Novel Cellular Factors Restricting Viral Membrane Fusion

描述（由申请人提供）：在这个R21申请中，我们的目标是确定新发现的细胞限制因子，称为干扰素诱导跨膜（IFITM）蛋白，如何调节病毒膜融合和进入，并在此过程中，帮助开发新的抗病毒治疗方法。本建议的具体目的如下。目的1：验证IFITM蛋白通过阻止半融合抑制膜融合的假设。我们将进行细胞-细胞和病毒粒子-细胞融合试验，包括使用单病毒颗粒成像技术，以确定被IFITM蛋白阻断的病毒膜融合过程的阶段。脂质类似物调节膜融合过程的不同阶段将被应用。我们将探讨IFITM蛋白限制细胞和发育性融合原诱导的膜融合的可能性。目的2：验证IFITM蛋白表达改变细胞膜脂质秩序并赋予正自发曲率的假设。我们将进行荧光膜标记实验，以验证IFITM蛋白导致细胞膜更有序，从而降低流动性，从而降低融合能力的假设。我们还将通过合成与IFITM蛋白功能域相对应的肽重构人工脂质体来确定IFITM蛋白对膜曲率的影响。冷冻电子显微镜断层扫描将用于直接观察IFITM肽插入人工脂质体囊泡后的曲率变化。IFITM蛋白是迄今为止已知的第一个也是唯一一个限制病毒膜融合和进入的细胞限制因子。拟议研究的结果将为IFITM蛋白如何限制病毒膜融合提供新的见解。这些结果将指导未来对IFITM蛋白生物学的全面研究，以及它们的作用机制

DESCRIPTION (provided by applicant): In this R21 application, we aim to determine how the newly identified cellular restriction factors, known as interferon-inducible transmembrane (IFITM) proteins, modulate viral membrane fusion and entry, and in doing so, aid the development of novel antiviral therapeutics. The specific aims of this proposal are as follows. Aim 1: Test the hypothesis that IFITM proteins inhibit membrane fusion by preventing hemifusion. We will perform cell-cell and virion-cell fusion assays, including the use of single viral particle imaging technique, to determine the stages of the viral membrane fusion process blocked by IFITM proteins. Lipid analogs that modulate distinct stages of the membrane fusion process will be applied. We will explore the possibility that IFITM proteins restrict membrane fusion induced by cellular and developmental fusogens. Aim 2: Test the hypothesis that expression of IFITM proteins changes the lipid order of cell membranes and confers positive spontaneous curvature. We will perform fluorescence membrane labeling experiments to test the hypothesis that IFITM proteins cause the cell membrane to be more ordered, resulting in reduced fluidity and therefore less competency for fusion. We will also determine the effect of IFITM proteins on membrane curvature by reconstituting artificial liposomes with synthetic peptides corresponding to the functional domains in IFITM proteins. Cryo-EM tomography will be applied to directly visualize the curvature changes upon insertion of IFITM peptides into the artificial liposome vesicles. IFITM proteins are the first and so far only cellular restriction fators that are known to restrict viral membrane fusion and entry. Results from the proposed studies will provide novel insight as to how IFITM proteins restrict viral membrane fusion. These results will guide future comprehensive investigations into the biology of IFITM proteins, as well as their critical roles in restricting viral entry and infection.