Electron microscopy studies of novel poliovirus genome-releasing intermediates

新型脊髓灰质炎病毒基因组释放中间体的电子显微镜研究

• 批准号: 7906887
• 负责人: HAZEL C LEVY
• 金额: $ 5.22万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7906887
• 关键词: Accounting; Antiviral Agents; Biochemical; Biological Models; Capsid; Capsid Proteins; Cardiovirus; Cell membrane; Cell physiology; Cells; Complex; Cues; Cytoplasm; Data; Data Analyses; Data Collection; Data Set; Development; Disease; Electron Microscopy; Enterovirus; Family; Family Picornaviridae; Foot-and-Mouth Disease Virus; Genetic Materials; Genome; Goals; Hepatitis A Virus; Heterogeneity; Human; Human poliovirus; Image; Infection; Knowledge; Lead; Life Cycle Stages; Liposomes; Maps; Membrane; Methodology; Methods; Microscope; Modeling; Molecular; Mutagenesis; Nature; Pathogenicity; Pathway interactions; Peptides; Polioviruses; Process; Protein Region; Protocols documentation; RNA; Resolution; Rhinovirus; Role; Sampling; Site; Staging; Structure; Surface; Techniques; Testing; Time; Tomogram; Viral; Virion; Virus; cold temperature; density; electron density; improved; novel; particle; poliovirus receptor; prevent; programs; public health relevance; reconstruction; research study; tomography; viral RNA; virus morphology

DESCRIPTION (provided by applicant): Picornaviruses are non-enveloped viruses and cannot rely on fusion of a viral membrane with a host cell membrane to gain access to the cytoplasm. They rely on the ability of capsid proteins to undergo as-yet-uncharacterized conformational changes for the viral RNA to breach the host-membrane barrier. These irreversible changes include externalization of peptides from the interior of the capsid shell. Poliovirus is the prototypical picornavirus and serves as an excellent model system. Our broad aim is to gain knowledge of viral structural dynamics associated with host-membrane attachment and genome release by using electron microscopy and three-dimensional data analysis (3DEM). The picornaviruses share a high degree of structural homology with one another and, therefore, the models and methodologies developed during this study will be relevant to other picornaviruses. The two specific aims of this proposed study are 1) to determine the 3DEM structures of poliovirus intermediates during genome-release, and 2) to determine 3DEM structures of such intermediates attached to liposomes. The experimental steps to be carried out are listed below: a. Optimize conditions for preparing vitrified EM grids enriched with poliovirus particles at a particular stage (early or late) of RNA-release. b. Collect EM data (both un-tilted and tomographic) using the Polara and Tecnai F30 microscopes. c. Determine both icosahedral and asymmetric 3D reconstructions of RNA-release intermediates from the 2D EM images using the program PFT for un-tilted data and IMOD for tomograms. d. Build pseudo-atomic models to fit the higher-resolution EM maps, to help account for density changes. PUBLIC HEALTH RELEVANCE: The Picornavirus family includes cardioviruses, foot-and-mouth disease virus, rhinoviruses, hepatitis A virus and enteroviruses. Among these, there are significant causes of human and veterinary diseases. Detailed knowledge of the molecular mechanisms that these viruses use for membrane attachment and genome release would help in the development of antiviral strategies to block the earliest stages of infection and reduce pathogenicity.

描述（由申请人提供）：picornavirus是非发育的病毒，不能依靠病毒膜与宿主细胞膜的融合来进入细胞质。他们依靠衣壳蛋白可以对病毒RNA进行尚未构象变化的能力，以违反宿主膜屏障。这些不可逆转的变化包括从衣壳壳内部对肽的外部化。脊髓灰质炎病毒是典型的picornavirus，它是出色的模型系统。我们的广泛目的是通过使用电子显微镜和三维数据分析（3DEM）获得与宿主 - 膜附着和基因组释放相关的病毒结构动力学知识。 PICORNAVIRES彼此具有高度的结构同源性，因此，本研究期间开发的模型和方法将与其他PICORNAVIRASS有关。这项拟议的研究的两个具体目的是1）确定基因组释放期间脊髓灰质炎病毒中间体的3DEM结构，以及2）确定附着在脂质体上的此类中间体的3DEM结构。要执行的实验步骤如下：优化在RNA释放的特定阶段（早期或晚期），制备富含脊髓灰质炎病毒颗粒的玻璃化EM电网的条件。 b。使用polara和tecnai F30显微镜收集EM数据（不倾斜和断层扫描）。 c。使用程序PFT使用程序PFT来确定来自2D EM图像的RNA释放中间体的二十面体和非对称3D重建，用于未倾斜的数据和IMOD用于断层图。 d。构建伪原子模型以适合高分辨率EM图，以帮助考虑密度变化。公共卫生相关性：Picornavirus系列包括心脏病病毒，脚和口病毒，鼻病毒，乙型肝炎病毒和肠病毒。其中，有重大的人类和兽医疾病原因。详细了解这些病毒用于膜附着和基因组释放的分子机制将有助于开发抗病毒药策略，以阻止最早的感染阶段并降低致病性。