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: 8260253
• 负责人: Benhur Lee
• 金额: $ 27.51万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8260253
• 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.

尼帕病毒和Hednra病毒是致命的副粘病毒，被归类为优先病原体和新兴病原体。 传染病病原体。我们提出了一个多学科的项目，利用优化的平台， PI（Dr. Benhur Lee）研究低于BSL 4水平的尼帕病毒和亨德拉病毒进入。项目 将PI的分子病毒学专业知识与共同PI的独特资源和专业知识相结合 (Dr.内森·沃尔夫和Z博士Hong Zhou），以阐明一种潜在的广谱人畜共患病病毒 病原体我们将首先关注亨尼帕病毒作为我们方法的原理证明，尽管我们 我希望能够将我们的分析扩展到其他人畜共患病，如丝状病毒，沙粒病毒，汉坦病毒和 非典三名调查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 口炎病毒（VSV）报告基因平台在两组互补研究中的应用：（1）结构病毒学 增强我们对亨尼帕病毒融合级联的理解的研究，以及（2）为了更高的通量 检测中和和/或交叉反应性抗体的分析，用于诊断、监测和疫苗 发展努力。我们建议的基本原理是血清中和试验和 用我们的VSV-rluc（海肾荧光素酶）报告基因假型进行的结构病毒学研究将提供 生物学相关信息比使用它们的活病毒对应物更有效和经济。为了实现上述目标，我们提出了以下具体目标：（1）利用冷冻电子显微镜（cryo-EM）和冷冻电子断层扫描（cryo-ET）观察尼帕病毒F和G寡聚体，获得对亨尼帕病毒融合级联的超分子纳米级理解 假型化到VSV上，和（2）研究来自全球病毒预测的相关血清样品 使用我们的亨尼帕病毒VSV假型。此外，我们最近开发了一种 基于催化改进的β-内酰胺酶-Nipah基质的检测，具有灵敏度、特异性和更高的通量 在BSL 2条件下天然亨尼帕病毒进入和出芽的分析。因此，我们还提出了一个具体的目标， (3)：使用我们的尼帕，鉴定和表征亨尼帕病毒出芽的小分子抑制剂 基于基质VLP的测定。我们的筛选将与胡安卡洛斯德拉托雷斯医生同时进行。 （我们病毒性人畜共患病专题组的另一位PI）努力筛选沙粒病毒的小分子抑制剂 芽殖和任何先导候选物可以在彼此的芽殖测定中交叉测试， 病毒出芽的广谱抑制剂。