猪圆环病毒2型编码蛋白5诱导宿主细胞内质网应激及自噬作用机制研究

It is a basis for revealing the pathogenesis of porcine circovirus type 2 (PCV2) by clarifying the role and mechanism of PCV2 encoded proteins. A new encoded protein has been identified in our research and tentatively named “ORF5”. We found that the marker genes related to cell endoplasmic reticulum stress and autophagy were upregulated, cell cycle arrest in cells expressed ORF5 protein of PCV2. As well known that the endoplasmic reticulum stress reactive can regulate the cell cycle and autophagy to maintain cellular homeostasis. However, those cellular effects also will be utilized by virus for its replication and proliferation. The role of ORF5 will be explored with endoplasmic reticulum stress, cell cycle arrest and autophagy induced by ORF5 protein as a starting point in this project. The role and mechanism of ORF5 protein in PCV2 infection will be revealed on molecular level by analyzing the mechanism of ORF5 protein, which induce endoplasmic reticulum stress-cell cycle-autophagy and those cellular effects in PCV2 replication and proliferation on cellular and organ level. The results have significant theoretical value and potential application value in illustrating the pathogenesis, developing approaches of prevention and control PCV2.

对猪圆环病毒2型（PCV2）编码蛋白功能和作用机制的阐明是揭示其致病机理的基础。我们发现和鉴定了一个新的PCV2编码产物，暂将其命名为“ORF5蛋白”。ORF5蛋白能诱导宿主细胞内质网应激和细胞自噬标志基因的上调、细胞周期阻滞。内质网应激反应可以调控细胞周期、介导细胞自噬，在维持细胞内环境稳定的同时也为病毒复制增殖提供了条件。本项目拟以PCV2 ORF5蛋白为研究对象，以ORF5蛋白诱导的内质网应激、细胞周期阻滞和细胞自噬为切入点，通过对ORF5蛋白在细胞和机体水平诱导内质网应激－细胞周期阻滞－细胞自噬的机制及对PCV2复制增殖影响的解析，从分子水平揭示ORF5蛋白在PCV2感染中的作用及机制，为PCV2致病机理阐明和防控方案制定提供新的科学资料，具有重要的理论意义和潜在的应用价值。

本项目以ORF5蛋白诱导宿主细胞的内质网应激（ERS）和细胞自噬作用为切入点，对ORF5蛋白在PCV2致病中的作用和机制进行深入研究。结果表明，ORF5蛋白可引起宿主细胞（特别是猪肺泡巨噬细胞，PAMs）多种蛋白、生命进程和信号通路改变，使细胞S期阻滞，诱导ERS和细胞自噬，促进PCV2的复制。ORF5蛋白是通过激活PERK、ATF6和IRE1等三条信号转导途径诱导宿主细胞ERS。ERS途径是ORF5诱导宿主细胞自噬的主要途径，通过激活PERK-eIF2α-ATF4和mTOR-ERK1/2-AMPK，诱导了细胞自噬；GPNMB和YWHAB是与ORF5互作的胞内蛋白，GPNMB使宿主细胞周期缩短，具有抑制细胞内质网应激、自噬和细胞凋亡作用，两者对PCV2的复制具有抑制作用；PCV2感染猪组织细胞中出现ERS和细胞自噬，且其诱导水平和病毒载量呈现相关性。.本项目研究探明了ORF5蛋白在PCV2致病中的作用，阐明了其诱导宿主细胞ERS和细胞自噬的信号通路及分子作用机制，发现了宿主细胞蛋白在PCV2感染中的作用，研究结果为PCV2致病机制的揭示提供了新的科学资料，为PCV2的防控提供了新的细胞靶点，具有潜在的应用价值。