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: 10021667
• 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/10021667
• 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.

Icosahedral Capsid组件是一个高度协调的过程，涉及多种蛋白的顺序添加， 最终导致具有适当大小和形态的感染性病毒。该项目的长期目标是 对蛋白质的机械理解：与衣壳组装有关的蛋白质相互作用。这 由于缺乏对病毒capsid蛋白的了解而阻碍了新的抗病毒药物的开发 编程以采用正确的构象来生产正确的组装产品。衣壳大会将 可以使用DSDNA病毒模型P22进行研究。在噬菌体P22中，疱疹病毒和许多其他 dsDNA病毒是由具有无处不在的HK97倍的外套蛋白形成的。初始组件 产品是Procapsid（PC）。脚手架蛋白（SP）指导涂层蛋白（CP）的适当组装形成PC。 SP还指导门蛋白复合物的掺入，这对于基因组封装至关重要。 噬菌体P22提供了出色的模型组装系统，因为体内过程很容易 在体外模仿。噬菌体P22的简单遗传学和良好的生物化学提供了重要的 优于复杂哺乳动物DSDNA病毒的组装模型。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伸长反应 为了形成适当的procapsid和病毒体。在此授予期内，我们将检验我们的中心假设 以以下目标。 AIM 1。将门户蛋白复合物的机理定义为PC。我们假设SP 通过与保守的皮带相互作用，可以控制PC组装过程中的门户蛋白掺入 门户环表面上的疏水残基。门户蛋白对于形成感染性至关重要 病毒粒子用于尾巴，疱疹病毒和腺病毒。尽管SP中突变体的表征以及 门户蛋白，以及针对门户或SP的SSRNA适体的使用，我们将阐明 组装期间的门户合并。 目的2。了解对衣壳形态的控制。我们假设特定的CP构象变化 由SP控制procapsid和衣壳形态诱导。我们将通过单个表征互动 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的大小和形状的突变体。 目标3。了解脚手架蛋白在PC组装中的功能。我们假设SPS有 本质上无序的段，使它们与组装所需的许多蛋白质伴侣相互作用 件。有关其结构的高分辨率信息，无论是在解决方案还是在PC中。我们 将使用最先进的NMR技术与突变分析相结合来表征 来自噬菌体P22和SF6的脚手架蛋白质。