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

项目摘要 严重急性呼吸综合征冠状病毒2（SARS-CoV-2）是一种新型冠状病毒， 持续的人类大流行病（COVID-19）已被列为国际公共卫生紧急事件， 世界卫生组织（WHO）的关注。对SARS-CoV-2研究的迫切需求是， 促进治疗的发展，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间传播。SARS和MERS等冠状病毒是 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。 它们被NIH归类为C类病原体，因为它们易于生产和传播， 潜在的高发病率和死亡率。SARS-CoV-2动态的详细机制研究 复制和病毒脱落（即，出芽）可以为病毒生命周期中新药物靶点的鉴定提供信息 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质来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， 生物化学，生物物理学和病毒组装的计算研究，将使用实验在体外和细胞 结合计算分析的研究，以调查这项资助的中心假设： 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， 二型冠状病毒在两个具体的目标，我们将（i）确定细胞和生物物理机制，通过SARS-CoV， 2 M形式的病毒颗粒在计算机，在体外和人类细胞和（ii）确定如何N脂质结合驱动 有助于形成新病毒颗粒的定位。这些研究将与结构 M（Browhan实验室）和N（Ollmann Saphire实验室）的生物学，并通过真实的 与库恩实验室合作，在BSL-3设施中进行SARS-CoV-2。 这些问题将研究在一个紧密结合的方法，使用结构和体外定量 评估脂质-蛋白质和蛋白质-蛋白质相互作用的技术和细胞测定， 病毒出芽和感染所必需的病毒蛋白相互作用的分子基础。在计算上， 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，通过全原子（AA）MD模拟进一步完善。这 创新和综合的方法将不仅提供结果的仔细验证，而且还提供详细的 对脂质-蛋白质和蛋白质-蛋白质相互作用的结构见解， SARS-CoV-2.在这些研究中确定了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。

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.