Active and arrested paramyxovirus fusion machinery visualized by cryo-electron to

描述（由申请人提供）：解决该领域挑战的新成像策略。副粘病毒亨德拉病毒和尼帕病毒（HeV、NiV）的致命性和传播性使这些病原体受到严重关注。这些也是在生物工程和病毒学之间开发创新成像平台的理想模型。尽管副粘病毒融合机制的晶体结构数据相对丰富，但其动态过程（从病毒与宿主细胞的结合开始到感染结束）却知之甚少。导致融合过程激活的包膜病毒糖蛋白之间的相互作用尚未得到严格的分析。对病毒融合纳米机械进行成像将加深我们对病毒膜融合的理解，并使我们能够确定干扰该过程和阻止感染周期的策略。我们建议捕获、固定和可视化融合过程的激活中间状态，暴露病毒进入所必需的保守结构域。虽然激活的构象通常仅存在于活细胞的表面，但我们建议使用工程脂质体来捕获和固定过渡态。我们将使用以仿生方式呈现在脂质体表面的受体分子来“触发”融合蛋白并激活病毒融合机制中的构象变化。然后，我们将使用融合抑制肽将融合机制阻止在其激活状态，从而用脂质体固定捕获的中间体。然后将通过冷冻电子断层扫描分析这些复合物。目标 1. 生成 NiV 聚变机构中间状态 1.1 捕获 NiV 聚变机构的中间状态。我们将在融合反应的中间阶段捕获 NiV 融合机制，首先使用脂质体上存在的细胞受体分子激活融合过程，然后用融合抑制肽捕获融合中间体，阻止完成膜融合所需的融合蛋白的构象重折叠。 1.2 融合过程不同状态的功能和形态学研究。荧光光谱和负染色透射电子显微镜将用于确定子目标 1.1 中捕获的复合物的稳定性。目标 2. 确定受体结合和融合蛋白在结合和融合激活过程中如何相互作用 NiV 融合机制的电子冷冻断层扫描 (ECT) 成像。使用 ECT，我们将可视化 NiV 受体结合蛋白 (G) 触发融合 (F) 蛋白介导的融合的过程和要求以及 F 结构重排的后续步骤。 公共健康相关性：该项目的目标是开发一种基于使用人工脂质体吸引和锁定病毒融合复合物中间状态的新型成像策略。拟议的研究旨在详细了解副粘病毒用来侵入宿主细胞的病毒融合纳米机器的功能。为了确定如何最好地干扰病毒感染周期，这种理解是必要的。

DESCRIPTION (provided by applicant): New imaging strategy to address challenges in the field. The lethal and transmissible nature of the paramyxoviruses Hendra virus and Nipah virus (HeV, NiV) makes these pathogens of serious concern. These are also ideal models for developing an innovative new imaging platform at the interface between bioengineering and virology. Despite the relative abundance of crystal structure data for the paramyxovirus fusion machinery, the dynamic processes - starting with the binding of virus to the host cell and ending with infection -- are poorly understood. The interplay between the envelope viral glycoproteins that leads to activation of the fusion process has eluded rigorous analysis. Imaging the viral fusion nano-machinery will deepen our understanding of viral membrane fusion and will allow us to identify strategies for interfering with the process and arresting the infection cycle. We propose to capture, immobilize, and visualize activated intermediate states of the fusion process, exposing the conserved domains that are essential for viral entry. While the activated conformation is normally present only at the surface of live cells, we propose to use engineered liposomes to capture and immobilize transitional states. We will use receptor molecules, presented in a biomimetic fashion on the surface of the liposomes, to "trigger" the fusion protein and activate the conformational change in the viral fusion machinery. We will then use fusion inhibitory peptides to arrest the fusion machinery in its activated state, and thus immobilize the captured intermediates with the liposomes. These complexes will then be analyzed by cryo-electron tomography. Aim 1. Generate NiV fusion machinery intermediate states 1.1 Trapping intermediate states of the NiV fusion machinery. We will capture the NiV fusion machinery at intermediate stages of the fusion reaction, by first activating the fusion process using cellular receptor molecules presented on liposomes, and then trapping fusion intermediates with fusion-inhibitory peptides that block the conformational refolding of the fusion protein that is required for completing membrane fusion. 1.2 Functional and morphological studies of different states of the fusion process. Fluorescence spectroscopy and negative-stain transmission electron microscopy will be used to determine the stability of the captured complexes from sub aim 1.1. Aim 2. Determine how the receptor binding and fusion proteins interact during binding and fusion activation Electron cryo-tomography (ECT) imaging of NiV fusion machinery. Using ECT we will visualize the process and requirements for the NiV receptor binding protein (G) to trigger fusion (F) protein-mediated fusion and subsequent steps in F structural rearrangement. PUBLIC HEALTH RELEVANCE: The goal of this project is to develop a novel imaging strategy based on the use of artificial liposomes to attract and lock intermediate states of viral fusion complexes. The proposed research seeks to gain a detailed understanding of the function of the viral fusion nano-machinery that paramyxoviruses use to invade host cells. Such an understanding is necessary in order to identify how best to interfere with the cycle of viral infection.