肠道病毒71型2C蛋白分选病毒核酸进入外泌体的作用机制研究

EV71是手足口病的主要病原体，其致病机制尚不明确。病毒可利用含有病毒成份的外泌体感染细胞，以扩大感染。前期发现EV71的3A蛋白通过与Rab27a结合增强外泌体的释放，然而病毒RNA进入外泌体的机制尚不清楚。预实验结果提示，EV71的非结构蛋白2C能促进病毒RNA进入细胞内体，增加外泌体中病毒RNA含量。且2C能与胞内的HSPA8蛋白直接作用，外泌体中有高水平HSPA8。由此推测，在EV71感染时，2C蛋白结合病毒RNA，在HSPA8的帮助下，分拣病毒RNA进入内体，随外泌体释放，促进病毒的散播。本项目将观察胞内病毒RNA、2C、HSPA8在内体中的共定位及时空关系，筛选HSPA8与2C蛋白的互作位点，确定2C蛋白分选病毒RNA的机制，并利用外泌体形成模型和动物模型加以验证，再分析外泌体与临床的相关性。本研究将阐明肠道病毒调控病毒RNA进入外泌体的机制，为病毒致病机理的研究提供新的切入点

EV71 is the main pathogen of hand, foot and mouth disease, but the pathogenic mechanisms remain largely unknown. The virus can expand the infection by exosomes containing viral components. We found previously that 3A protein of EV71 could enhance the release of exosomes by binding to Rab27a. However, the mechanism of sorting viral RNA into exosomes remains unclear. The preliminary data suggested that the viral non-structural protein 2C could promote the sorting of viral RNA into the late endosome and increase the content of viral RNA in exosomes. Moreover, 2C can directly interact with intracellular HSPA8 protein, and there is a high level of HSPA8 in exosomes. Therefore, it is speculated that 2C protein binding to viral RNA, with the help of HSPA8, sorts viral RNA into the endosome which is released in exosomes later and promotes the spread of viruses. This project will observe the co-localization and spatiotemporal relationship of intracellular viral RNA, 2C, and HSPA8 in the endosome, and screen the interaction sites between HSPA8 and 2C proteins, so as to determine the mechanism of 2C proteins sorting viral RNA into exosomes. Exosome formation model and animal model were used to verify the mechanism of 2C protein. The correlation of exosome and disease severity in EV71 infected patients will be analyzed also. This study will elucidate the mechanism by which enterovirus regulates sorting of viral RNA into exosomes and provide a new insight for the study of viral pathogenesis.