水泡性口炎病毒致脑损伤过程中互作蛋白的筛选及其功能分析

Deciphering host–pathogen protein–protein interactions (PPI) is a way to understand how pathogens exploit host cell machinery and assess the pathophysiology of infectious diseases in molecular terms. Vesicular stomatitis virus (VSV) causes severe central nervous system (CNS) damage when administered to mice. However, it is unclear which host factors involved in vsv infected CNS. To understanding pathogenesis of VSV, high-throughput screening (HTS) projects- yeast two-hybrid system (Y2H) that aim at exploring systematically the PPI networks of CNS and VSV will be performed. Then the target genes correlated with VSV infection were confirmed in the neuron cells and mice by RNA interference, over-expression, and western blot assays, and its functional analysis were performed. The prospective results will help us to understand deeply the regulation mechanism on PPI correlated with VSV infection and select potential targets for antiviral drug therapy.

VSV感染可致小鼠CNS损伤而表现出神经症状，但脑组织中特异性的宿主蛋白在抗病毒感染或促进病毒侵嗜等过程中发挥的作用目前还不明确，这一科学问题的解决将对揭示VSV的神经毒性具有重要意义。本项目以VSV侵嗜CNS为切入点，通过酵母双杂交系统从小鼠脑组织中筛选出与该病毒相互作用的宿主蛋白；通过RNAi、基因过表达等实验技术进一步鉴定这些宿主因子对病毒吸附、侵染、转录和复制、释放等不同环节的影响，分析这些宿主蛋白在病毒复制、抵御病毒感染等方面的作用；经生物信息学分析后western blot检测，验证宿主蛋白在VSV感染神经细胞过程中参与的细胞信号通路，解析宿主蛋白影响VSV复制的机制。预期成果不仅为深入阐明VSV致CNS损伤及逃避中枢系统免疫防御的分子机制奠定基础，同时为筛选抗VSV感染的药物靶点以及开发安全性较高的VSV载体提供重要的参考价值。

在本项目研究中，我们克隆并表达了VSV-M蛋白，同时制备了M蛋白的多克隆抗体。我们通过酵母双杂交系统，发现了p8、eIF3i等宿主蛋白同M蛋白相互作用，并通过GST pull-down及激光共聚焦等方法进行了验证。进而解析了M蛋白上与p8及eIF3i蛋白相互作用的关键氨基酸残基位点。同时明确了p8及eIF3i对VSV在宿主细胞中转录、复制及滴度的影响。不仅如此，我们还探究了宿主细胞内受VSV侵染影响的AKT信号通路，并证实了eIF3i在其中起到的重要作用。本项目的实施为解析VSV侵染宿主细胞的分子机制奠定了基础。