Biophysical studies of viral membrane fusion proteins

病毒膜融合蛋白的生物物理学研究

• 批准号: 10798382
• 负责人: James B Munro
• 金额: $ 5.78万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10798382
• 关键词: 2019-nCoV; Address; Administrative Supplement; Biological Assay; Biological Models; Cell surface; Cells; Cellular Membrane; Chimeric Proteins; Cryo-electron tomography; Custom; Data; Endocytosis; Ensure; Environment; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Funding; Glycoproteins; Hemagglutinin; Image; Influenza; Influenza A virus; Influenza Hemagglutinin; Institution; Laboratories; Life Cycle Stages; Mediating; Membrane Fusion; Microscope; Molecular Conformation; Research; Sialic Acids; Surface; Time; Viral; Virion; Virus; biophysical analysis; design; endosome membrane; equipment acquisition; experimental study; late endosome; medical schools; premature; single molecule

SUMMARY Hemagglutinin (HA) is an envelope glycoprotein that resides on the surface of influenza A virus (IAV) particles. HA mediates attachment of IAV virions to the cell surface through interaction with sialic acid (SA) moieties. Following internalization by endocytosis, the acidic environment of the late endosome triggers conversion of HA from a metastable prefusion conformation to a highly stable postfusion coiled coil. This cascade of conformational changes in HA results in fusion of the viral and endosomal membranes. Importantly, exposure of IAV to acidic pH prior to attachment to a cellular membrane prematurely converts HA to the postfusion conformation, which renders IAV noninfectious. Therefore, ensuring proper timing of HA conformational changes is critical to the life cycle of IAV. While endpoints of the HA conformational changes during membrane fusion are well characterized, recent structural data is beginning to identify intermediate states. However, the HA conformational trajectory that leads from prefusion to postfusion is not yet validated, nor has the timing of membrane fusion with respect to HA conformation been determined. The funded project leverages the combined strengths of the Munro and Somasundaran laboratories in cutting-edge single-molecule Förster resonance energy transfer (smFRET) imaging, single-virion fusion assays, cryoelectron tomography (cryoET), and computational approaches to address these questions in viral fusion. The funded project relies heavily on the application of advanced, quantitative fluorescence microscopy within the Munro laboratory. With institutional support from UMass Chan Medical School, the Munro laboratory has custom built a microscope specifically designed to conduct the proposed experiments. The requested Administrative Supplement for Equipment Purchases will provide upgrades to the existing microscope. These upgrades will address shortcomings of the microscope, which limit the ability to successfully complete the proposed Aims, and which were not foreseen at the time of application.

概括 血凝素 (HA) 是一种存在于甲型流感病毒 (IAV) 颗粒表面的包膜糖蛋白。 HA 通过与唾液酸 (SA) 部分相互作用介导 IAV 病毒颗粒附着到细胞表面。 通过内吞作用内化后，晚期内体的酸性环境触发 HA从亚稳态融合前构象转变为高度稳定的融合后卷曲线圈。这个级联 HA 的构象变化导致病毒和内体膜的融合。重要的是曝光度 在附着到细胞膜之前将 IAV 调节至酸性 pH 值会过早地将 HA 转化为融合后的物质 构象，使 IAV 不具有传染性。因此，确保HA构象的正确时机 变化对于 IAV 的生命周期至关重要。虽然HA构象的终点在膜过程中发生变化 融合已经得到很好的表征，最近的结构数据开始识别中间状态。然而， 从灌注前到灌注后的 HA 构象轨迹尚未得到验证，也没有验证其发生的时间。 确定了HA构象的膜融合。资助项目利用 Munro 和 Somasundaran 实验室在尖端单分子 Förster 领域的综合优势 共振能量转移 (smFRET) 成像、单病毒粒子融合测定、冷冻电子断层扫描 (cryoET)、 和计算方法来解决病毒融合中的这些问题。资助项目很大程度上依赖于 Munro 实验室内先进的定量荧光显微镜的应用。和 在麻省大学陈医学院的机构支持下，Munro 实验室定制了一台显微镜 专门设计用于进行所提出的实验。所要求的行政补充 设备采购将对现有显微镜进行升级。这些升级将解决 显微镜的缺点限制了成功完成所提出的目标的能力，并且 申请时并未预见到。

项目成果

hACE2-Induced Allosteric Activation in SARS-CoV versus SARS-CoV-2 Spike Assemblies Revealed by Structural Dynamics.

• DOI: 10.1021/acsinfecdis.3c00010
• 发表时间: 2023-06-09
• 期刊: ACS INFECTIOUS DISEASES
• 影响因子: 5.3
• 作者: Chen, Chengbo;Zhu, Richard;Hodge, Edgar A.;Diaz-Salinas, Marco A.;Nguyen, Adam;Munro, James B.;Lee, Kelly K. K.
• 通讯作者: Lee, Kelly K. K.