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（由申请人提供）：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，这些高度专业化的包涵结构集中了病毒复制蛋白和核酸，防止细胞固有的激活。尽管包涵体在病毒复制中很重要，但关于这些细胞器如何形成和介导其功能的知识仍存在关键差距。易于处理的实验模型显示了溶瘤和疫苗应用的前景，可阐明双链 (ds) RNA 病毒包涵体形成、基因组复制和子代颗粒释放的机制，就像其他 dsRNA 病毒一样，其中包括动物的重要病原体（环状病毒）和人类（轮状病毒）中，呼肠孤病毒包涵体由病毒非结构蛋白成核，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。子代呼肠孤病毒颗粒通过一种不会引起细胞裂解的机制从内含物中运输并从某些类型的细胞中释放出来，以填补有关呼肠孤病毒内含物生物发生和功能的关键知识空白。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的潜在形成，并将使用药理学来确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，呼肠孤病毒非结构蛋白 σNS（一种结合病毒 RNA 和 GTP 的重要呼肠孤病毒包含成分）的功能将通过 RNA 结合和激酶活性的体外测定来确定。单独的σNS以及与其配体的复合物将使用X射线晶体学和冷冻电子显微镜来确定。呼肠孤病毒复制中的σNS活性将使用具有以下特征的病毒来确定。通过反向遗传学和 σNS GTP 结合活性抑制剂设计的 σNS 中的结构引导突变在特定目标 3 中，将使用电子断层扫描和目标特异性分子探针阐明呼肠孤病毒退出受感染细胞的途径。使用RNAi、药理学抑制剂和显性失活突变体测试呼肠孤病毒排出的功能，将使用基于RNAi的病毒来鉴定呼肠孤病毒排出的新宿主介质。这些研究将增强对致病病毒改变细胞结构以设计包涵细胞器、复制其基因组并退出受感染细胞的机制的基本了解，我们预计这些信息将促进阻碍这些重要病毒复制的抗病毒药物的开发。步骤并加强呼肠孤病毒作为溶瘤治疗剂的使用。