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Cell Biology of Reovirus Infection

呼肠孤病毒感染的细胞生物学

基本信息

批准号：
9278678
负责人：
TERENCE S. DERMODY
金额：
$ 42.51万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-06-01 至 2019-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9278678
关键词：
Animals Antiviral Agents Apical Apoptotic Architecture Binding Biochemical Biogenesis Biotechnology Cell Death Cell membrane Cells Cellular biology Clinical Trials Collaborations Complex Coupled Cryoelectron Microscopy Cytolysis Cytoplasm Data Development Disease Dominant-Negative Mutation Double Stranded RNA Virus Electron Microscopy Engineering Epithelial Cells Experimental Models Fostering GTP Binding GTP-Binding Proteins Gene Deletion Genome Guanosine Triphosphate Health Human Image Infection Knowledge Laboratories Ligands Lipids Mediating Mediator of activation protein Medicine Membrane Molecular Chaperones Molecular Probes Morphogenesis Morphology Mus Mutation Nature Necrosis Nonstructural Protein Nucleic Acids Oncolytic Orbivirus Organelles Pathway interactions Phosphotransferases Production Proteins RNA Binding RNA Interference RNA interference screen Recruitment Activity Reoviridae Infections Reovirus Research Rotavirus Site Structure Study models Surface Testing Therapeutic Universities Vaccines Viral Viral Genome Viral Nonstructural Proteins Viral Pathogenesis Viral Structural Proteins Virion Virus Virus Replication Work X-Ray Crystallography base brain endothelial cell cancer therapy cell transformation cell type cellular imaging college drug development electron tomography genetic inhibitor in vitro Assay inhibitor/antagonist killings light microscopy lipid biosynthesis novel particle pathogen prevent protein function research study reverse genetics viral RNA

项目摘要

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.

描述（由申请方提供）：大多数在宿主细胞的细胞质中复制的病毒形成新细胞器，作为病毒基因组复制和颗粒组装的位点。这些高度特化的包涵体结构浓缩病毒复制蛋白和核酸，防止细胞内在防御的激活，并协调子代颗粒的释放。尽管包合复合物在病毒复制中的重要性，但关于这些细胞器如何形成和介导其功能的知识存在关键空白。拟议的研究使用呼肠孤病毒，一种遗传上易于处理的实验模型，显示出溶瘤和疫苗应用的前景，以阐明双链（ds）RNA病毒包涵体形成，基因组复制和子代颗粒释放的机制。与包括动物（环状病毒）和人类（轮状病毒）的重要病原体的其他dsRNA病毒一样，呼肠孤病毒包涵体由病毒非结构蛋白成核，所述病毒非结构蛋白募集病毒结构蛋白用于基因组复制和颗粒组装。我们已经发现呼肠孤病毒包涵体包埋在脂质中，并且后代呼肠孤病毒颗粒使用不引起细胞裂解的机制从包涵体转运并从某些类型的细胞释放。提出了三个综合的具体目标，以填补呼肠孤病毒包涵体生物起源和功能的关键知识空白。在具体目标1中，将使用相关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潜在机制。将使用药理学抑制剂和候选宿主分子的基因缺失来鉴定呼肠孤病毒包涵体形成所需的膜生物合成途径。在特定目标2中，将使用RNA结合和激酶活性的体外测定来定义呼肠孤病毒非结构蛋白σNS（一种结合病毒RNA和GTP的呼肠孤病毒包涵体必需组分）的功能。将使用X射线晶体学和低温电子显微镜测定单独的σNS和与其配体复合的σNS的结构。将使用通过反向遗传学工程化的σNS中具有结构引导突变的病毒和σNS GTP结合活性的抑制剂来确定σNS在呼肠孤病毒复制中的活性。在特定目标3中，将使用电子断层扫描和靶特异性分子探针阐明呼肠孤病毒退出感染细胞的途径。将使用RNAi、药理学抑制剂和显性阴性突变体测试候选囊泡途径在呼肠孤病毒排出中的功能。呼肠孤病毒退出的新宿主介质将使用基于RNAi的病毒流出筛选来鉴定。这些研究将增强对致病病毒改变细胞结构以工程化包涵体细胞器、复制其基因组并退出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发展，阻止这些重要的病毒复制步骤，并加强使用呼肠孤病毒作为溶瘤治疗。