SUBUNIT INTERACTIONS DURING ICOSAHEDRAL CAPSID ASSEMBLY

二十面体衣壳组装过程中的亚基相互作用

• 批准号: 2185414
• 负责人: Peter E. Prevelige
• 金额: $ 18.25万
• 依托单位: BOSTON BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2185414
• 关键词: Raman spectrometry; analytical ultracentrifugation; bacteriophage P22; capsid; circular dichroism; conformation; cryoscopy; electron microscopy; fluorescence spectrometry; intermolecular interaction; polymerization; protein purification; protein structure function; sedimentation velocity; stoichiometry; temperature sensitive mutant; thermostability; virion; virus assembly; virus protein

The long term objective of this project is to elucidate the molecular mechanism controlling the assembly of icosahedral virus capsids. The key questions that we wish to address are: 1) What is the pathway by which viral protein subunits assemble into icosahedral capsids, and 2) What controls the conformation switching of the subunits that is required for proper assembly. Our approach is to study the in vitro assembly of the procapsid of wild type and temperature sensitive bacteriophage P22 strains. In this application I propose: 1) To dissect the initiation complex for the in vitro polymerization of procapsids. the presence of the phage encoded pilot protein increases the rate of assembly by stabilizing the initiation complex I will (a) characterize, and isolate the complex of pilot protein with coat protein responsible for stabilizing the initiation complex and (b) I will purify assembly competent chemically crosslinked coat protein oligomers and directly test their ability to initiate the assembly reaction, and interact with the scaffolding protein. 2) To detect, characterize and isolate the "building blocks" for procapsid assembly. The two potential candidates are (a) an oligomer of scaffolding protein, and (b) a coat/protein scaffolding protein hetero- oligomer. The nature of the scaffolding oligomer will be defined by analytical ultracentrifugation. Mixed oligomers will be described by fluorescence, analytical ultracentrifugation, and cryo-electron microscopy. The use of bis-ANS to trap assembly intermediates will be examined. 3) To detect the conformation changes in the coat proteins subunits accompanying assembly. These conformation changes are required for successful polymerization of coat protein subunits into procapsids. The changes in secondary structure will be detected by circular dichroism, and changes in side chain environment by both CD and fluorescence. The stabilization afforded to the subunit upon polymerization will be assessed by thermal studies, and the origin of the morphology change following in vitro polymerization determined by cryoelectron microscopy and image reconstruction. Development of therapeutic agents targeted at direct inhibition of subunit assembly during viral morphogenesis is a promising though relatively unexplored arena. The increasing research effort devoted to the development of viruses as delivery vehicles for therapeutics, be they DNA, protein, or chemotherapeutic agents suggests that the ability to assemble virions i vitro from their proteins subunits in a controlled fashion will ultimately to be essential. It is expected that these studies will contribute to the conceptual framework required to realized these medically important goals.

这个项目的长期目标是阐明分子 二十面体病毒衣壳组装的控制机制。钥匙 我们想要解决的问题是：1)通过什么途径 病毒蛋白亚基组装成二十面体衣壳，以及2)什么 控制所需亚基的构象切换 适当的装配。我们的方法是研究细胞的体外组装 野生型温度敏感型噬菌体P22的普鲁卡菌素 菌株。在本申请中，我建议： 1)剖析了体外聚合的引发络合物。 普罗卡西德。噬菌体编码的引导蛋白的存在增加 稳定引发络合物的组装速度I将(A) 引导蛋白与外壳蛋白复合体的鉴定和分离 负责稳定起始物和(B)我会提纯 组装能力强的化学交联外壳蛋白低聚物和 直接测试它们启动组装反应的能力，以及 与支架蛋白相互作用。 2)检测、表征和隔离 普罗卡西德组合物。这两个潜在的候选者是(A)一种低聚物 支架蛋白，以及(B)外壳/蛋白质支架蛋白杂合体。 齐聚物。支架齐聚物的性质将通过以下方式定义 分析超速离心法。混合低聚物的描述如下 荧光、分析超速离心法和低温电子 显微镜。使用BIS-ANS捕获组装中间体将是 检查过了。 3)检测外壳蛋白亚基的构象变化 随附组件。这些构象变化是必需的 成功地将外壳蛋白亚基聚合成普罗帕斯。这个 二级结构的变化将通过圆二色谱来检测， Cd和荧光对侧链环境的影响。这个 聚合时给亚基提供的稳定性将是 通过热研究进行评估，以及形态变化的来源 冷冻电子显微镜测定的体外聚合反应 和图像重建。 以直接抑制为靶点的治疗药物的研究进展 病毒形态发生过程中的亚基组装是一种很有前途的方法 相对未开发的竞技场。越来越多的研究工作致力于 病毒作为治疗载体的发展，无论是它们 DNA、蛋白质或化疗药物表明， 在受控条件下从病毒蛋白亚基体外组装病毒粒子 时尚最终将是必不可少的。预计这些 研究将有助于实现所需的概念框架 这些医学上重要的目标。