Platforms for structure-function studies of entry and budding of viral zoonotic

人畜共患病毒进入和出芽的结构功能研究平台

• 批准号: 7675173
• 负责人: Benhur Lee
• 金额: $ 27.47万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7675173
• 关键词: Antibodies; Arenavirus; Basic Science; Biological Assay; Cryoelectron Microscopy; Diagnostic; Emerging Communicable Diseases; Hantavirus; Hendra Virus; Henipavirus; Infectious Diseases Research; Lead; Life; Light; Molecular Virology; Nipah Virus; Paramyxovirus; Renilla Luciferases; Reporter; Research Personnel; Resources; Sampling; Serum; Severe Acute Respiratory Syndrome; Strategic Planning; Structure; Technology; Testing; Update; Vesicular stomatitis Indiana virus; Viral; Virus; Virus Inhibitors; Zoonoses; base; beta-Lactamase; biodefense; electron tomography; high throughput analysis; high throughput screening; improved; inhibitor/antagonist; multidisciplinary; nanoscale; pathogen; small molecule; vaccine development; virology

Nipah and Hednra viruses are lethal paramyxoviruses classified as Priority Pathogens and Emerging Infectious Disease agents. We propose a multidisciplinary project that capitalizes on a platform optimized by the PI (Dr. Benhur Lee) to study Nipah and Hendra virus entry at less than BSL4 levels. The project synergizes the molecular virology expertise of the PI with the unique resources and expertise of the co-Pis (Dr. Nathan Wolfe and Dr. Z. Hong Zhou) to shed light on a potentially broad spectrum of zoonotic viral pathogens. We will first focus on the henipaviruses as proof of principle for our approach, although we expect to be able to expand our analyses to other zoonoses like filioviruses, arenaviruses, hantaviruses and SARS. All three investigators are at UCLA. The objective of our proposal is to use our optimized Vesicular Stomatitis Virus (VSV) reporter platform in two complementary sets of studies: (1) for structural virology studies that enhance our understanding of the henipaviral fusion cascade, and (2) for higher throughput assays to detect neutralizing and/or cross-reactive antibodies for diagnostics, surveillance, and vaccine development efforts. The underlying rationale for our proposal is that serum neutralization assays and structural virology studies done with our VSV-rluc (Renilla luciferase) reporter pseudotypes will provide biologically relevant information more efficiently and economically than using their live viral counterparts. To accomplish our stated objectives, we propose the following Specific Aims: (1) To gain a supramolecular nanoscale understanding of the henipavirus fusion cascade using cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) to visualize Nipah virus F and G oligomers pseudotyped onto VSV, and (2) To study relevant serum samples from the Global Viral Forecasting Initiative using our henipavirus VSV pseudotypes. Additionally, we have recently developed a catalytically improved beta-lactamase-Nipah matrix based assay for sensitive, specific and higher throughput analysis of native henipavirus entry and budding at BSL2 conditions. Thus, we also propose a Specific Aim (3): To identify and characterize small molecule inhibitors of henipavirus budding using our Nipah matrix VLP based assay. Our screen will be performed concurrently with Dr. Juan Carlos de la Torres' (another PI in our viral zoonoses thematic group) efforts to screen for small molecule inhibitors of arenavirus budding and any lead candidates can be cross-tested in each other's budding assay in order to identify putative broad spectrum inhibitors of viral budding.

NIPAH和HEDNRA病毒是归类为优先病原体和新兴的致命性帕托病毒 传染病药物。我们提出了一个多学科项目，该项目在优化的平台上大写 PI（Benhur Lee博士）以低于BSL4水平研究NIPAH和HENDRA病毒进入。项目 与Co-Pis的独特资源和专业知识协同PI的分子病毒学专业知识 （内森·沃尔夫（Nathan Wolfe）博士和Z. Hong Zhou博士）揭示了潜在的人畜共患病毒 病原体。尽管我们 希望能够将我们的分析扩展到其他人畜共患 SARS。这三位调查人员都在加州大学洛杉矶分校。我们建议的目的是使用我们优化的囊泡 两种互补的研究集中的气孔炎病毒（VSV）记者平台：（1）结构病毒学 增强我们对HENIPAVIRAL FUSION CASCADE的理解的研究，以及（2）以更高的吞吐量 检测诊断，监视和疫苗的中和和/或交叉反应抗体的测定 发展工作。我们提出的基本原理是血清中和测定法和 通过我们的VSV-RLUC（Renilla Luciferase）记者假型进行的结构病毒学研究将提供 与使用其活病毒对应物相比，在生物学上相关的信息更有效，更经济。为了实现我们既定的目标，我们提出了以下具体目的：（1）使用冷冻电子显微镜（Cryo-EM）和冷冻电子层析成像（Cryo-EM）和Cryo-Electron proghrography（Cryo-et）获得对HENIPAVIRUS融合级联的超分子纳米级理解，以可视化Nipah Virus F和Goligomers。 假型在VSV上，（2）研究全局病毒预测的相关血清样品 使用我们的HENIPAVIRUS VSV伪型的倡议。此外，我们最近开发了 催化改进的基于敏感，特异性和更高吞吐量的基于β-内酰胺酶-NIPAH基质的测定 在BSL2条件下对本地HENIPAVIRUS进入和萌芽的分析。因此，我们也提出了一个特定的目标 （3）：使用我们的nipah识别和表征Henipavirus芽的小分子抑制剂 基于矩阵VLP的测定法。我们的屏幕将与Juan Carlos de la Torres博士同时进行 （我们病毒式人畜共患主题组中的另一个PI）筛选小分子抑制剂的努力 萌芽和任何主要候选人可以在彼此的萌芽测定中进行跨测试，以便识别 推定的病毒萌芽的广泛频谱抑制剂。