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

描述(由申请人提供)：这项建议的具体目标是全面了解一种无包膜节肢动物传播病毒-蓝舌病毒(BTV)的感染过程，该病毒是呼肠孤病毒科的成员，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与其他几种在科学和医学上具有重要意义的病毒(例如轮状病毒)具有家族关系。BTV作为一种重要经济疾病的病原体已被广泛研究，也越来越多地被用作相关病毒的模型系统。在分子、生化和结构领域取得了实质性进展；然而，由于没有适当的工具和检测系统，BTV和相关病毒的某些领域的生物学和结构仍然不清楚。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个节段的双链RNA基因组，但病毒基因组如何包装到其保护性衣壳中或每个片段的拷贝被结合以使病毒存活的机制仍不清楚。在这项建议中，我们旨在保持和扩大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。特别是，这项应用将利用我们最近(通过我们目前的NIH资金)开创的突破性技术进步(即，第一个复杂dsRNA病毒的体外包装系统)，了解这种复杂衣壳病毒的基因组包装和复制事件，并推断所有多片段RNA病毒。在具体目标1中，我们将确定10个单链RNA的包装顺序和BTV颗粒组装所涉及的信号。在特定目标2中，我们将专注于鉴定对单链RNA包装至关重要的蛋白质和蛋白质复合体，而在特定目标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结构的复制能力。目前的数据表明，单链RNA驱动BTV转录复合体(TC)的组装，导致衣壳(核心)组装，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，精确地为后续转录做好准备。在特定目标4中，我们将使用最先进的单粒子冷冻电子显微镜和冷冻断层扫描，在包装粒子的顶点寻找TC组织的直接结构证据，使用天然和突变粒子来确定突变的后果。这些目标的完成将为合理设计抑制病毒复制的药物铺平道路，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): 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.