Mechanisms of Enterovirus Entry

描述(申请人提供)：利用结构方法(冷冻电子显微镜和X射线结晶学)，我们将研究柯萨奇病毒B3(CVB3)通过结合两个受体(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。CVB3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。我们提出的关于CVB3进入的研究是与费城儿童医院传染病科杰弗里·伯格尔森博士富有成效合作的继续。传统和创新方法的结合将被用来可视化受体结合和导致进入和去涂层的由此产生的构象变化。特别令人感兴趣的是一种从未被观察到的新型进入中间体(目标1)。这种颗粒已经被分离出来，从衣壳中伸出指状密度突起，这些密度被假设为用于将颗粒锚定到宿主膜上的多肽。我们将研究这种新颗粒(和其他进入中间体)与受体结合并感染宿主的能力。除了在溶液中用纯化的病毒和重组受体蛋白进行常规结构研究(AIM 2)外，全长受体将锚定在脂质双层圆盘上，形成病毒-受体复合体，用于原位不对称研究(AIM 3)。这 Approach模拟了生理结合事件，在该事件中，病毒与其衣壳上单个聚焦区域的受体相互作用。这种不对称方法的初步结果表明，与许多可溶性受体相比，病毒与少数膜结合受体相互作用时的行为不同。具体地说，我们的原位A-颗粒保留了一个完整的基因组(就像A-颗粒在感染中所做的那样)，并且与用可溶性受体或加热处理的颗粒相比，它经历了不同的蛋白质重排。这项拟议的研究旨在了解肠道病毒衣壳在成功发现并原位附着于受体后必须经历的结构变化。

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.