小反刍兽疫病毒诱导细胞自噬的机制研究

Peste des petits ruminants (PPR) is an economically significant infectious disease caused by peste des petits ruminants virus (PPRV). Since its first outbreak in Tibet in July 2007, PPR was temporarily controlled in this region by immunizing all susceptible animals in the epidemic areas and the surrounding areas with a live-attenuated vaccine of PPRV Nigeria 75/1 strain. However, several outbreaks of PPR were reported in more than 10 provinces such as Xinjiang and Gansu in late 2013 and early 2014, which makes the prevention and control of this disease extremely severe for China. This research project is based on the applicant’s new discovery that PPRV infection can trigger autophagy in Vero cells and that PPRV exploits the cellular autophagy machinery for replication. However, the underlying mechanism needs to be further investigated. In the present study, PPRV Nigeria 75/1 strain and Vero cells will be used to elucidate the molecular mechanism of autophagy induced by PPRV infection. First, the protein of PPRV involved in autophagy induction will be identified. Next, the key signal molecules of several major signaling pathways of autophagy will be determined. In addition, the interaction between the encoding proteins of PPRV and the relevant signal molecules, as well as the interaction between the relevant signal molecules will be analyzed. The results of this study will provide valuable scientific evidence for designing an immune-effect better attenuated vaccine through PPRV infectious clone construction and designing novel antiviral strategies by means of regulating the autophagic activity of host cells.

小反刍兽疫（PPR）是由小反刍兽疫病毒（PPRV）引起的有重大经济影响的家畜传染病。自2007年7月西藏首次暴发以来，我国通过对疫区及周边地区易感动物进行PPRV弱毒活疫苗免疫的方式使疫情暂时控制在西藏。然而2013年底和2014年初，新疆和甘肃等十余省份相继暴发数起PPR疫情，导致PPR防控异常严峻。本项目基于申请者最近首次证实PPRV感染能诱导宿主细胞发生自噬，且PPRV利用自噬机制促进自身复制。但是，PPRV诱导细胞自噬的机制尚不清楚。本项目拟以PPRV Nigeria 75/1株和Vero细胞为材料，在鉴定PPRV自噬诱导蛋白的基础上，通过对几个主要自噬信号通路关键信号分子的检测、对PPRV编码蛋白与相关信号分子以及相关信号分子之间互作情况的分析，阐明PPRV诱导自噬的分子机制，为通过感染性克隆构建免疫效果更佳的PPRV弱毒疫苗以及探索通过调控细胞自噬水平设计新型抗病毒策略提供理论依据。

小反刍兽疫（PPR）是由小反刍兽疫病毒（PPRV）引起的有重大经济影响的家畜传染病，开展PPRV与宿主细胞相互作用的分子机制研究，有助于更好地防控PPR。本项目在前期研究首次证实PPRV感染诱导细胞自噬、且利用自噬机制促进病毒复制的基础上，利用慢病毒包装系统成功构建了自发绿色荧光稳定表达自噬体标志分子—微管相关蛋白轻链3（LC3）的Vero细胞系（Vero-EGFP-LC3β）。以PPRV Nigeria 75/1疫苗株和Vero/Vero-EGFP-LC3β细胞为材料，借助Western blot、激光共聚焦免疫荧光和细胞转染等技术进一步证实，异位表达PPRV核衣壳蛋白（N）和磷蛋白（P）蛋白均能显著提高Vero细胞内LC3-I向LC3-II的转化以及p62蛋白的降解，表明PPRV N蛋白和P蛋白参与诱导细胞自噬。借助Western blot和药物处理等技术进一步发现，PPRV N蛋白和P蛋白均可以激活内质网应激（Endoplasmic Reticulum Stress，ERS）反应，并通过双链RNA依赖的蛋白激酶样内质网激酶（PKR-like ER kinase，PERK）信号通路诱导未折叠蛋白反应（Unfolded protein response，UPR），最终诱导PPRV感染的Vero细胞发生自噬。PPRV诱导自噬的分子机制的阐明，为通过感染性克隆构建免疫效果更佳的PPRV弱毒疫苗以及探索通过调控细胞自噬水平设计新型抗病毒策略提供了科学依据。