Coronavirus Porcine Epidemic Diarrhea Virus Nucleocapsid Protein Interacts with p53 To Induce Cell Cycle Arrest in S-Phase and Promotes Viral Replication
Coronavirus Porcine Epidemic Diarrhea Virus Nucleocapsid Protein Interacts with p53 To Induce Cell Cycle Arrest in S-Phase and Promotes Viral Replication
复制标题
冠状病毒猪流行性腹泻病毒核衣壳蛋白与p53相互作用诱导细胞周期停滞在S期并促进病毒复制
DOI：
10.1128/jvi.00187-21
复制
发表时间：
2021-08-01
影响因子：
5.4
通讯作者：
Sun, Dongbo
中科院分区：
文献类型：
--
作者：
Su, Mingjun;Shi, Da;Sun, Dongbo
破坏宿主细胞周期以促进病毒复制是冠状病毒感染的一个共同特征。冠状病毒核衣壳（N）蛋白可以调节宿主细胞周期，但其机制细节仍然很大程度上未知。在这里，我们研究了猪流行性腹泻病毒（PEDV）N蛋白的操作对细胞周期的影响以及对病毒复制的影响。结果表明，PEDV N 诱导 Vero E6 细胞周期停滞在 S 期，从而促进病毒复制（P < 0.05）。 S期停滞依赖于N蛋白核定位信号S(71)NWHFYYLGTGPHADLRYRT(90)以及N蛋白和p53之间的相互作用。在细胞核中，N蛋白与p53的结合维持了p53的持续高水平表达，从而激活了p53-DREAM通路。 N蛋白与p53相互作用的关键结构域是S(171)RGNSQNRGNNQGRGASQNRGGNN(194)(NS171-N194)，其中G(183)RG(185)是核心残基。 NS171-N-194 和 G(183)RG(185) 对于 N 诱导的 S 期停滞至关重要。此外，通过分子对接筛选了针对PEDV N蛋白NS171-N-194结构域的小分子药物。金丝桃苷可通过干扰N蛋白与p53之间的相互作用来拮抗N蛋白诱导的S期阻滞，抑制病毒复制（P < 0.05）。上述实验也在猪肠细胞中得到验证，数据与Vero E6细胞中的结果一致。因此，这些结果表明PEDV N蛋白与p53相互作用，激活p53-DREAM途径，随后诱导S期阻滞，为病毒复制创造有利的环境。这些发现为 PEDV 与宿主相互作用以及针对 PEDV 的新型抗病毒策略的设计提供了新的见解。 重要性 许多病毒会破坏宿主细胞周期，以创造促进病毒生长的细胞环境。 PEDV 是一种新出现和重新出现的冠状病毒，已导致全球养猪业遭受重大经济损失。我们的研究首次证明 PEDV N 诱导的 S 期细胞周期停滞可促进病毒复制。我们发现了 PEDV N 诱导 S 期停滞的新机制，其中 PEDV N 蛋白与 p53 的结合使细胞核中 p53 的表达持续保持高水平，从而通过激活 p53-DREAM 途径介导 S 期停滞。此外，一种小分子化合物金丝桃苷靶向 PEDV N 蛋白，干扰 N 蛋白和 p53 之间的相互作用，更重要的是，它通过拮抗细胞周期停滞来抑制 PEDV 复制。该研究揭示了PEDV与宿主相互作用的新机制，也为PEDV提供了新的抗病毒策略。这些数据为进一步研究冠状病毒-宿主相互作用奠定了基础。
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 (P < 0.05). S-phase arrest was dependent on the N protein nuclear localization signal S(71)NWHFYYLGTGPHADLRYRT(90) 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(171)RGNSQNRGNNQGRGASQNRGGNN(194) (NS171-N194), in which G(183)RG(185) are core residues. NS171-N-194 and G(183)RG(185) were essential for N-induced S-phase arrest. Moreover, small molecular drugs targeting the NS171-N-194 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 (P < 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.IMPORTANCE 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 antagonizing cell cycle arrest. This study reveals a new mechanism of PEDV-host interaction and also provides a novel antiviral strategy for PEDV. These data provide a foundation for further research into coronavirus-host interactions.