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且未经测试的靶标， 并且可能是利用病毒作为纳米技术平台的一种手段。然而，人们对汇编知之甚少。病毒衣壳是由数十至数千个蛋白质组成的复合物，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增加。小核糖核酸病毒被认为提供了衣壳组装的最简单的例子。根据估计的沉降系数，推测小核糖核酸病毒由十二个五聚体组装而成。 （十二面体与二十面体共享点对称性，并且与二十面体对偶。）我们选择检查牛肠道病毒 (BEV)，它是脊髓灰质炎病毒和人类肠道病毒 71 的非致病关系。即使在流行的牛身上，BEV 也不具有致病性。除了易于操作外，BEV 在培养过程中还能产生高产率的 14S 中间体和高产率的空未成熟衣壳 (80S)；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 建立为一个易于处理的实验系统。我们开发了一种有效的 BEV 过度表达系统。我们准确测量了14S和80S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 14S 可逆地组装成 80S。在本提案中，我们将定义 BEV 的体外组装机制，并将其用作理解组装在 BEV 复制中的作用的工具。在目标 1 中，我们将观察溶液中的组装，表征组装能量学和动力学，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产生的影响。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，以便我们可以将体外活性与培养中的突变体活性进行比较；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。这些结果将有助于建立小核糖核酸病毒组装的体外模型以及组装生物物理学与小核糖核酸病毒复制的相关性。总而言之，这些努力将使人们更好地了解体外和体内病毒组装的过程。