Defining the cis and trans acting factors required for assembly of infectious Blu

描述（由申请方提供）：本提案的具体目的是完全了解无包膜节肢动物传播病毒蓝舌病毒（BTV）感染过程的长期目标的一部分，蓝舌病毒是呼肠孤病毒科的一个成员，包括感染广泛宿主的病毒，包括人类、动物、植物、真菌和细菌。因此，BTV与其他几种科学和医学上重要的病毒（例如轮状病毒）有着家族关系。BTV作为经济上重要的疾病的病原体已经被广泛研究，并且越来越多地作为相关病毒的模型系统。在分子、生物化学和结构领域已经取得了实质性的进展;然而，由于缺乏适当的工具和测定系统，BTV和相关病毒的生物学和结构的某些领域仍然不清楚。BTV和其他成员（轮状病毒和呼肠孤病毒）是复杂的多层衣壳病毒，具有10-12个分段的双链RNA基因组，但病毒基因组如何包装到其保护性衣壳中的细节或每个片段的拷贝并入病毒以使其存活的机制仍然难以捉摸。在这项提案中，我们的目标是保持和扩大BTV的领先地位，以实现我们的长期目标，即全面了解病毒的感染过程。特别地，本申请将利用突破性的技术进步（即，第一个复杂dsRNA病毒的体外包装系统），以了解这种复杂衣壳病毒的基因组包装和复制事件，并推断所有多节段RNA病毒。在具体目标1中，我们将确定十个ssRNA的包装顺序和BTV颗粒组装中涉及的信号。在具体目标2中，我们将专注于鉴定ssRNA包装所必需的蛋白质和蛋白质复合物，而在具体目标3中，将使用我们建立的反向遗传学系统在细胞培养中评估突变体构建体的复制能力。目前的数据表明，ssRNA驱动BTV转录复合物（TC）的组装，导致衣壳（核心）组装，并且包装的基因组前体RNA分子位于组装的二十面体衣壳的12个顶点处，精确地引发它们用于随后的转录。在具体目标4中，我们将使用最先进的单颗粒冷冻电子显微镜和冷冻断层扫描，使用天然和突变颗粒来确定突变的后果，在包装颗粒的顶点处寻找TC组织的直接结构证据。这些目标的完成将为合理设计抑制剂以阻断病毒复制铺平道路，并为对人类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): The specific aims of this proposal are part of a long term goal to understand completely the infection course of a non-enveloped arthropod-borne virus, Bluetongue virus (BTV), a member of the Reoviridae family that include viruses infecting a wide range of hosts, including human, animals, plants, fungi, and bacteria. As such BTV shares a family relationship with several other scientifically and medically important viruses (e.g. Rotaviruses). BTV has been studied extensively as an agent of economically important disease and also, increasingly, as a model system for related viruses. Substantial progress has been achieved in the molecular, biochemical and structural fields; however, certain areas of BTV and related viruses' biology and structure remain unclear due to the unavailability of appropriate tools and assay systems. BTV and other members (rotavirus and reoviruses) are complex, multi-layered capsid viruses with 10-12 segmented double-stranded RNA genomes but the details of how virus genomes are packaged into their protective capsids or the mechanism by which a copy of each segment is incorporated for the virus to be viable remain elusive. In this proposal we aim to maintain and extend the leading position of BTV towards our long term goal of understanding, in totality, the infection course of the virus. In particular, this application wll utilize a groundbreaking technological advance (i.e., the first in vitro packaging system for a complex dsRNA virus) recently pioneered by us (through our current NIH funding), to understand the genome packaging and replication events of this complex capsid virus and by inference for all multi-segmented RNA viruses. In specific aim 1 we will determine the packaging order of ten ssRNAs and the signals involved in assembly of BTV particles. In specific aim 2 we will focus on identification of the protein and protein complexes essential for ssRNA packaging while in the specific aim 3, the replication competence of mutant constructs will be assessed in cell culture using our established reverse genetics system. Current data suggest that ssRNAs drive assembly of the BTV transcription complex (TC), leading to capsid (core) assembly and that packaged genome precursor RNA molecules are positioned at the 12 vertices of the assembling icosahedral capsid, precisely priming them for subsequent transcription. In specific aim 4 we will seek direct structural evidence of the TC organization at the vertices of the packaged particles, using both native and mutant particles to determine the consequences of mutation, using the state-of-the art single particle cryo-electron microscopy and cryo-tomography. Completion of these goals will pave the way for the rational design of inhibitors to block virus replication and provide lead compounds for related viruses of pathogenic significance to man and animals.