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 严重的急性呼吸综合征冠状病毒2（SARS-COV-2）是一种新型冠状病毒 正在进行的人类大流行（Covid-19）已被归类为国际公共卫生紧急情况 世界卫生组织（WHO）的关注。迫切需要SARS-COV-2研究 促进疗法的发展，了解病毒复制和发病机理，并确定如何 病毒从细胞到细胞以及患者传播到患者。冠状病毒（例如SARS和MERS） 地球上最危险的病原体，死亡率很高，缺乏可行的治疗剂或疫苗。 由于NIH的易于生产和传播，它们被NIH归类为C类病原体 高发病和死亡率的潜力。 SARS-COV-2动力学的详细机械研究 复制和病毒脱落（即萌芽）可以告知病毒生命周期中新药物靶标的识别 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建立病毒脂质包膜的理解。 Stahelin和Voth Laboratories，建立在彼此的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实验性的体外和细胞 与计算分析集成的研究，研究了这笔赠款中的中心假设：选择性 脂质 - 蛋白相互作用驱动Sars-的M（膜）和N（核蛋白）的组装和萌芽 COV-2。在两个具体的目标中，我们将（i）确定SARS-COV-的细胞和生物物理机制 2 m在硅，体外和人类细胞中形成病毒颗粒，（ii）确定n脂质结合驱动 有助于形成新病毒颗粒的本地化。这些研究将与结构集成 M（Browhan实验室）和N（Ollmann Saphire实验室）的生物学，也可以用真实的验证 与Kuhn实验室合作的BSL-3设施中的SARS-COV-2。 这些问题将使用结构和体外定量进行紧密整合的方法研究 评估脂质 - 蛋白质和蛋白质 - 蛋白质相互作用和细胞测定的技术，以分开 病毒萌芽和感染所必需的病毒蛋白相互作用的分子基础。计算， 我们将使用粗粒（CG）分子动力学（MD）模拟来表征在 膜并确定一组模型，以通过全原子（AA）MD模拟进一步完善。这 创新和综合的方法不仅会仔细验证结果，而且还提供详细的结果 对脂质 - 蛋白质和蛋白质 - 蛋白质相互作用的结构见解，用于组装和萌芽 SARS-CoV-2。在这些研究中鉴定的M和N的蛋白质界面，这将是病毒组装的关键 并扩散将告知未来针对SARS-COV-2和其他冠状病毒的药物。