辅伴侣蛋白BAG3抑制狂犬病病毒复制的功能及分子机制研究

Rabies virus (RABV) is a member of the genus Lyssavirus belonging to the family Rhabdoviridae, and its matrix protein is a multifunctional protein, which plays an important role in regulating viral transcription and replication, promoting the assembly and budding of virus particles, inducing cell apoptosis and affecting virus virulence. In this study, based on our preliminary findings of RABV replication inhibited by overexpressing the co-chaperone BAG3, and the interaction between RABV M protein and BAG3, techniques of virology, molecular biology, cell biology and biochemistry were used to determine the function of BAG3 during RABV infection. In addition, a tandem affinity purification coupled with mass spectrometry approach were used to screen and identify the host proteins which exhibited an increased or reduced interaction with BAG3 in the presence of RABV M protein, and the effect of these host proteins on the RABV replication regulated by BAG3 was further analyzed by using the above techniques, which elucidated molecular details of the role of BAG3 during RABV infection. The study on the interaction between RABV M protein and BAG3 in the mechanism of RABV replication is useful for the research and development of new drug targets for RABV, which has important scientific significance and theoretical value.

狂犬病病毒（Rabies virus, RABV）为弹状病毒科狂犬病病毒属成员，其基质蛋白（M蛋白）是一种多功能蛋白，在调控病毒转录与复制、促进病毒粒子组装和出芽、诱导细胞凋亡和影响病毒致病力方面发挥着重要作用。本研究在我们初步发现过表达辅伴侣蛋白BAG3抑制RABV复制以及BAG3与病毒M蛋白相互作用的前期工作基础上，借助病毒学、分子生物学、细胞生物学以及生物化学等技术，确定BAG3在RABV感染过程的具体作用方式。同时采用串联亲和纯化与质谱联用方法筛选并鉴定在病毒M蛋白参与的情况下与BAG3相互作用发生增强或减弱的宿主蛋白，借助上述技术分析这些宿主蛋白对BAG3调控RABV复制的作用，阐明BAG3在RABV感染过程中作用的分子细节。本研究对病毒M蛋白与BAG3相互作用在RABV复制机制中的深入研究有助于抗RABV新药物靶标的研究开发，具有重要的科学意义及理论价值。

狂犬病是我国严重的公共卫生问题之一，严重威胁着人民群众的生命安全和心理健康，同时也给社会造成沉重的经济负担。狂犬病病毒（RABV）是引起狂犬病的致病因子，揭示RABV在感染细胞中的复制机理有助于挖掘新的狂犬病潜在药物靶点。本项目以RABV感染的细胞为研究模型，首先制备了针对病毒基质蛋白（M）的兔源性和鼠源性多克隆抗体，证明了它们在免疫印迹、免疫荧光及免疫共沉淀实验中与不同RABV毒株M蛋白均具有良好反应性，为挖掘M蛋白与宿主蛋白互作关系及研究RABV致病机制提供重要的生物学工具；阐明了病毒M蛋白通过不依赖蛋白酶体途径和不依赖巨自噬-溶酶体途径的其他溶酶体途径发生自然降解的分子细节，紧接着揭示了BAG3通过与M蛋白互作并经该途径对其降解进而负调控RABV复制周期的分子机制；鉴定出在M蛋白参与的情况下与BAG3互作减弱的宿主蛋白Stk38，解析了Stk38通过与M蛋白互作引导M蛋白定位于线粒体、Stk38通过抑制BAG3与M蛋白的互作以促进M蛋白表达及其影响RABV复制周期的分子机理。总之，本项目对BAG3与RABV互作关系分子细节的研究，丰富了RABV复制机制的理论体系，为狂犬病新药物靶标的研发提供理论依据。此外，借助本项目资助，进一步发现病毒M蛋白通过与分子伴侣自噬途径（CMA）（溶酶体途径的一种）的关键蛋白Hsc70、LAMP2A互作经CMA途径发生自然降解，而BAG3是否通过CMA途径降解病毒M蛋白以及Stk38如何与BAG3、CMA途径相互协调进而调控RABV复制周期，接下来还需继续进行深入研究。