Cell Biology of Reovirus Infection

描述(申请人提供)：在宿主细胞的细胞质中复制的大多数病毒形成新的细胞器，作为病毒基因组复制和颗粒组装的地点。这些高度专业化的包涵体结构集中了病毒复制蛋白质和核酸，防止激活细胞固有防御，并协调后代颗粒的释放。尽管包涵体复合体在病毒复制中很重要，但在这些细胞器如何形成和调节其功能方面，仍存在关键的知识空白。这项拟议的研究使用呼肠孤病毒，这是一种遗传上易于处理的实验模型，显示出在溶瘤和疫苗应用方面的前景，以阐明双链(DS)RNA病毒包涵体形成、基因组复制和后代粒子释放的机制。与其他dsRNA病毒一样，包括动物(环状病毒)和人类(轮状病毒)的重要病原体，呼肠孤病毒包涵体是由病毒非结构蛋白核化的，这些非结构蛋白招募病毒结构蛋白进行基因组复制和颗粒组装。我们发现呼肠孤病毒包涵体嵌入脂质中，后代呼肠孤病毒颗粒从包涵体中运输，并使用一种不会导致细胞溶解的机制从某些类型的细胞中释放。提出了三个综合的具体目标，以填补有关呼肠孤病毒包涵体、生物发生和功能的关键知识空白。在具体目标1中，将使用相关的光学和电子显微镜结合电子断层扫描和生化分析来确定呼肠孤病毒包涵体形成的机制。形成呼肠孤病毒包涵体所需的膜生物合成途径将使用药物抑制剂和候选宿主分子的基因删除来鉴定。在具体目标2中，呼肠孤病毒非结构蛋白σNS是一种结合病毒核糖核酸和GTP的重要呼肠孤病毒包涵体成分，其功能将通过体外核糖核酸结合和激酶活性分析来确定。σNS单独和与其配体形成的络合物的结构将用X射线结晶学和冷冻电子显微镜确定。σNS在呼肠孤病毒复制中的活性将使用由反向遗传学设计的σNS结构引导突变的病毒和σNS GTP结合活性的抑制剂来确定。在具体目标3中，将使用电子断层扫描和靶标特异性分子探针来阐明呼肠孤病毒退出受感染细胞的途径。候选的囊泡通路将使用RNAi、药物抑制剂和显性-阴性突变体来测试呼肠孤病毒出口的功能。将使用基于RNAi的病毒出口筛查来识别呼肠孤病毒出口的新宿主介体。这些研究将加强对致病病毒改变细胞结构以设计包涵体细胞器、复制其基因组和退出受感染细胞的机制的基本理解。我们预计，这些信息将促进抗病毒药物的开发，这些药物可以阻止这些关键的病毒复制步骤，并加强呼肠孤病毒作为溶瘤治疗的使用。

DESCRIPTION (provided by applicant): Most viruses that replicate in the cytoplasm of host cells form neoorganelles that serve as sites of viral genome replication and particle assembly. These highly specialized inclusion structures concentrate viral replication proteins and nucleic acids, prevent activation of cell-intrinsic defenses, and coordinate release of progeny particles. Despite the importance of inclusion complexes in viral replication, there are key gaps in knowledge about how these organelles form and mediate their functions. The proposed research uses reovirus, a genetically tractable experimental model that shows promise for oncolytic and vaccine applications, to elucidate mechanisms of double-stranded (ds) RNA virus inclusion formation, genome replication, and progeny particle release. Like other dsRNA viruses, which include important pathogens of animals (orbiviruses) and humans (rotaviruses), reovirus inclusions are nucleated by viral nonstructural proteins that recruit viral structural proteins for genome replication and particle assembly. We have discovered that reovirus inclusions are embedded in lipid and that progeny reovirus particles are transported from inclusions and released from some types of cells using a mechanism that does not cause cell lysis. Three integrated specific aims are proposed to fill key knowledge gaps about reovirus inclusion biogenesis and function. In Specific Aim 1, mechanisms underlying formation of reovirus inclusions will be determined using correlative light and electron microscopy coupled with electron tomography and biochemical analyses. Membrane-biosynthetic pathways required for reovirus inclusion formation will be identified using pharmacologic inhibitors and gene deletions of candidate host molecules. In Specific Aim 2, functions of reovirus nonstructural protein σNS, an essential reovirus inclusion component that binds viral RNA and GTP, will be defined using in vitro assays of RNA binding and kinase activity. The structure of σNS alone and in complex with its ligands will be determined using X-ray crystallography and cryo-electron microscopy. Activities of σNS in reovirus replication will be determined using viruses with structure-guided mutations in σNS engineered by reverse genetics and inhibitors of σNS GTP-binding activity. In Specific Aim 3, pathways used by reovirus to exit infected cells will be elucidated using electron tomography and target-specific molecular probes. Candidate vesicular pathways will be tested for function in reovirus egress using RNAi, pharmacologic inhibitors, and dominant-negative mutants. New host mediators of reovirus exit will be identified using an RNAi-based viral egress screen. These studies will enhance a basic understanding of mechanisms by which pathogenic viruses alter cellular architecture to engineer inclusion organelles, replicate their genomes, and exit infected cells. We anticipate that this information will foster development of antiviral drugs that impede these essential viral replication steps and enhance the use of reovirus as an oncolytic therapeutic.