Active and arrested paramyxovirus fusion machinery visualized by cryo-electron to

通过冷冻电子可视化活跃和停滞的副粘病毒融合机制

基本信息

批准号：
8540333
负责人：
Matteo Porotto
金额：
$ 22.1万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-07 至 2015-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8540333
关键词：
Address Architecture Automobile Driving Binding Binding Proteins Biochemical Biological Assay Biomedical Engineering Biomimetics Biophysics Cell fusion Cell membrane Cells Cellular Membrane Chemistry Chimeric Proteins Cholesterol Complex DNA Sequence Rearrangement Data Development Dominant-Negative Mutation Electrons Engineering Family Fluorescence Spectroscopy Freezing G-substrate Glycoproteins Goals Hendra Virus Image Infection Invaded Life Liposomes Location Mediating Membrane Membrane Fusion Membrane Glycoproteins Membrane Proteins Modeling Molecular Conformation Nature Negative Staining Nipah Virus Paramyxovirus Pathogenesis Peptides Process Protein Region Proteins Reaction Relative (related person)Research Staging Structure Surface Time Translations Transmembrane Domain Transmission Electron Microscopy Viral Viral Fusion Proteins Viral Physiology Virion Virus Virus Diseases Virus Receptors base design electron tomography fundamental research i-cholesterol influenzavirus inhibitor/antagonist innovation molecular dynamics nano novel pathogen prevent receptor receptor binding tomography virology

Address Architecture Automobile Driving Binding Binding Proteins Biochemical Biological Assay Biomedical Engineering Biomimetics Biophysics Cell fusion Cell membrane Cells Cellular Membrane Chemistry Chimeric Proteins Cholesterol Complex DNA Sequence Rearrangement Data Development Dominant-Negative Mutation Electrons Engineering Family Fluorescence Spectroscopy Freezing G-substrate Glycoproteins Goals Hendra Virus Image Infection Invaded Life Liposomes Location Mediating Membrane Membrane Fusion Membrane Glycoproteins Membrane Proteins Modeling Molecular Conformation Nature Negative Staining Nipah Virus Paramyxovirus Pathogenesis Peptides Process Protein Region Proteins Reaction Relative (related person)Research Staging Structure Surface Time Translations Transmembrane Domain Transmission Electron Microscopy Viral Viral Fusion Proteins Viral Physiology Virion Virus Virus Diseases Virus Receptors base design electron tomography fundamental research i-cholesterol influenzavirus inhibitor/antagonist innovation molecular dynamics nano novel pathogen prevent receptor receptor binding tomography virology

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项目摘要

DESCRIPTION (provided by applicant): New imaging strategy to address challenges in the field. The lethal and transmissible nature of the paramyxoviruses Hendra virus and Nipah virus (HeV, NiV) makes these pathogens of serious concern. These are also ideal models for developing an innovative new imaging platform at the interface between bioengineering and virology. Despite the relative abundance of crystal structure data for the paramyxovirus fusion machinery, the dynamic processes - starting with the binding of virus to the host cell and ending with infection -- are poorly understood. The interplay between the envelope viral glycoproteins that leads to activation of the fusion process has eluded rigorous analysis. Imaging the viral fusion nano-machinery will deepen our understanding of viral membrane fusion and will allow us to identify strategies for interfering with the process and arresting the infection cycle. We propose to capture, immobilize, and visualize activated intermediate states of the fusion process, exposing the conserved domains that are essential for viral entry. While the activated conformation is normally present only at the surface of live cells, we propose to use engineered liposomes to capture and immobilize transitional states. We will use receptor molecules, presented in a biomimetic fashion on the surface of the liposomes, to "trigger" the fusion protein and activate the conformational change in the viral fusion machinery. We will then use fusion inhibitory peptides to arrest the fusion machinery in its activated state, and thus immobilize the captured intermediates with the liposomes. These complexes will then be analyzed by cryo-electron tomography. Aim 1. Generate NiV fusion machinery intermediate states 1.1 Trapping intermediate states of the NiV fusion machinery. We will capture the NiV fusion machinery at intermediate stages of the fusion reaction, by first activating the fusion process using cellular receptor molecules presented on liposomes, and then trapping fusion intermediates with fusion-inhibitory peptides that block the conformational refolding of the fusion protein that is required for completing membrane fusion. 1.2 Functional and morphological studies of different states of the fusion process. Fluorescence spectroscopy and negative-stain transmission electron microscopy will be used to determine the stability of the captured complexes from sub aim 1.1. Aim 2. Determine how the receptor binding and fusion proteins interact during binding and fusion activation Electron cryo-tomography (ECT) imaging of NiV fusion machinery. Using ECT we will visualize the process and requirements for the NiV receptor binding protein (G) to trigger fusion (F) protein-mediated fusion and subsequent steps in F structural rearrangement.

描述（由申请人提供）：应对现场挑战的新成像策略。 hendra病毒和尼帕病毒（HEV，NIV）的致命性和可传染性质使这些病原体引起了严重关注。这些也是在生物工程和病毒学之间开发创新的新成像平台的理想模型。尽管帕托及病毒融合机械的晶体结构数据相对丰富，但动态过程 - 从病毒与宿主细胞结合并以感染结束的动态过程 - 知识渊博。融合过程激活的包膜病毒糖蛋白之间的相互作用已避免了严格的分析。成像病毒融合纳米机械将加深我们对病毒膜融合的理解，并使我们能够确定干扰该过程并阻止感染周期的策略。我们建议捕获，固定和可视化融合过程的激活的中间状态，以暴露对病毒进入必不可少的保守域。尽管通常仅存在活性构象，但我们建议使用工程脂质体捕获和固定过渡状态。我们将使用脂质体表面以仿生方式呈现的受体分子来“触发”融合蛋白，并激活病毒融合机械的构象变化。然后，我们将使用融合抑制性肽将融合机器以其活化状态阻止，从而将捕获的中间体与脂质体固定。然后，这些复合物将通过冷冻电子层析成像进行分析。 AIM 1。生成NIV融合机械中间状态1.1 NIV融合机械的中间状态。我们将通过首先使用脂质体上呈现的细胞受体分子激活融合过程，然后将融合融合中间体与融合抑制性抑制性的肽一起捕获融合过程，从而捕获NIV融合机制，从而捕获融合过程。 1.2融合过程的不同状态的功能和形态学研究。荧光光谱和负染色的透射电子显微镜将用于确定Sub AIM 1.1捕获的配合物的稳定性。 AIM 2。确定在NIV融合机械的结合和融合激活电子冷冻术（ECT）成像过程中，受体结合和融合蛋白如何相互作用。使用ECT，我们将可视化NIV受体结合蛋白（G）的过程和要求，以触发融合（F）蛋白介导的融合以及F结构重排的后续步骤。