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

阐明 SARS-CoV-2 的组装和出芽机制

• 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
• 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

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.

项目摘要 严重急性呼吸综合征冠状病毒2(SARS-CoV-2)是一种新的冠状病毒，可引起 被列为国际突发公共卫生事件的正在进行的人类大流行(新冠肺炎) 受到世界卫生组织(世卫组织)的关注。对SARS-CoV-2的研究迫切需要 促进治疗学的发展，了解病毒复制和发病机制，并确定如何 病毒在细胞间和病人之间传播。SARS和MERS等冠状病毒属于 是地球上最危险的病原体，死亡率高，缺乏可行的治疗方法或疫苗。 由于它们易于产生和传播，被美国国家卫生研究院列为C类病原体 潜在的高发病率和高死亡率。SARS-CoV-2病毒动力学的详细机制研究 复制和病毒脱落(即萌芽)可以帮助识别病毒生命周期中的新药物靶点 并丰富了我们对这种人畜共患病病原体如何利用宿主细胞脂类来构建病毒脂膜的理解。 斯塔林和沃斯实验室，建立在彼此的合作和特定的专业知识基础上 生物化学、生物物理学和计算机研究病毒的组装，将使用体外和细胞实验 与计算分析相结合的研究，以调查这项拨款中的中心假设：选择性 脂-蛋白相互作用驱动SARS病毒M(膜)和N(核蛋白)的组装和萌发- CoV-2。在两个特定的目标中，我们将(I)确定SARS冠状病毒通过其细胞和生物物理机制 2M在硅胶、体外和人类细胞中形成病毒颗粒，以及(Ii)确定N脂结合如何驱动 有助于形成新的病毒颗粒的定位。这些研究将与结构性研究相结合 M(Browhan实验室)和N(Ollmann Sphire实验室)的生物学，并与正品进行验证 与库恩实验室合作，在BSL-3设施中使用SARS-CoV-2病毒。 这些问题将用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和细胞分析以梳理 病毒萌发和感染所必需的病毒蛋白相互作用的分子基础。从计算上讲， 我们将使用粗粒度(CG)分子动力学(MD)模拟来表征 通过全原子(AA)分子动力学模拟，确定一组模型，以进一步完善膜。这 创新和综合的方法不仅将提供仔细的结果验证，而且还将提供详细的 对脂-蛋白质和蛋白质-蛋白质相互作用的结构洞察 SARS-CoV-2。在这些研究中确定的M和N的蛋白质界面，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靶向提供信息。