Cell Biology of Enterovirus Infection in Polarized Epithelial Cells

描述（由申请人提供）：肠病毒通过粪-口途径传播，必须穿过肠粘膜才会引起感染。肠内排列着极化的上皮细胞，具有明显的顶端和底侧表面；细胞间紧密连接防止病毒接近基底膜上的受体。许多肠病毒，包括许多柯萨奇B病毒（CBV）和埃可病毒（EV），似乎已经独立地进化到结合衰减加速因子（DAF），这是一种在极化上皮表面表达的分子。我们认为这些病毒利用DAF与肠腔上皮细胞相互作用。DAF不仅仅是为病毒附件提供一个站点。我们最近发现，在极化细胞中，它启动了CBV进入和感染所需的至少两条细胞内信号通路：Abl激酶的激活导致肌动蛋白重排和病毒向紧密连接的运动，在那里与柯萨奇病毒和腺病毒受体（CAR）相互作用启动释放病毒RNA的脱膜过程；Fyn激酶的激活触发病毒在囊泡内的内化，并传递到内质网（ER），在那里在复制之前完成脱膜。我们最近已经确定，病毒从紧密连接进入与紧密连接蛋白occludin的内化有关，并且病毒进入和occludin的内化都是通过一个过程发生的，该过程结合了囊泡内吞作用和巨噬细胞增多症的特征。这些结果表明极化细胞的感染是一个复杂的过程，可能涉及独特的细胞生物学机制。我们提出了三组实验来检查极化上皮感染的细胞生物学。1. 我们将定义daf结合的EV在标记病毒进入后进入极化上皮的途径，并使用特定的抑制剂（药物，sirna）来剖析潜在的细胞过程。2. 我们将确定病毒进入过程中肌动蛋白重排的功能和微绒毛结构的变化，并确定在病毒- daf相互作用下肌动蛋白重组的机制。3. 我们将筛选siRNA文库，以确定CBV进入极化上皮所需的信号分子，并确定特定信号分子在感染过程中的作用。公共卫生相关性：肠病毒是病毒性脑膜炎的主要原因，在美国是心肌炎的第二大原因，与极化上皮相互作用是感染的第一步。了解肠病毒感染极化细胞的特殊机制将揭示新的潜在药物靶点。

DESCRIPTION (provided by applicant): Enteroviruses are transmitted by the fecal-oral route, and must cross the intestinal mucosa to initiate infection. The intestines are lined by polarized epithelial cells, with distinct apical and basolateral surfaces; intercellular tight junctions prevent virus access to receptors on basolateral membranes. Many enteroviruses, including many coxsackie B viruses (CBV) and echoviruses (EV), appear to have evolved independently to bind to decay accelerating factor (DAF), a molecule expressed on the surface of polarized epithelium. We believe that these viruses use DAF to interact with epithelial cells in the intestinal lumen. DAF does more than simply provide a site for virus attachment. We recently found that it initiates at least two intracellular signaling pathways required for CBV entry and infection in polarized cells: activation of Abl kinase leads to actin rearangements and virus movement to the tight junction, where interaction with the coxsackievirus and adenovirus receptor (CAR) initiates the uncoating process that releases viral RNA; activation of Fyn kinase triggers virus internalization in caveolar vesicles, and delivery to the endoplasmic reticulum (ER) where uncoating is completed before replication ensues. We have more recently determined that virus entry from the tight junction is linked to the internalization of a tight junction protein, occludin, and that both virus entry and occludin internalization occur by a process that combines aspects of caveolar endocytosis with features characterisitc of macropinocytosis. These results suggest that infection of polarized cells is a complex process likely to involve distinctive cell biological mechanisms. We propose three sets of experiments to examine the cell biology of infection in polarized epithelium. 1. We will define the pathway by which DAF-binding EV enter polarized epithelium, following the entry of labeled virus, and using specific inhibitors (drugs, siRNAs) to dissect the underlying cellular processes. 2. We will determine the function of actin rearrangements and changes in microvillus architecture during virus entry, and identify the mechanism by which actin reorganization is initiated in response to virus-DAF interaction. 3. We will screen a siRNA library to identify signaling molecules required for CBV entry into polarized epithelium, and define the role of specific signaling molecules in the process of infection. PUBLIC HEALTH RELEVANCE: Enteroviruses are the major cause of viral meningitis, and the second major cause of myocarditis in the US, and interaction with polarized epithelium is the first step in infection. Understanding the special mechanisms by which enteroviruses infect polarized cells will reveal new potential drug targets.