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: 10707286
• 负责人: Robert Virgil Stahelin
• 金额: $ 76.67万
• 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/10707286
• 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; Classification; 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; Lipid Binding; Lipids; Measurement; Mediating; Membrane; Middle East Respiratory Syndrome; Modeling; Molecular; Molecular Conformation; Morbidity - disease rate; Nucleocapsid; Nucleoproteins; PIK3CG gene; Pathogenesis; Patients; Phosphatidylinositols; Planet Earth; Play; Process; Production; Property; Protein Analysis; Protein C; 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 Packaging; Viral Pathogenesis; Viral Proteins; Virion; Virus; Virus Assembly; Virus Replication; Virus Shedding; World Health Organization; Zoonoses; biophysical analysis; biosafety level 3 facility; computer studies; experimental study; in silico; inhibitor; innovation; insight; molecular dynamics; mortality; multiple myeloma M Protein; multitask; new therapeutic target; novel coronavirus; pandemic disease; particle; pathogen; programs; protein function; protein protein interaction; protein structure; 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.

项目总结