Biophysical studies of viral membrane fusion proteins

摘要 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病毒粒子与细胞表面的附着。 在内吞作用内化之后，晚期内吞体内的酸性环境触发了 HA从亚稳的融合前构象到高度稳定的融合后盘绕线圈。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 IAV在附着到细胞膜之前的酸性pH过早地将HA转化为融合后的HA 构象，这使得IAV不具传染性。因此，确保HA构象的适当时机 变化对IAV的生命周期至关重要。而HA的构象终点在膜过程中发生变化 聚变的特征很好，最近的结构数据开始识别中间状态。然而， 从融合前到融合后的HA构象轨迹尚未得到验证，也没有确定 确定了与HA构象有关的膜融合。受资助的项目利用 门罗实验室和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、单病毒粒子融合分析、低温电子断层扫描(CryoET)、 以及在病毒融合中解决这些问题的计算方法。受资助的项目严重依赖于 先进的定量荧光显微镜在门罗实验室的应用。使用 在马萨诸塞州大学医学院的机构支持下，门罗实验室定制了一台显微镜 专门设计来进行拟议中的实验。所要求的行政补充资料 购买设备将对现有显微镜进行升级。这些升级将解决 显微镜的缺陷，这限制了成功完成拟议目标的能力，以及 在申请时没有预见到。

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.