Assembly of a Dodecahedral Virus

十二面体病毒的组装

• 批准号: 8415339
• 负责人: Adam Zlotnick
• 金额: $ 7.31万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8415339
• 关键词: Antiviral Agents; Baculovirus Expression System; Biology; Biophysics; Bovine Enterovirus; Capsid; Capsid Proteins; Cattle; Cell Culture Techniques; Complement; Complex; Data; Defect; Enterovirus 71; Family; Family Picornaviridae; Foot-and-Mouth Disease; Genome; Hepatitis A Virus; Hepatitis B Virus; Human poliovirus; In Vitro; Infection; Ionic Strengths; Kinetics; Lead; Literature; Measurement; Measures; Modeling; Molecular Weight; Mutation; Nanotechnology; Nucleic Acids; Physics; Picornaviridae Infections; Plaque Assay; Play; Process; Production; Proteins; Reaction; Relative (related person); Rhinovirus; Role; Solutions; Structure; System; Testing; Thermodynamics; Time; Viral Packaging; Virion; Virus; Virus Assembly; Virus Replication; base; design; in vitro Model; in vitro activity; in vivo; mathematical model; milligram; mutant; particle; pathogen; public health relevance; receptor binding; response; sedimentation coefficient; small molecule; tool; trafficking

DESCRIPTION (provided by applicant): Assembly of a Dodecahedral Virus. The assembly mechanism of a virus is critical to its biology, may offer new and untested targets for antivirals, and may be a means to leverage viruses as platforms for nanotechnology. However, assembly is poorly understood. Virus capsids are complexes of tens to thousands of proteins, usually arranged with icosahedral point symmetry. The number of possible assembly intermediates grows combinatorially with the number of units involved in the reaction. Picornaviruses are believed to provide the simplest example of capsid assembly. Based on estimated sedimentation coefficients, it was postulated that picornaviruses are assembled from twelve pentamers. (Dodecahedra share point symmetry with, and are dual to, icosahedra.) We have chosen to examine Bovine Enterovirus (BEV) a non-pathogenic relation of Poliovirus and Human Enterovirus-71. BEV is non-pathogenic even in cattle, where it is endemic. In addition to its ease of handling, in culture BEV produces high yields of 14S intermediates and high yields of empty immature capsid (80S); the presence of these species are what makes it a valuable model for assembly studies. In preliminary data, we establish BEV as a tractable experimental system. We have developed an effective BEV over-expression system. We have accurately measured the molecular weights of 14S and 80S and found they correspond to pentamer and pentagonal dodecahedron, respectively. We have shown that 14S reversibly assembles into 80S. In this proposal, we will define the in vitro assembly mechanism of BEV and use that as a tool for understanding the roles of assembly in BEV replication. In aim 1, we will observe assembly in solution, characterizing assembly energetics and kinetics, fitting kinetics to rigorous mathematical models of assembly, and determining the structures of assembly reactants and products. In aim 2, we will perturb assembly and examine its effect on virus production. Using structures determined in aim 1 and crystal structures from the literature, we will design assembly mutants so that we can compare in vitro activity with mutant activity in culture; we will also examine (for the first time) the effect of small molecules on the picornavirus assembly reaction. These results will contribute an in vitro model of picornavirus assembly and a correlation of assembly biophysics with picornavirus replication. Taken together, these efforts will lead to a far better understanding of the process of virus assembly in vitro and in vivo.

描述（由申请人提供）：十二面体病毒的组装。病毒的装配机制对其生物学至关重要，可能为抗病毒药提供了新的和未经测试的靶标， 并且可能是将病毒作为纳米技术平台的一种手段。但是，集会知之甚少。病毒衣壳是数十万至数千种蛋白质的复合物，通常与二十六角点对称性排列。可能的组装中间体的数量与反应中涉及的单位数量合并。据信PICORNAVIRAS提供了最简单的衣壳组件示例。基于估计的沉积系数，据推测，picornavirus是从十二个五聚体中组装的。 （Dodecahedra与Icosahedra的共享点对称性。）我们选择检查牛肠病毒（BEV）脊髓灰质炎病毒和人肠病毒-71的非致病关系。 BEV即使在流行的牛中也是非致病的。除了易于处理之外，在培养中，BEV还产生了14S中间体的高收率和空的未成熟帽壳（80s）的高收率。这些物种的存在使其成为组装研究的宝贵模型。在初步数据中，我们将BEV建立为可聊天的实验系统。我们已经开发了一个有效的BEV过表达系统。我们已经准确地测量了14s和80s的分子量，发现它们分别对应于五角星和五角形十二面体。我们已经证明14s可逆地组装成80s。在此提案中，我们将定义BEV的体外装配机理，并将其用作理解组装在BEV复制中的作用的工具。在AIM 1中，我们将在解决方案中观察组装，表征组装能量和动力学，将动力学拟合到严格 组装的数学模型，并确定组装反应物和产品的结构。在AIM 2中，我们将扰动组装并检查其对病毒生产的影响。使用在AIM 1和文献中确定的结构，我们将设计组装突变体，以便我们可以将体外活性与培养中的突变活性进行比较。我们还将（这是第一次）小分子对Picornavirus组装反应的影响。这些结果将促进Picornavirus组装的体外模型，以及组装生物物理学与Picornavirus复制的相关性。综上所述，这些努力将导致对体外和体内病毒组装过程的更好理解。