Molecular Basis of Alphavirus Assembly

甲病毒组装的分子基础

• 批准号: 7228116
• 负责人: Richard J. Kuhn
• 金额: $ 28.69万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7228116
• 关键词: Alphavirus; Amino Acids; Animals; Antiviral Agents; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Biological Models; Cells; Cellular Membrane; Collection; Complement; Complex; Core Assembly; Cryoelectron Microscopy; Ensure; Environment; Event; Glycoproteins; Group Structure; Human; In Vitro; Life Cycle Stages; Lipids; Macromolecular Complexes; Membrane Proteins; Molecular; Molecular Genetics; Morphogenesis; Nucleic Acid Biochemistry; Nucleic Acids; Nucleocapsid; Nucleocapsid Proteins; Pathway interactions; Process; Protein Array; Proteins; RNA; RNA Viruses; Reagent; Resolution; Role; Site-Directed Mutagenesis; Structural Biochemistry; Structure; Structure-Activity Relationship; Techniques; Viral; Viral Proteins; Virion; Virus; Virus Assembly; Virus Replication; Work; X-Ray Crystallography; analytical ultracentrifugation; base; design; genetic analysis; human disease; image reconstruction; in vitro Assay; in vivo; insight; macromolecular assembly; mutant; novel; particle; pathogen; protein function; structural biology

DESCRIPTION (provided by applicant): Virus assembly is the final step in the life cycle of a virus prior to its release from the infected cell. Enveloped virus assembly is a complex process involving multiple viral and possibly cellular proteins that result in the release of infectious virions by budding at a cellular membrane. Our previous work has provided significant insights into the assembly process of the plus strand RNA enveloped alphaviruses. These viruses have a worldwide distribution and are responsible for severe forms of animal and human disease. Our focus has been in describing the molecular mechanism by which the viral nucleocapsid core is formed. We propose to continue these studies with a set of three specific aims that are directed at: 1) determining the structure and function of the initial building block of the alphavirus nucleocapsid core; 2) elaborating the complete pathway for core assembly; and 3) defining the molecular interactions between the E2 glycoprotein and the nucleocapsid core that are responsible for forming an icosahedral enveloped virus. A multi-disciplinary approach will use molecular genetics, biochemistry and structural techniques to probe the mechanism of virus assembly and to ultimately describe the process in atomic detail. Using cryo-electron microscopy image reconstructions of the whole virus, and the atomic structure of the nucleocapsid protein and the E1 and E2 glycoproteins as a guide, site-directed mutagenesis will be carried out to probe structure-function relationships. The resulting mutants will be studied by a variety of in vivo and in vitro biochemical assays that will examine protein-protein and protein-nucleic acid interactions involved in nucleocapsid core formation and interactions of the nucleocapsid core with the icosahedral-organized glycoproteins. Ultimately, understanding this process will aid in the design of novel antivirals and provide a greater appreciation of protein and nucleic acid interactions that are necessary for forming complex macromolecular assemblies.

描述（由申请人提供）：病毒组装是病毒从感染细胞释放之前的生命周期的最后一步。包裹的病毒组装是一个复杂的过程，涉及多种病毒和可能的细胞蛋白，可通过在细胞膜上萌芽来释放传染性病毒。我们以前的工作为加上链RNA包裹的α病毒的组装过程提供了重大见解。这些病毒具有全球分布，并负责严重的动物和人类疾病。我们的重点是描述形成病毒核素核的分子机制。我们建议通过针对以下三个特定目的继续进行这些研究：1）确定α核蛋白酶核心α的初始构件的结构和功能； 2）详细说明核心组件的完整途径； 3）定义E2糖蛋白与核皮质核之间的分子相互作用，这些糖蛋白负责形成二十健康的包膜病毒。多学科方法将使用分子遗传学，生物化学和结构技术来探测病毒组装的机制，并最终以原子细节描述该过程。使用整个病毒的冷冻电子显微镜图像重建，以及核素蛋白的原子结构以及E1和E2糖蛋白作为指导，将进行定位的诱变，以探测结构 - 函数函数函数关系。将通过各种体内和体外生化测定法对所得突变体进行研究，该测定将检查蛋白质 - 蛋白质蛋白和蛋白质核酸相互作用，涉及核蛋白酶核心形成以及核蛋白酶核与Icosahecapsid core与Icosahedral-Organized glycoproteins的相互作用。最终，了解这一过程将有助于设计新型抗病毒药，并为形成复杂的大分子组件所必需的蛋白质和核酸相互作用提供更大的欣赏。