Interactions of proteins in the Bluetongue virus core

描述(申请人提供)：一旦细胞感染，呼肠孤病毒衣壳永远不会完全分解，病毒转录本从二十面体核心颗粒中挤出。因此，核心必须包含转录和封端所需的所有酶反应。病毒蛋白质之间以及这些蛋白质与核心的病毒基因组之间的相互作用是高度特异的，核心可以被认为是一台精确的分子机器。该家族的三个成员(蓝舌病毒、水稻矮缩病毒和哺乳动物正病毒)的核心的原子结构是可用的，并且是通过X射线衍射解决的最大结构之一。然而，BTV(和RDV)的核心架构与呼肠孤病毒不同，只有呼肠孤病毒有定义的盖子转塔。对于BTV，封顶蛋白与RNA依赖的RNA聚合酶(RdRp)共同定位在核心的五个顶点。虽然核内的转录复合体已经定位，但不可能从晶体结构中解析其结构细节。本申请的目的是为BTV转录复合体的功能提供一个完整的结构和生化理解。这一目标的完成将为合理设计抑制环状病毒复制的抑制剂铺平道路，并为具有致病意义的相关病毒提供先导化合物。此外，这项工作将有助于理解转录的结构基础和理解如何从基于蛋白质的模块化单位构建复杂的机器的日益增长的领域。BTV在传递这一信息方面处于独特的地位。它是呼肠孤病毒科中唯一可以获得纯化重组酶蛋白的原子核结构和完整的体外活性的非转塔成员。拟议的研究将建立在这些试剂和我们最近的BTV封端酶的晶体结构的基础上，以了解酶功能的分子基础以及核心的酶蛋白如何协同作用实现转录。在具体目标1中，我们将进行详细的诱变、生化和生物物理研究，以了解封闭酶四种酶活性的结构与功能关系。具体目标2将侧重于Capping和RdRp蛋白之间的蛋白质-蛋白质相互作用，以及这些蛋白质与核心主要结构蛋白之间的相互作用。具体目标3将侧重于核心相关病毒解旋酶蛋白的结构-功能研究，以了解该蛋白是否在病毒基因组的转录或包装中发挥功能。最后，在特定的目标4重组蛋白将在体外重建病毒转录复合体，以了解这些蛋白是如何相互作用和影响彼此的活性。在所有方法中，我们将结合使用结构方法(核磁共振、低温电子显微镜、X射线结晶学、动态和静态光散射)和生化方法(单分子动力学研究、诱变、拉曼和红外光谱、高效液相色谱、酶分析跟踪动力学、表面等离子体共振)。公共卫生相关性：该项目旨在提供对复杂病毒如何作为纳米机器发挥作用的完整了解。这一目标的完成将为合理设计抑制病毒(特别是动物病毒)复制的抑制剂铺平道路，并为对人和动物具有致病意义的相关病毒提供先导化合物。

DESCRIPTION (provided by applicant): Upon cell infection, Reoviridae capsids never completely disassemble, and viral transcripts are extruded from the icosahedral core particle. As a consequence the core must contain all the necessary enzymatic reactions for transcription and capping. The interactions between viral proteins and between these proteins and the viral genome in the core, is highly specific and the core may be considered to function as a precise molecular machine. Atomic structures for the cores of three members of this family (Bluetongue virus, Rice dwarf virus and mammalian orthoreovirus) are available, and are among the largest structures solved by X- ray diffraction. However, the core architecture of BTV (and RDV) is different with only reovirus having defined capping turrets. For BTV, capping protein is co-localised with the RNA-dependent-RNA-polymerase (RdRp) at the fivefold vertices of the core. Although the transcription complex within the core has been localised, it was not possible to resolve its structural details from the crystal structure. The objective of this application is to provide a complete structural and biochemical understanding of how the BTV transcription complex functions. Completion of this goal will pave the way for the rational design of inhibitors to block orbivirus replication and provide lead compounds for related viruses of pathogenic significance. Furthermore, the work will contribute to the growing areas of understanding the structural basis of transcription and for understanding how complex machines can be constructed from modular protein-based units. BTV is uniquely placed to deliver this information. It is the only non-turreted member of the Reoviridae for which an atomic core structure and complete in vitro activity of purified recombinant enzymatic proteins is available. The proposed research will build on these reagents and on our recent crystal structure of the BTV capping enzyme to understand the molecular basis of enzyme function and how the enzymatic proteins of the core act in concert to achieve transcription. In Specific Aim 1 we will undertake a detailed program of mutagenesis, biochemical and biophysical studies to understand the structure-function relationships of the four enzymatic activities of the capping enzyme. Specific Aim 2 will focus on the protein-protein interactions between the capping and RdRp proteins, and between these proteins and the major structural proteins of the core. Specific Aim 3 will focus on structure-function studies of the core associated viral helicase protein to understand if this protein is functional in transcription or packaging of the viral genome. Finally, in Specific Aim 4 recombinant proteins will be used to reconstitute viral transcription complexes in vitro to understand how these proteins interact with and affect the activity of each other. In all approaches we will use a combination of structural (NMR, cryo-EM, X-ray crystallography, dynamic and static light scattering) and biochemical (single molecule kinetic studies, mutagenesis, Raman and IR spectroscopy, HPLC, enzyme assays to follow kinetics, surface plasmon resonance) approaches. PUBLIC HEALTH RELAVANCE: The project aims to provide a complete understanding of how a complex virus functions as a nanomachine. Completion of this goal will pave the way for the rational design of inhibitors to block virus (in particular, an animal virus) replication and provide lead compounds for related viruses of pathogenic significance to humans and animals.