Inhibition of Ebolavirus Entry by IFITM2 Protein

描述(申请人提供)：埃博拉病毒(EBOV)进入宿主细胞是由其唯一的糖蛋白(GP)介导的。GP及其相关的EBOV进入事件具有许多不同寻常的特征，为我们对病毒进入的基本理解提供了新的见解。在这个R21项目中，我们的目标是阐明新发现的细胞限制因子，即干扰素诱导的跨膜蛋白(IFITM)，特别是IFITM2是如何有效和特异地抑制EBOV进入的，并通过这样做，帮助开发新的抗病毒疗法。目的1：建立EBOV的单一病毒融合试验，并对IFITM2抑制的膜融合阶段进行分析。我们将利用EBOV GP可以有效地整合到由VP40基质蛋白形成的病毒样颗粒(VLP)中的这一事实，并开发一个单一的病毒成像和融合系统来确定IFITM2如何抑制EBOV在内溶酶体中的融合。目的2：阐明IFITM2特异性抑制EBOV gp介导的进入的分子和生化机制。我们将测试中心假设，即IFITM2通过干扰其细胞内受体Niemann-Pick C1(NPC1)的触发能力和/或胆固醇运输活性来深刻地抑制EBOV的进入。我们将使用一系列的生化和新型荧光脂质标记技术来评估IFITM2对EBOV gp的胆固醇含量、膜流动性和构象变化的影响。EBOV是一种高致病性丝状病毒，可引起人类严重出血热，病死率高达90%。这些研究的结果将为IFITM2如何限制EBOV GP介导的膜融合和进入提供重要的新见解，并促进我们对IFITM阻止病毒进入的一般机制的理解。

DESCRIPTION (provided by applicant): Entry of Ebolavirus (EBOV) into host cells is mediated by its sole glycoprotein, known as GP. The GP and its associated EBOV entry events possess many unusual features that provide novel insights into our fundamental understanding of viral entry. In this R21 project, we aim to elucidate how the newly identified cellular restricton factors, known as interferon-inducible transmembrane (IFITM) proteins, especially IFITM2, potently and specifically inhibit EBOV entry, and in doing so, aid the development of novel antiviral therapeutics. Aim 1: Establish a single virus fusion assay for EBOV and dissect the stages of membrane fusion inhibited by IFITM2. We will take advantage of the fact that EBOV GP can be efficiently incorporated into its virus-like particles (VLPs) formed by the VP40 matrix protein, and develop a single virus imaging and fusion system to determine how IFITM2 inhibits EBOV fusion in endolysosomes. Aim 2: Elucidate the molecular and biochemical mechanisms by which IFITM2 specifically inhibits EBOV GP-mediated entry. We will test the central hypothesis that IFITM2 profoundly inhibits EBOV entry by disturbing the triggering capability and/or the cholesterol transport activity of its intracellular receptor, Niemann-Pick C1 (NPC1). A series of biochemical and novel fluorescence lipid labeling techniques will be used to assess the effect of IFITM2 on cholesterol content, membrane fluidity, and conformational changes of EBOV GP. EBOV is a highly pathogenic filovirus that causes severe hemorrhagic fever in humans, with a fatality rate of up to 90%. Results from the proposed studies will provide critical novel insight into how IFITM2 restricts EBOV GP-mediated membrane fusion and entry, as well as advance our understanding of the general mechanism of IFITMs that block viral entry.