Inhibition of viral entry by interferon-induced proteins

干扰素诱导的跨膜蛋白(IFITM)在细胞培养和体内均显示出广泛的抗病毒活性。 活着。IFITM的表达已被证明可以预防包括流感在内的重要人类病原体的感染， 埃博拉、西尼罗河和SARS病毒，通过阻止病毒与目标细胞融合而发挥作用。抗IFITM病毒的存在 病毒，如小鼠白血病和拉萨病毒，表明限制发生在一个细胞隔间- 具体的方式。IFITM抑制多种病毒融合的机制及其逃逸 IFITM限制的机制尚不清楚，部分原因是与可视化相关的困难 它们在活细胞中的动态分布。我们制备并验证了功能性荧光标记的IFITM蛋白 并首次对单一病毒/IFITM共同贩运和融合进行了成像。初步实验表明，病毒 与IFITM的共同流量被困在半融合阶段，不能形成融合孔，而抵抗 病毒似乎是通过缺乏IFITM的内体传播的。我们还发现了一个新的角色 病毒融合中的肌醇磷脂和IFITM限制表型。我们的工作假设是IFITM 通过以下方式阻止病毒进入：(I)通过改变以下特性在死端半融合阶段捕获病毒融合 细胞膜；以及(Ii)有利于与扩大的多囊泡内的腔内小泡进行非生产性融合 车厢。我们将使用一组经过验证的IFITM敏感和抗IFITM组件来测试工作假设 伪病毒和病毒学、细胞生物学、膜生物物理学和高级成像的强大组合 技巧。具体地说，我们将：(1)阐明IFITM与病毒的动态共存是否是一种 限制的先决条件；(2)确定IFITM是否通过改变机械性能来抑制病毒融合 不利于从半融合到完全融合和/或通过转移病毒的细胞膜的 进入非生产途径；以及(3)描述磷脂酰肌醇在IFITM限制中的作用 表型。深入了解IFITM介导的病毒限制机制将为以下方面提供重要线索 细胞产生有效的抗病毒反应，并提出预防感染的新策略。

Interferon-inducible transmembrane proteins (IFITMs) exhibit broad antiviral activity, both in cell culture and in vivo. IFITM expression has been shown to prevent infection of important human pathogens, including influenza, Ebola, West Nile and SARS viruses, by blocking viral fusion with target cells. The existence of IFITM-resistant viruses, such as Murine Leukemia and Lassa viruses, suggests that restriction occurs in a cellular compartment- specific manner. The mechanism by which fusion of diverse viruses is inhibited by IFITMs and the escape mechanisms from IFITM restriction are not understood, due in part to difficulties associated with visualization of their dynamic distribution in living cells. We made and validated functional fluorescently-tagged IFITM proteins and imaged, for the first time, single virus/IFITM co-trafficking and fusion. Pilot experiments suggest that viruses that co-traffic with IFITMs are trapped at a hemifusion stage, unable to form a fusion pore, whereas resistant viruses appear to be transported through endosomes devoid of IFITMs. We also discovered a novel role for phosphoinositides in viral fusion and the IFITM restriction phenotype. Our working hypothesis is that IFITMs block virus entry by: (i) trapping viral fusion at a dead-end hemifusion stage through by altering the properties of cell membranes; and (ii) favoring non-productive fusion with intralumenal vesicles within enlarged multivesicular compartments. We will test the working hypothesis using a panel of validated IFITM-sensitive and –resistant pseudoviruses and a powerful combination of virology, cell biology, membranebiophysics and advanced imaging techniques. Specifically, we will: (1) elucidate whether dynamic colocalization of IFITMs with viruses is a prerequisite for restriction; (2) determine whether IFITMs inhibit viral fusion by altering the mechanical properties of cell membranes that disfavor the transition from hemifusion to full fusion and/or through diverting the virus entry to a non-productive pathway; and (3) delineate the role of phosphoinositides in the IFITM restriction phenotype. Insights into the mechanism of IFITM-mediated virus restriction will provide important clues on how cells mount efficient antiviral responses and suggest new strategies for preventing infection.