Elucidation of Assembly and Budding Mechanisms of SARS-CoV-2

阐明 SARS-CoV-2 的组装和出芽机制

基本信息

批准号：
10595342
负责人：
Robert Virgil Stahelin
金额：
$ 77.24万
依托单位：
PURDUE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-19 至 2027-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10595342
关键词：
2019-nCoV Address Automobile Driving Behavior Binding Biochemical Biochemistry Biological Models Biophysical Process Biophysics C-terminal COVID-19 Category C pathogen Cell membrane Cells Cellular Assay Collaborations Computer Analysis Computer Models Coronavirus Dangerousness Data Drug Targeting Fatality rate Future Genome Goals Golgi Apparatus Grain Grant Healthcare Systems Human In Vitro Infection International Laboratories Life Cycle Stages Light Lipid Binding Lipids Measurement Mediating Membrane Membrane Biology Membrane Lipids Membrane Proteins Middle East Respiratory Syndrome Modeling Molecular Molecular Conformation Morbidity - disease rate Nucleocapsid Nucleocapsid Proteins Nucleoproteins Pathogenesis Patients Phosphatidylinositols Planet Earth Play Process Production Property Protein Analysis Proteins Public Health RNA Research Research Personnel Role Severe Acute Respiratory Syndrome Site Sphingolipids Structural Models Structural Protein Structure System Techniques Testing Therapeutic United States National Institutes of Health Vaccines Validation Viral Viral Genome Viral Pathogenesis Viral Proteins Virion Virus Virus Assembly Virus Replication Virus Shedding World Health Organization Zoonoses base biophysical analysis biosafety level 3 facility computer studies experimental study in silico inhibitor innovation insight membrane assembly molecular dynamics mortality multitask new therapeutic target novel coronavirus pandemic disease particle pathogen programs protein protein interaction protein structure function public health emergency recruit structural biology therapeutic development therapeutic target tool viral envelope lipids virology

项目摘要

Project Summary Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus responsible for the ongoing human pandemic (COVID-19) that has been classed as a Public Health Emergency of International Concern by the World Health Organization (WHO). There is an urgent demand for SARS-CoV-2 research to facilitate the development of therapeutics, understand viral replication and pathogenesis, and determine how the virus spreads from cell-to-cell as well as patient to patient. Coronaviruses such as SARS and MERS are among the most dangerous pathogens on Earth, with high fatality rates and lack of viable therapeutics or vaccines. They are classified as category C pathogens by the NIH due to their ease of production and dissemination with the potential of high morbidity and mortality. Detailed mechanistic studies on the dynamics of SARS-CoV-2 replication and viral shedding (i.e., budding) may inform identification of new drug targets in the viral life cycle and enrich our understanding of how this zoonotic pathogen utilizes host cell lipids to build the viral lipid envelope. The Stahelin and Voth laboratories, building on collaborations with each other and specific expertise in biochemistry, biophysics and computational studies of virus assembly, will use experimental in vitro and cellular studies integrated with computational analysis to investigate the central hypothesis in this grant: that selective lipid-protein interactions drive the assembly and budding of the M (membrane) and N (nucleoprotein) of SARS- CoV-2. In two specific aims, we will (i) determine the cellular and biophysical mechanisms by which SARS-CoV- 2 M form virus particles in silico, in vitro and in human cells and (ii) determine how N lipid binding drives localization that contributes to formation of new viral particles. These studies will be integrated with structural biology of M (Browhan laboratory) and N (Ollmann Saphire laboratory) and also be validated with authentic SARS-CoV-2 in a BSL-3 facility in collaboration with the Kuhn laboratory. These questions will be studied in a tightly integrated approach using structural and in vitro quantitative techniques to assess lipid-protein and protein-protein interactions and cellular assays to tease apart the molecular underpinnings of viral protein interactions necessary for viral budding and infection. Computationally, we will use coarse-grained (CG) molecular dynamics (MD) simulations to characterize the assembly process on the membrane and to identify a set of models for further refinement through all-atom (AA) MD simulations. This innovative and integrated approach will not only provide careful validation of the results, but also provide detailed structural insights into the lipid-protein and protein-protein interactions governing the assembly and budding of SARS-CoV-2. The protein interfaces of M and N identified in these studies, which will be key for virus assembly and spread, will inform future drug targeting against SARS-CoV-2 and other coronaviruses.

项目概要严重急性呼吸综合征冠状病毒 2 (SARS-CoV-2) 是一种新型冠状病毒，可导致持续的人类大流行（COVID-19）已被列为国际突发公共卫生事件世界卫生组织（WHO）的关注。迫切需要 SARS-CoV-2 研究促进治疗方法的开发，了解病毒复制和发病机制，并确定如何病毒在细胞与细胞之间以及患者与患者之间传播。 SARS 和 MERS 等冠状病毒属于地球上最危险的病原体，死亡率很高，而且缺乏可行的治疗方法或疫苗。由于它们易于产生和传播，因此被 NIH 归类为 C 类病原体潜在的高发病率和死亡率。 SARS-CoV-2 动力学的详细机制研究复制和病毒脱落（即出芽）可能有助于识别病毒生命周期中的新药物靶点并丰富我们对这种人畜共患病原体如何利用宿主细胞脂质构建病毒脂质包膜的理解。 Stahelin 和 Voth 实验室建立在相互合作和特定专业知识的基础上病毒组装的生物化学、生物物理学和计算研究，将使用体外和细胞实验研究与计算分析相结合，以调查本次资助的中心假设：选择性脂质-蛋白质相互作用驱动 SARS 的 M（膜）和 N（核蛋白）的组装和出芽冠状病毒-2。在两个具体目标中，我们将 (i) 确定 SARS-CoV- 的细胞和生物物理机制。 2 M 在计算机、体外和人类细胞中形成病毒颗粒，并 (ii) 确定 N 脂质结合如何驱动有助于形成新病毒颗粒的定位。这些研究将与结构研究相结合 M（Browhan 实验室）和 N（Ollmann Saphire 实验室）的生物学，并通过真实的验证与库恩实验室合作在 BSL-3 设施中检测 SARS-CoV-2。这些问题将通过结构和体外定量的紧密结合的方法进行研究评估脂质-蛋白质和蛋白质-蛋白质相互作用的技术以及区分脂质-蛋白质和蛋白质-蛋白质相互作用的细胞分析技术病毒出芽和感染所需的病毒蛋白相互作用的分子基础。计算上，我们将使用粗粒度（CG）分子动力学（MD）模拟来表征组装过程膜并确定一组模型，以便通过全原子 (AA) MD 模拟进一步细化。这创新和综合的方法不仅会仔细验证结果，而且会提供详细的对脂质-蛋白质和蛋白质-蛋白质相互作用的结构见解控制着组装和出芽 SARS-CoV-2。这些研究中确定的 M 和 N 的蛋白质界面，这将是病毒组装的关键和传播，将为未来针对 SARS-CoV-2 和其他冠状病毒的药物提供信息。