EV71病毒诱导细胞应激颗粒形成的分子机制及其生物学功能

At the early stages of viral infection, the induced mammalian cell cytoplasmic stress granule (SG) are strongly associated with quick shutdown of host mRNA translation. By contrast, some virus can hijack even utilize this stress granule to complete virus complication for their own benefit. Enterovirus 71 (EV71) is a non-enveloped single-(+) strand RNA virus of the Picornaviridae family, genus Enterovirus. EV71 infection mainly caused several epidemics of hand-foot-and-mouth in young children. Our previous study suggested that EV71-induced SG is not only the viral replication platform, but also is the signaling hubs. These results suggested SG can be used as a drug target for anti-EV71 virus. In this study, the main component of the EV71-induced SG will be firstly isolated and identified with mass spectrometry (MS). The interaction among those components will be analyzed. Then the effects of viral proteins, cytoskeletal proteins, p38 MAPK pathway on the mRNP migration and aggregation, as well as their interacting relationship, will be further studied. Subsequently, molecular mechanism that the activation of p38 MAPK pathway induced SG migration and aggregation along the microtubule will be further illuminated. At last, the influence of SG on EV71 virus replication will be investigated. The interaction between SG and signal molecules (p38 MAPK, PI3K/Akt, JNK, RLRs) and their regulation mode will be elucidated. Through elucidation of the molecular mechanism of SG formation and its biological function during EV71 infection, we will not only add newly important theoretical theory basis about the interaction between the EV71 virus and host cells, but also point out a new research direction of EV71 virus.

细胞在病毒感染早期会停止翻译，mRNP在胞质聚集形成应激颗粒拮抗病毒，但有些病毒也会劫持利用应激颗粒。EV71属于单股正链RNA病毒，引起婴幼儿严重手足口病，我们前期发现，应激颗粒不仅是EV71复制的平台，而且是信号转导的枢纽，提示应激颗粒可以作为抗EV71药物研发的靶点，但其形成机制及生物学功能未深入研究。本研究将首次分离鉴定EV71诱导的应激颗粒主要成份，分析这些成份之间的互作关系；然后研究病毒蛋白、细胞骨架蛋白、p38 MAPK通路等在mRNP迁移聚集中的作用，以及互作关系，分析EV71诱导应激颗粒微管蛋白依赖性的形成机制；最后解析应激颗粒与EV71互作对病毒复制的影响，与相关信号分子(p38 MAPK、PI3K/Akt、JNK、RLRs)的相互作用，以及对这些信号通路的调控方式；最终阐明EV71诱导应激颗粒形成的分子机制及其生物学功能，为EV71与宿主的互作提供新的理论和研究方向。

本研究聚焦EV71病毒与宿主互作的前沿科学问题，深入研究EV71病毒与应激颗粒相互作用关系，解析EV71病毒诱导宿主细胞应激颗粒(SGs)迁移，聚集，及其解聚的分子机制，以及细胞应激颗粒的生物学功能。.本研究首先发现，EV71病毒感染早期诱导宿主细胞核蛋白Sam68迁移至细胞质，然后被招募至应细胞激颗粒并参与应激颗粒的形成。应用截短突变分析发现，Sam68的KH结构域在招募过程中发挥至关重要的作用。如果沉默Sam68蛋白的表达并不影响SGs的形成，说明Sam68并不是应激颗粒必须的组成性成份。随后研究发现，如果破坏微管结构会显著抑制应激颗粒的迁移聚集，直至形成，说明EV71病毒诱导的应激颗粒的形成依赖于微管功能的完整性。.进一步研究发现，在EV71病毒感染后期，不仅会解聚并抑制应激颗粒的形成，而且还可以解聚并抑制亚砷酸盐诱导形成的应激颗粒。过表达应激颗粒标示性蛋白G3BP1，不仅会促进应激颗粒的形成，同时会抑制EV71病毒的复制，说明应激颗粒在拮抗EV71病毒复制过程中发挥一定的作用。.这些实验结果说明，在EV71病毒感染早期，EV71病毒感染诱导相关应激颗粒蛋白(如Sam68、G3BP1等)迁移聚集至细胞质，随着微管迁移聚集形成更大的应激颗粒，发挥一定的抗病毒作用。然而，在病毒感染后期，EV71病毒会解聚应激颗粒，从而逃避宿主的抗病毒作用，促进自身的复制。本研究首次解析了EV71病毒诱导宿主应激颗粒迁移聚集，以及解聚应激颗粒的机制与生物学功能，丰富了EV71病毒与宿主互作的相关基础理论，具有重要的科学意义。