狂犬病病毒基质蛋白-宿主细胞蛋白互作组鉴定与作用机制研究

Rabies is a devastating zoonotic disease caused by the Rabies virus. According to statistics from WHO, rabies kills around 60000 people globally each year. China is one of the countries which are seriously affected by rabies with the human rabies cases ranking second over the world. Thus, rabies is an important threat that cannot be ignored by public health security. As obligate intracellular pathogens, viruses hijack host cell systems to complete replication cycle through the complicated interactions between viral proteins and host cell proteins. Matrix protein (M) is the smallest viral protein in rabies virus and plays multiple roles during the viral life cycle, including mediation of the virions assembly and egress, regulation of the balance of virus transcription and replication and involvement in virus pathogenesis and immune evasion. The host factors contribute to the diverse functions of the M protein. Hence, we focus on the host cell proteins interactome of M. Consequently, a number of new-found M-interacting host cell proteins were preliminarily screened out. For the purpose of illuminating the virus-host sophisticated interaction mechanisms and regulatory networks, we seek to identify the M-host cell proteins interactome and investigate the proteins interactive pattern and the impact to viral life cycle, map the functional domains and interaction sites of M and host proteins. The results of this study will provide new insights into the development of novel antiviral targets and therapeutic strategies to fight rabies.

狂犬病是一种由狂犬病病毒(Rabies virus, RABV)引起的重要人兽共患传染病，据WHO统计，全球每年约6万人死于该病。中国狂犬病发病死亡人数居世界第二位，该病是我国公共卫生安全领域不可忽视的威胁。病毒是专性细胞内病原体，其生命进程需要通过病毒蛋白与宿主蛋白发生广泛的相互作用，以利用宿主系统完成增殖传播。基质蛋白M是最小的狂犬病病毒蛋白，却在病毒生命循环中扮演着多种重要角色，负责介导病毒出芽释放、调控病毒转录复制平衡、参与决定病毒致病性和免疫逃逸过程，M蛋白多种功能的实现都需要宿主因素的参与。本项目以M蛋白为研究对象，初步筛选出若干新发现的与M结合的宿主细胞蛋白。在此基础上，进一步鉴定分析M-宿主蛋白互作组，研究两者相互作用模式及对病毒生命进程的影响，定位蛋白功能域与互作位点，揭示病毒与宿主间精密的作用机制与调控网络，为研发创新性抗病毒靶标与治疗策略提供依据。

狂犬病病毒（RABV）基质蛋白M是多功能的结构蛋白，除了在病毒感染复制循环中负责介导病毒粒子装配和出芽释放外，还参与调控病毒的转录复制效率，病毒免疫逃逸机制以及决定病毒致病性。基质蛋白M与功能各异的宿主细胞蛋白的相互作用，是其在病毒生命循环中扮演多种重要角色的基础。籍由病毒蛋白-宿主蛋白间的相互作用网络，RABV得以调控和利用细胞信号通路和生理机制，创造利于病毒感染、增殖、传播的宿主细胞环境。本研究以RABV-M及其互作宿主蛋白为研究对象，通过GST-Pulldown串联质谱技术鉴定了RABV易感细胞内与M蛋白潜在互作的宿主蛋白组，并从中筛选出Ⅲ型中间丝结蛋白Desmin和波形蛋白Vimentin，证实两者均与RABV-M互作并促进M蛋白所介导的病毒粒子出芽释放。同时，证实中间丝骨架结构的完整性对RABV的有效增殖具有重要作用，并且感染RABV的小鼠脑内Ⅲ型中间丝蛋白Desmin和Vimentin表达水平显著上调，提示了Ⅲ中间丝骨架结构对RABV感染增殖的生物学意义。另一方面，鉴定宿主蛋白真核翻译起始因子eIF4A，热休克蛋白Hsp90与RABV-M互作，并正调控病毒感染复制。其中，过表达eIF4A显著提升了RABV-M的转录翻译效率，增加了子代病毒产量。此外，小鼠感染RABV弱毒株后第7天，脑内eIF4A与Hsp90表达水平出现上调。综上所述，本研究完成了易感细胞内RABV-M互作蛋白组的初步鉴定，并筛选到了4个功能各异的，在病毒感染复制循环中具备重要生物学意义的RABV-M互作宿主蛋白，为深入描绘病毒-宿主相互调控网络及新型抗病毒宿主靶标的开发提供了关键信息。