Assembly of a Dodecahedral Virus

十二面体病毒的组装

• 批准号: 8600240
• 负责人: Adam Zlotnick
• 金额: $ 6.55万
• 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 2016-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8600240
• 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。病毒的组装机制对其生物学至关重要，可能为抗病毒药物提供新的未经测试的靶点， 并且可能是利用病毒作为纳米技术平台的一种手段。然而，对组装的理解却很少。病毒衣壳是由数十到数千个蛋白质组成的复合体，通常以二十面体点对称排列。可能的组装中间体的数量随着参与反应的单元的数量而组合地增长。据信小核糖核酸病毒提供衣壳组装的最简单实例。基于估计的沉降系数，假定小核糖核酸病毒由12个五聚体组装而成。（十二面体与二十面体共享点对称，并且是二十面体的对偶）。我们选择检查牛肠道病毒（BEV），这是脊髓灰质炎病毒和人肠道病毒-71的非致病性关系。BEV即使在牛中也是非致病性的，在那里它是地方性的。除了易于处理之外，在培养物中BEV产生高产率的14 S中间体和高产率的空的未成熟衣壳（80 S）;这些物种的存在使其成为组装研究的有价值的模型。在初步的数据中，我们建立了BEV作为一个易处理的实验系统。我们已经开发了一种有效的BEV过表达系统。我们精确地测定了14 S和80 S的分子量，发现它们分别对应于五聚体和五角十二面体。我们已经证明14 S可逆地组装成80 S。在本提案中，我们将定义BEV的体外组装机制，并将其作为理解组装在BEV复制中的作用的工具。在aim 1中，我们将观察溶液中的组装，表征组装的能量学和动力学，将动力学拟合为严格的 组装的数学模型，并确定组装反应物和产物的结构。在目标2中，我们将扰乱组装并检查其对病毒生产的影响。使用目标1中确定的结构和文献中的晶体结构，我们将设计组装突变体，以便我们可以比较体外活性与培养中的突变体活性;我们还将（首次）研究小分子对小核糖核酸病毒组装反应的影响。这些结果将有助于在体外模型的小核糖核酸病毒组装和组装生物物理学与小核糖核酸病毒复制的相关性。总之，这些努力将导致更好地了解病毒组装过程在体外和体内。