Mechanisms of Enterovirus Entry

描述（由申请方提供）：我们将使用结构方法（冷冻电子显微镜和X射线晶体学）研究柯萨奇病毒B3（CVB 3）通过接合两种受体（衰变加速因子和柯萨奇病毒和腺病毒受体）进入宿主细胞的机制。CVB 3是一种人类病原体，可引起心肌炎、胰腺炎，并与青少年糖尿病的发病有关。我们提出的CVB 3进入的研究是与费城儿童医院传染病科Jeffery Bergelson博士富有成效的合作的延续。传统和创新方法的组合将用于可视化受体结合和导致进入和脱膜的构象变化。特别令人感兴趣的是一种从未被观察到的新的进入中间体（目标1）。该颗粒已被分离，具有从衣壳突出的指状密度突起，并且这些密度被假设为用于将颗粒锚到宿主膜的肽。我们将研究这种新粒子（和其他进入中间体）结合受体并感染宿主的能力。除了用纯化的病毒和重组受体蛋白在溶液中进行的常规结构研究（目标2）外，全长受体将锚定在脂质双层圆盘上，形成病毒-受体复合物，用于原位不对称研究（目标3）。这 该方法模拟生理结合事件，其中病毒在其衣壳上的单个集中区域与受体相互作用。这种不对称方法的初步结果表明，与许多可溶性受体相比，病毒与一些膜结合受体相互作用时表现不同。具体来说，我们的原位A颗粒保留了完整的基因组（如A颗粒在感染中所做的那样），并且与用可溶性受体或热处理的颗粒相比，经历了不同的蛋白质重排。拟议的研究旨在了解肠道病毒衣壳在原位成功找到并附着于受体后必须经历的结构改变。

DESCRIPTION (provided by applicant): Using structural approaches (cryo-electron microscopy and x-ray crystallography), we will investigate the mechanisms used by coxsackievirus B3 (CVB3) to enter host cells by engaging two receptors (decay accelerating factor and coxsackievirus and adenovirus receptor). CVB3 is a human pathogen that causes myocarditis, pancreatitis, and has been implicated in the onset of juvenile diabetes mellitus. Our proposed studies of CVB3 entry are a continuation of a productive collaboration with Dr. Jeffery Bergelson, Division of Infectious Diseases at Children's Hospital at Philadelphia. A combination of traditional and innovative methods will be used to visualize both receptor binding and the resulting conformational changes that lead to entry and uncoating. Of particular interest is a novel entry intermediate that has never been observed (Aim 1). This particle has been isolated with fingerlike density projections extruding from the capsid, and these densities are hypothesized to be peptides used to anchor the particle to the host membrane. We will investigate the capability of this new particle (and other entry intermediates) to bind receptors and infect the host. In addition to conventional structural studies made with purified virus and recombinant receptor proteins in solution (Aim 2), the full-length receptor will be anchored onto a lipid bilayer disc to form virus-receptor complexes for an in situ asymmetric study (Aim 3). This approach simulates the physiological binding event, in which the virus interacts with receptors at a single, focused region on its capsid. Preliminary results with this asymmetric approach suggest the virus behaves differently when interacting with a few membrane-bound receptors compared to many soluble receptors. Specifically our in situ A-particle retains an intact genome (as the A-particle does in an infection) and undergoes different protein rearrangements compared to particles that are treated with soluble receptors or heat. The proposed study is directed at understanding the structural alterations that an enterovirus capsid must undergo to uncoat after successfully finding and attaching to the receptor in situ.