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.

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， 最终导致具有感染性的病毒粒子具有适当的大小和形态。这个项目的长期目标是 实现对蛋白质的机械理解：涉及衣壳组装的蛋白质相互作用。这个 由于缺乏对病毒衣壳蛋白的了解，新的抗病毒药物的开发受到阻碍 被编程为采用正确的构象来生产正确的组装产品。衣壳组件将 用噬菌体P22，一种dsDNA模型病毒进行研究。在噬菌体P22、疱疹病毒和许多其他 双链DNA病毒，衣壳是由一种具有普遍存在的HK97折叠的外壳蛋白形成的。初始程序集 产品是Proapsid(PC)。支架蛋白(SP)指导外壳蛋白(CP)的正确组装形成PC。 SP还指导门户蛋白复合体的掺入，这是基因组封装所必需的。 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 在体外被模仿。噬菌体P22的简单遗传学和成熟的生物化学提供了重要的 与复杂的哺乳动物dsDNA病毒相比，作为组装模型的优势。我们的中心假设是 特定的弱蛋白质：蛋白质相互作用调节组装、成核和延伸反应 以形成合适的前衣壳和病毒粒子。在这一授权期内，我们将检验我们的中心假设 目标有以下几点。 目的1.明确门脉蛋白复合体进入PC的机制。我们假设SP 在PC组装过程中，通过与一条保守的 门静脉环表面有疏水残留物。门静脉蛋白是形成感染性的 病毒粒子用于尾状噬菌体、疱疹病毒和腺病毒。通过对SP和Sp突变体的鉴定 门静脉蛋白，以及使用针对门静脉或SP的单链RNA适配子，我们将阐明其机制。 组装过程中的门户合并。 目的2.了解衣壳形态的控制。我们假设特定的CP构象变化 SP诱导的前壳蛋白和衣壳形态。我们将用单项来描述这种相互作用 分子荧光法。我们将研究CP是如何通过表征CP来控制衣壳形态的 改变PC大小和形状的突变体。 目的3.了解支架蛋白在PC组装中的作用。我们假设SPS有 固有的无序片段，使它们能够与组装所需的许多蛋白质伙伴相互作用 个人电脑。关于它们的结构的高分辨率信息非常少，无论是在溶液中还是在PC中。我们 将使用最先进的核磁共振技术结合突变分析来表征 噬菌体P22和SF6的支架蛋白。