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.

总结 血凝素（HA）是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）部分的相互作用介导IAV病毒粒子附着到细胞表面。 在通过内吞作用内化之后，晚期内体的酸性环境触发内吞体的转化。 HA从亚稳定的融合前构象转变为高度稳定的融合后卷曲螺旋。这一连串的 HA的构象变化导致病毒和内体膜的融合。重要的是，暴露 在附着到细胞膜之前，IAV的酸性pH过早地将HA转化为融合后的HA。 构象，这使得IAV无感染性。因此，确保HA构象形成的适当时机， 这些变化对IAV的生命周期至关重要。而膜过程中HA构象变化的终点 虽然核聚变的特征很好，但最近的结构数据开始识别中间状态。但 导致从融合前到融合后的HA构象轨迹尚未得到验证，也没有时间 相对于HA构象的膜融合被确定。该资助项目利用 Munro和Somasundaran实验室在尖端单分子Förster方面的综合优势 共振能量转移（smFRET）成像，单病毒体融合测定，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。该资助项目在很大程度上依赖 先进的定量荧光显微镜在Munro实验室的应用。与 在马萨诸塞大学陈医学院的机构支持下，芒罗实验室定制了一台显微镜， 专门用来进行实验的所要求的行政补充 设备采购将提供现有显微镜的升级。这些升级将解决 显微镜的缺点，这限制了成功完成拟议目标的能力， 在申请时没有预见到。

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.