细胞自噬对猪传染性胃肠炎病毒感染的抑制作用研究

Autophagy is a lysosomal-dependent cellular pathway that eliminates contains of autophagosomes. Autophagy plays an important role in maintaining cell homeostasis, defense of pathogens infection and immune response. Previous studies have demonstrated that autophagy can regulate viral infection and replication, and plays an important role in viral pathogenicity. At present time, transmissible gastroenteritis virus (TGEV) is one of the most important pathogens resulting in serious damage to the pork industry in China. The mortality rate may reach up to 90% in suckling piglets without immunoprotection. Our preliminary study found that, TGEV infection can induced significant autophagy activity in swine testicle (ST) and porcine kidney (PK15) cells, and treatments with specific autophagy-modulating drugs can affect the replication and proliferation of TGEV. However, the underlying mechanism how autophagy regulates the TGEV infection and replication still remains unknown. In this project, we will further investigate the molecular mechanism of the altered autophagy activity in TGEV infected cells in vitro and by specific pathogen free (SPF) bama pigs in vivo, illuminate the role of autophagy in TGEV infection and replication by specific autophagy-modulating drugs and genetically modifying the autophage-related genes, and find the viral proteins that has interaction with autophagy-related moleculars. This project will elucidate the mechanism of autophagy regulation on TGEV infection and replication from the perspective of protein interactions.

细胞自噬是保守的降解自噬内容物的过程，在维持细胞内环境稳态、抵抗病原感染和免疫反应中扮演重要角色。研究表明，自噬可以影响并调节病毒感染复制，对病毒的致病性具有重要作用。目前，猪传染性胃肠炎病毒（TGEV）是危害我国养猪业的重要病原之一，未获得免疫保护的哺乳仔猪致死率高达90%。前期研究发现，TGEV感染能够显著促进猪睾丸细胞ST和猪肾细胞PK15自噬活性；调节细胞自噬水平可以显著影响病毒复制增殖水平。然而，细胞自噬对TGEV感染复制的调控及其作用机制尚不清楚。所以本项目拟在前期研究基础上通过体外和SPF巴马小型猪体内进一步确定TGEV引起的宿主细胞自噬；通过自噬药物和基因干扰调节自噬水平，揭示自噬在TGEV感染复制中的作用；筛选与自噬分子相互作用的TGEV编码蛋白，从蛋白互作层面阐释自噬对TGEV感染复制调控的分子机制和生物学意义。

自噬，作为一种古老并不断进化的通路应答，在抗病毒感染等宿主先天性免疫应答过程中发挥重要作用。然而，自噬对猪传染性胃肠炎病毒（transmissible gastroenteritis virus，TGEV）感染的调节作用尚无报道。通过本项目，我们研究发现TGEV感染能够诱导宿主细胞胞质内具有单层膜或者双层膜特征的自噬小体数量增多，这些自噬小体的诱导形成与细胞自噬有关。TGEV感染能够诱导自噬小体标志分子LC3-II的表达，此外TGEV感染也能诱导细胞自噬流的产生，表明TGEV感染能够诱导宿主细胞产生完整的细胞自噬应答反应。细胞自噬抑制剂（wortmannin和LY294002）处理细胞能够抑制细胞自噬并促进TGEV感染复制。三种细胞自噬必需基因（LC3B、ATG5和ATG7）的特异性siRNA分别处理细胞，能够促进TGEV复制。自噬诱导药物（rapamycin）处理细胞，能够明显提高宿主细胞自噬水平，显著抑制TGEV复制。我们通过siRNA技术减少自噬抑制蛋白p300的表达，引起三种自噬必需基因（LC3B、ATG5和ATG7）表达增多，进而明显抑制TGEV复制水平。总之，我们结果表明TGEV感染能够诱导细胞自噬，进而细胞自噬能够抑制TGEV复制，这是宿主细胞拮抗TGEV感染的先天性免疫调节手段。