猪流行性腹泻病毒nsp1抑制宿主蛋白合成的分子机制研究

Porcine epidemic diarrhea virus (PEDV) is one of the major pathogens of diarrhea in piglets which has greatly threatened the global swine industry. PEDV can synthesize certain proteins immediately after it invades into the host cell, then the circumstance of the host cell will be changed to enhance viral replication. As one of the first translated proteins during the life cycle of PEDV, nsp1 plays a substantial role for the suppression of host protein synthesis, but the detailed mechanisms are still unclear. In this study, to determine the pathway of nsp1 inhibition host protein synthesis, whether nsp1 affects host mRNA stability or protein translation will be explored by RNA sequencing, fluorescence quantitative RT-PCR and ribopuromycilation assay. Then, we will screen and identify the nsp1 interacting proteins which participate in suppression of host gene expression by using techniques of tandem affinity purification, mass spectrometry, and mammalian two-hybrid system. Finally, we will determine the structure of nsp1 complexed with the interacting protein by X-ray crystallography and clarify the structure basis for the host cell factor participating in nsp1 regulating the host protein translation. We will further explore the function of PEDV nsp1 in virulence by reverse genetic system and piglet model. Overall, the mechanism of nsp1 inhibition host gene expression will be thoroughly clarified, which will provide a theoretical basis for development of novel vaccine against PEDV.

猪流行性腹泻病毒（PEDV）是严重危害养猪业的重要病原体。病毒入侵宿主细胞后会迅速表达某些蛋白，改变宿主细胞内环境，促进病毒复制。非结构蛋白1（nsp1）是PEDV最先翻译出的蛋白质之一，前期研究表明猪流行性腹泻病毒nsp1在抑制宿主蛋白合成中发挥重要作用，但其途径与机制尚不清楚。本项目拟利用转录组测序、荧光定量RT-PCR和Ribopuromycilation试验等方法研究nsp1对宿主mRNA稳定性以及蛋白翻译的影响，确定nsp1抑制宿主蛋白合成的途径；利用串联亲和纯化结合质谱分析、哺乳动物双杂交等方法筛选和鉴定参与nsp1抑制宿主基因表达的互作蛋白，进而解析nsp1与宿主互作蛋白的复合物晶体结构，阐明宿主因子在nsp1调控宿主蛋白翻译中的作用机制；利用反向遗传操作系统，在病毒水平验证nsp1的功能，从而深入诠释其调控宿主蛋白合成的分子机制，为PEDV新型疫苗的研制提供新思路。

猪流行性腹泻病毒（PEDV）是严重危害养猪业的重要病原体。本项目围绕PEDV nsp1调控宿主蛋白合成的分子机理，利用结构生物学、生物化学、 免疫学和病毒学等方法阐明其生物学功能，具体研究成果如下：（1）发现PEDV等α属冠状病毒 nsp1蛋白能够显著抑制宿主蛋白表达；（2）解析了PEDV、TGEV等 nsp1高分辨率全长晶体结构，证实了Motif（91-95aa）是其抑制宿主翻译的重要区域；（3）发现PEDV nsp1抑制宿主天然免疫应答因子的表达，并且能够调控细胞周期，使宿主细胞停留在G0/G1期；（4）利用反向遗传操作系统，在病毒水平验证了nsp1抑制宿主蛋白表达的功能，进一步研究发现nsp1 91-95aa突变后病毒毒力减弱，为猪肠道冠状病毒新型疫苗的研制提供新思路。成果被 JBC 杂志主编 Craig Cameron 选为 2003 年至2020 年该领域最具代表性进展 (总共 35 篇入选)。项目成果获得湖北省科技进步一等奖1项，发表项目标注SCI论文9篇，其中PLoS Pathog（1篇）、J Virol（3篇）、Emerg Microbes Infect（1篇）、J Biol Chem（1篇）、 Viruses（2篇）和Virology（1篇）；培养博士生4名，硕士生1名。