( 0.05). The above-described experiments were also validated in porcine intestinal cells, and data were in line with results in Vero E6 cells. Therefore, these results reveal the PEDV N protein interacts with p53 to activate the p53-DREAM pathway, and subsequently induces S-phase arrest to create a favorable environment for virus replication. These findings provide new insight into the PEDV-host interaction and the design of novel antiviral strategies against PEDV. Many viruses subvert the host cell cycle to create a cellular environment that promotes viral growth. PEDV, an emerging and reemerging coronavirus, has led to substantial economic loss in the global swine industry. Our study is the first to demonstrate that PEDV N-induced cell cycle arrest during the S-phase promotes viral replication. We identified a novel mechanism of PEDV N-induced S-phase arrest, where the binding of PEDV N protein to p53 maintains consistently high levels of p53 expression in the nucleus to mediate S-phase arrest by activating the p53-DREAM pathway. Furthermore, a small molecular compound, hyperoside, targeted the PEDV N protein, interfering with the interaction between the N protein and p53 and, importantly, inhibited PEDV replication by a