Understanding the Protein: Protein Interactions Required for Virus Assembly

了解蛋白质：病毒组装所需的蛋白质相互作用

• 批准号: 10217668
• 负责人: CAROLYN M TESCHKE
• 金额: $ 6.78万
• 依托单位: UNIVERSITY OF CONNECTICUT STORRS
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-15 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10217668
• 关键词: Abbreviations; Adenoviruses; Adopted; Affect; Amino Acid Substitution; Antiviral Agents; Bacteriophage P22; Bacteriophages; Basic Amino Acids; Biochemistry; Biological Assay; Biological Models; Capsid; Capsid Proteins; Complex; DNA; Development; Disease; Double Stranded DNA Virus; Electrostatics; Environment; Fluorescence; Genetic; Genome; Goals; Grant; Helix-Turn-Helix Motifs; Herpesviridae; Hydrophobicity; In Vitro; Individual; Lead; Methods; Modeling; Molecular Conformation; Morphology; Mutation Analysis; Outcome; Pharmaceutical Preparations; Pliability; Process; Protein Analysis; Protein Conformation; Proteins; Public Health; Reaction; Research; Resolution; Role; Scaffolding Protein; Shapes; Site; Specificity; Structure; Surface; System; Tail; Techniques; Testing; Viral; Viral Proteins; Virion; Virus; Virus Assembly; Work; aptamer; base; design; in vivo; monomer; mutant; novel; particle; protein complex; protein function; protein protein interaction; single molecule; therapeutic target; virus morphology

Icosahedral capsid assembly is a highly coordinated process involving sequential addition of multiple proteins, ultimately leading to an infectious virion of proper size and morphology. The long-term goal for this project is to achieve a mechanistic understanding of the protein:protein interactions involved in capsid assembly. The development of new anti-viral drugs is impeded by a lack of understanding of how viral capsid proteins are programmed to adopt the correct conformations to produce the correct assembly product. Capsid assembly will be investigated using bacteriophage P22, a model dsDNA virus. In phage P22, herpesvirus and many other dsDNA viruses, the capsid is formed from a coat protein having the ubiquitous HK97 fold. The initial assembly product is a procapsid (PC). Scaffolding protein (SP) directs proper assembly of coat protein (CP) to form PCs. SP also directs the incorporation of the portal protein complex, which is essential for genome encapsidation. Phage P22 provides an excellent model assembly system because complex in vivo processes are easily mimicked in vitro. The simple genetics and well-established biochemistry of phage P22 offers significant advantages as an assembly model over complex mammalian dsDNA viruses. Our central hypothesis is that specific weak protein:protein interactions regulate the assembly nucleation and elongation reactions in order to form proper procapsids and virions. In this granting period we will test our central hypothesis with the following aims. Aim 1. Define the mechanism of portal protein complex incorporation into PC. We hypothesize that SP controls portal protein incorporation during PC assembly through interaction with a conserved belt of hydrophobic residues on the surface of the portal rings. The portal protein is essential to form an infectious virion for the tailed phages, herpesviruses and adenoviruses. Though characterization of mutants in SP and portal protein, and the use of ssRNA aptamers specific for portal or SP, we will elucidate the mechanism of portal incorporation during assembly. Aim 2. Understand control of capsid morphology. We hypothesize specific CP conformational changes induced by SP control procapsid and capsid morphology. We will characterize the interaction by single molecule fluorescence methods. We will investigate how CP controls capsid morphology by characterizing CP mutants that change the size and shape of PCs. Aim 3. Understand how scaffolding protein functions in PC assembly. We hypothesize that SPs have intrinsically disordered segments to allow them to interact with the many protein partners required to assemble PCs. There is very little high-resolution information about their structures, either in solution or within PCs. We will use state-of-the-art NMR techniques combined with mutational analysis to characterize the structure of scaffolding proteins from phages P22 and Sf6.

二十面体衣壳组装是一个高度协调的过程，涉及顺序添加多种蛋白质， 最终产生具有适当大小和形态的感染性病毒粒子。该项目的长期目标是 实现对衣壳组装中涉及的蛋白质：蛋白质相互作用的机制理解。这 由于缺乏对病毒衣壳蛋白如何运作的了解，新的抗病毒药物的开发受到阻碍 编程以采用正确的构造来生产正确的组装产品。衣壳组装将 使用噬菌体 P22（一种双链 DNA 病毒模型）进行研究。在噬菌体 P22、疱疹病毒和许多其他病毒中 双链 DNA 病毒的衣壳由具有普遍存在的 HK97 折叠的外壳蛋白形成。初始组装 产品是原衣壳(PC)。支架蛋白 (SP) 指导外壳蛋白 (CP) 正确组装以形成 PC。 SP 还指导门户蛋白复合物的掺入，这对于基因组衣壳化至关重要。 Phage P22 提供了出色的模型组装系统，因为复杂的体内过程很容易 体外模拟。噬菌体 P22 简单的遗传学和完善的生物化学提供了重要的意义 与复杂的哺乳动物双链 DNA 病毒相比，作为组装模型具有优势。我们的中心假设是 特定的弱蛋白：蛋白质相互作用调节组装成核和延伸反应 以形成适当的原衣壳和病毒体。在这个授权期间，我们将检验我们的中心假设 具有以下目标。 目标 1. 明确门静脉蛋白复合物掺入 PC 的机制。我们假设 SP 通过与保守的带相互作用，控制 PC 组装过程中的门蛋白掺入 门环表面的疏水残基。门蛋白对于形成感染性至关重要 有尾噬菌体、疱疹病毒和腺病毒的病毒颗粒。尽管SP和突变体的表征 门静脉蛋白，以及使用针对门静脉或 SP 的 ssRNA 适体，我们将阐明其机制 组装过程中的门户合并。 目标 2. 了解衣壳形态的控制。我们假设特定的 CP 构象变化 由SP控制原衣壳和衣壳形态诱导。我们将通过单一的交互来表征 分子荧光方法。我们将通过表征 CP 来研究 CP 如何控制衣壳形态 改变 PC 大小和形状的突变体。 目标 3. 了解支架蛋白在 PC 组装中如何发挥作用。我们假设 SP 有 本质上无序的片段使它们能够与组装所需的许多蛋白质伙伴相互作用 件。无论是在解决方案中还是在电脑中，关于它们结构的高分辨率信息都非常少。我们 将使用最先进的核磁共振技术结合突变分析来表征 来自噬菌体 P22 和 Sf6 的支架蛋白。