乌鳢水泡病毒M蛋白诱导细胞凋亡的机制研究

Snakehead fish vesiculovirus (SHVV) infection has caused great economic loss in the snakehead fish culture in China. Understanding the pathogenetic mechanism of SHVV infection is the fundamental for the prevention of the SHVV infection. An SHVV strain has been isolated from the diseased snakehead fish and the complete genome of the SHVV has been determined in our lab. We showed that SHVV was able to infect and replicate very well in SNN-1 cells which was derived from snakehead fish. In addition, SHVV infection could induce cellular apoptosis in SSN-1 cells. Further more, the cellular apoptosis was also induced when the M protein of SHVV protein was over expressed in SSN-1 cells, indicating that the M protein alone was able to induce the apoptosis of SSN-1 cells. However, the mechanism underlying the cellular apoptosis induced by the M protein remains enigmatic. The goal of this proposal is to elucidate the mechanism of the apoptosis induced by the M protein. The major experiments are as follows: 1. A reverse genetic system for SHVV will be established.2. The domains and the amino acids of the M protein which are critical for the induction of apoptosis will be mapped.3. The apoptosis pathways induced by the M protein (mitochondrial pathway and /or death receptor pathway) will be elucidated.4. The effective proteins involved in the apoptosis pathways will be characterized. Based on the above results, we will propose a model for the cellular apoptosis induced by SHVV M protein. The obtained results will shed a new light on the pathogenesis of SHVV infection. The established reverse genetic system of SHVV will be a powerful tool for the studies of SHVV life cycle and the development of effective vaccine for SHVV.

乌鳢水泡病毒（SHVV）感染导致我国乌鳢养殖业重大经济损失。阐明SHVV的致病机理是防治该病毒病的重要基础。我们分离了SHVV病毒株，测定其全长基因组。证明SHVV可在乌鳢细胞株SSN-1中高效复制，并可诱导SSN-1细胞凋亡。进一步研究表明，单独过度表达SHVV编码M蛋白，也可导致SSN-1细胞凋亡，但其机制还不清楚。本项目在前期工作基础上，通过构建SHVV反向遗传系统和体外转染M基因mRNA方法，在转染和感染条件下研究M蛋白诱导SSN-1细胞凋亡的机制。主要内容有：1)、建立SHVV反向遗传系统；2)、鉴定M蛋白诱导细胞凋亡的结构域和关键氨基酸位点；3)、剖析SHVV诱导细胞凋亡的途径。4)、研究细胞凋亡途径效应蛋白的功能。研究结果将阐明SHVV编码M蛋白诱导细胞凋亡的机制，了解SHVV致病机理。建立SHVV反向遗传系统将为研究该病毒的生活史和高效疫苗的研发提供基础。

乌鳢水泡病毒（SHVV）感染导致我国乌鳢养殖业重大经济损失。阐明SHVV的致病机理是防治该病毒病的重要基础。我们建立了SHVV的反向遗传系统，开展 SHVV感染SSN-1细胞的microRNAs组学分析，在此基础上，筛选出宿主miR-214可以抑制病毒的复制。并进一步深入解读其抑制的双重机制：一方面，它可以通过靶向病毒的核蛋白（N）和磷酸化蛋白（P）来直接抑制病毒复制，领一方面，它可以通过靶向宿主蛋白，促进干扰素的表达来抑制病毒的复制。研究SHVV感染和细胞自吞噬的关系。SHVV高效复制需要谷氨酰胺，而且依赖三羧酸循环途径。 缺乏谷氨酰胺可以干扰了细胞内部氧化型和还原型谷胱甘肽的库存平衡，启动细胞自噬，导致细胞凋亡。开展 SHVV感染SSN-1细胞的转录组学分析，在此基础上，发现病毒感染激活RIG-I、Toll-like receptor, NF-kB途径，进一步筛选出宿主IFI35蛋白可以促进病毒的复制。由于SHVV和感染黄鳝的弹状病毒Monopterus albus rhabdovirus (MoARV)在核苷酸水平上有94%的相似性，我们开展 SHVV感染宿主范围研究，发现它可以感染除了乌鳢以外的黄鳝。有理由认为这2种病毒存在同一个种的可能性。SHVV感染可以导致细胞凋亡，M蛋白单独可以诱导细胞凋亡，还没有发现独特的结构域和特别的氨基酸位点在细胞凋亡中起到关键作用。M蛋白通过激活Caspase9，不是Caspase8，表明细胞凋亡途径是线粒体的内途径，不是通过死亡受体的外途径。本研究结果为防控SHVV感染提供了新的依据。