禽HEV ORF3蛋白对鸡肝细胞线粒体功能的影响及机制研究

Avian Hepatitis E virus (HEV) infection caused liver injury, which is the main reason of egg production decreasing and mortality increasing. However, because of the difficulty in isolation and multiplication of avian HEV, it seriously hinders the pathogenesis of the virus. Previously, we found mitochondria swelling in avian HEV infected chicken’s liver and ORF3 protein mainly expressed on mitochondria. Then in the project, based on these findings, the influence of avian HEV ORF3 protein to mitochondrial function of liver cells will be analyzed by in vitro and in vivo experiments. Subsequently, the mitochondrial protein (Mt-X) that interacted with ORF3 protein will be determined through GST-Pull down and SHOTGUN technique. And the key amino acids of the interaction between Mt-X and avian HEV ORF3 proteins will be determined using Co-IP, BiFC, confocal and point mutation. Finally, avian HEV ORF3 protein interacting with Mt-X protein affected mitochondrial function of liver cells would be confirmed. And also the mechanism of avian HEV ORF3 protein affected the mitochondrial function of liver cells will be expounded from affecting Mt-X expression and blocking interact between Mt-X and its’ downstream proteins. The research of the project will pay the way for the research of molecular mechanisms of liver injury caused by avian HEV infection, virus pathogenesis and provide the relevant measures for disease controlling and prevention.

禽戊型肝炎病毒（Hepatitis E virus, HEV）感染引发肝损伤是鸡死淘率升高和产蛋率下降的主要原因。由于该病毒分离和增殖极其困难，严重阻碍该病致病机理研究。前期我们发现禽HEV感染后肝脏线粒体肿胀，且ORF3蛋白主要表达在线粒体上。本项目将在此基础上，首先通过体内和体外实验分析病毒ORF3蛋白对线粒体功能的影响，然后通过GST-Pull down和SHOTGUN技术确定与其互作的肝细胞线粒体蛋白Mt-X；通过Co-IP、BiFC、激光共聚焦和点突变技术确定两者互作及互作的关键氨基酸；最后通过体外和体内实验确认禽HEV ORF3与Mt-X蛋白互作影响肝细胞线粒体功能，并且从影响Mt-X表达量和阻断其与下游蛋白互作两个方面，阐明禽HEV ORF3蛋白对鸡肝细胞线粒体功能影响的机制，以期为禽HEV感染诱发肝损伤的分子机制和病毒致病机制的研究奠定基础，并为该病的防控提供针对性的措施。

禽戊型肝炎病毒（Hepatitis E virus, HEV）感染引发肝损伤是鸡死淘率升高和产蛋率下降的主要原因。由于该病毒分离和增殖极其困难，严重阻碍该病致病机理研究。本项目首先通过动物攻毒实验和临床收集的疑似禽HEV感染的病料，确认禽HEV感染引起鸡只肝脏损害和细胞器损伤，而且病毒的ORF3蛋白表达主要集中在肝细胞线粒体部位，ORF3蛋白的表达主要导致细胞增殖水平降低、细胞凋亡比例增加和细胞内ATP水平降低；进一步筛选得到16个肝细胞线粒体蛋白与ORF3蛋白互作；初步选定IDH2、PHB、ETFA和VDAC3四个线粒体蛋白与ORF3蛋白进行互作的验证和功能分析；激光共聚焦和CO-IP技术确认四个线粒体蛋白分别与ORF3蛋白共定位和互作；通过荧光定量PCR方法，发现ORF3转染组LMH细胞内PHB的蛋白和mRNA表达量升高；继而在LMH细胞内过表达PHB蛋白，结果引发LMH细胞的增殖水平明显降低，细胞凋亡比例升高，同时转染后细胞内ATP水平升高，分析可能是由于ORF3蛋白与其他线粒体蛋白互作的原因导致。该项目为禽HEV致病机制和对宿主病理损伤机制的研究奠定基础，并为禽HEV的防控提供理论支持。