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: 10194510
• 负责人: CAROLYN M TESCHKE
• 金额: $ 55.57万
• 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/10194510
• 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，涉及多个蛋白质的顺序添加， 最终导致具有适当大小和形态的感染性病毒体。该项目的长期目标是 实现蛋白质的机械理解：蛋白质相互作用参与衣壳装配。的 新的抗病毒药物的开发由于缺乏对病毒衣壳蛋白如何 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（一种模型dsDNA病毒）进行研究。在噬菌体P22中，疱疹病毒和许多其他病毒 在dsDNA病毒中，衣壳由具有普遍存在的HK97折叠的外壳蛋白形成。述初始组装 产物是原衣壳（PC）。支架蛋白（SP）指导外壳蛋白（CP）正确组装形成PC。 SP还指导门户蛋白复合物的掺入，这是基因组整合所必需的。 噬菌体P22提供了一个很好的模型组装系统，因为复杂的体内过程很容易 在试管中模仿。噬菌体P22的简单遗传学和良好建立的生物化学提供了重要的 作为组装模型优于复杂的哺乳动物dsDNA病毒。我们的核心假设是， 特异性弱蛋白：蛋白质相互作用调节组装成核和延伸反应 以形成合适的原衣壳和病毒体。在这段时间里，我们将检验我们的核心假设。 具有以下目的。 目标1.明确门脉蛋白复合物掺入PC的机制。我们假设SP 在PC组装过程中，通过与保守的 疏水性残基在门静脉环的表面上。门脉蛋白是形成感染性 病毒体用于尾腺病毒、疱疹病毒和腺病毒。虽然在SP中突变体的表征和 门蛋白，以及门或SP特异性ssRNA适体的使用，我们将阐明 组装期间的门户并入。 目标2.了解衣壳形态的控制。我们假设特定的CP构象变化 SP诱导控制原衣壳和衣壳形态。我们将通过单个 分子荧光法我们将研究CP如何控制衣壳形态的特点CP 改变PC大小和形状的突变体。 目标3.了解支架蛋白在PC组装中的功能。我们假设SP具有 内在无序的片段，使它们能够与组装所需的许多蛋白质伴侣相互作用， PCs.无论是在溶液中还是在PC中，关于它们结构的高分辨率信息都很少。我们 将使用最先进的核磁共振技术结合突变分析来表征 支架蛋白来自于CMP22和Sf6。