肠道病毒71型神经细胞中复制中心形成机制的研究

Enterovirus 71 (EV71) is a single-stranded RNA icosahedral virus 30nm in diameter belonging to the genus Enterovirus within the Picornaviridae family. In young children, its infection usually causes hand, foot and mouth disease (HFMD). EV71 infections are usually accompanied by severe neurological complications such as aseptic meningitis, acute flaccid paralysis, encephalitis and other rarer manifestations. Although the initial viral illness often is self-limited, EV71 infection may result in long term neurologic and psychiatric effects on the central nervous system (CNS) in children. Members of the Picornaviridae have similar particle morphology and genome organization, but several studies have revealed important differences in the replication center of picornaviruses from different genera. However, little is known about the development of a specialized area for virus replication in EV71 infected cells. In previous study, we investigated the involvement of vimentin in EV71 replication and the effects of virus infection on vimentin structure in human astrocytoma cells. We showed that EV71 infection activated Calmodulin-dependent Protein Kinase II （CaMK-II）, resulting in the phosphorylation of vimentin at serine 82. The serine 82 phosphorylated vimentin was rearranged, leading to the formation of virus replication centers near the perinuclear region.Phosphorylation of vimentin and the formation of aggresomes were required for the replication of EV71 since the latter was decreased markedly after phosphorylation was blocked by KN93, a CaMK-II inhibitor. Thus, as one of the consequences of CaMK-II activation, vimentin phosphorylation and rearrangement may support virus replication by playing a structural role for the formation of the replication factories. Despite we have proved that the formation of EV71 replication centers is CaMK-II activation dependent，and EV71 VP1 protein is responsible for the activation of CaMK-II. But how EV71 VP1 expression lead to the activation of CaMK-II is still unknown. In the next step, by analyzing the influences of EV71 VP1 on the signal pathways that involved in CaMK-II activation, we will determine the detailed mechanisms of how EV71 VP1 expression activates CaMK-II and help to form viral replication centers. In another study, we observed that EV71 infection recruited mitochondria，as reflected by numerous mitochondria arranged surround the viral replication centers. But how the mitochondria were recruited during EV71 infection still require further analysis. Thus,in present study，we will determine the reasons for mitochondria reclustering,and at the same time, study the influences of mitochondria rearrangement on EV71 replication. Furthermore, we will indeed analyze the other host factors that involved in EV71 replication centers formation.

肠道病毒71型(EV71)属小RNA病毒科肠道病毒属，是人手足口病的一种主要病原病毒。与其他手足口病病原病毒不同，EV71的感染除引导致足口病外，通常还会引起严重的神经系统病变。目前认为，EV71对神经细胞的感染所引起细胞凋亡或坏死是其导致神经系统病变的诱因。病毒感染宿主细胞后，通常会在细胞内一个特殊的区域产生病毒的复制中心。这种复制中心的形成对于病毒组分的产生，病毒粒子的组装及成熟至关重要。通过研究，我们发现EV71感染人神经细胞后，通过其VP1蛋白激活钙依赖性蛋白激酶II（CaMK-II），导致中间丝Vimentin的解聚，并在细胞的特殊区域形成vimentin依赖性的聚集体。进一步研究显示，这种聚集体结构是EV71在神经细胞中的复制中心。本研究中，我们将进一步探索EV71在神经细胞中是如何激活CaMK-II，如何形成病毒复制中心等科学问题。以期为EV71抑制类药物的研究提供一定基础。

EV71是人手足口病的主要病原病毒之一，其感染通常会导致被感染儿童出现严重的神经系统病变并可能导致神经系统的不可逆损伤。近几年，我国大陆地区的手足口病感染呈上升趋势，由EV71感染导致的死亡病例也逐年增加。病毒感染宿主细胞后， 通常会在细胞内一个特殊的区域产生病毒的复制中心。 这种复制中心的形成对于病毒组分的产生，病毒粒子的组装及成熟至关重要。之前，关于EV71在宿主细胞内是否存在复制中心，复制中心的结构及其形成机制等并无相关研究。本课题中，我们通过电子显微镜及共聚焦显微技术等，分析了EV71在人神经细胞中的复制中心的位置及结构，并阐述了复制中心形成的机理。在EV71复制中心中，我们观察到了膜结构及其表面的纤维结构的蛋白。EV71可以通过其VP1结构蛋白来促进Vimentin的解聚，Vimentin会进一步结合到膜结构表面并招募附近的VP1结构蛋白，从而形成一个组装位点，并进一步结合其他结构蛋白来形成病毒的复制中心。在另外一项研究中，我们发现EV71感染后，可以通过其3C非结构蛋白的入核，并促进细胞核内的Sam68蛋白向胞质的扩散。Sam68蛋白会结合到复制中心EV71基因组的5’端的IRES区，并进行病毒蛋白的翻译。此外，我们还发现，EV71会通过其2BC蛋白前体促进细胞内线粒体向复制中心的聚集，并以此为病毒复制提供更多的能量和原料。总之，通过我们一系列的研究，揭示了EV71病毒复制中心形成的机制，鉴定出了一些在病毒复制中心形成中极为关键的细胞及病毒组分。这些为我们进一步了解EV71的复制机制及致病机理提供了重要的理论基础。