Inhibition of viral entry by interferon-induced proteins

干扰素诱导的跨膜蛋白（IFITMs）在细胞培养物和细胞培养物中都表现出广泛的抗病毒活性。 vivo. IFITM表达已被证明可以预防重要的人类病原体的感染，包括流感， 埃博拉、西尼罗河和SARS病毒，通过阻断病毒与靶细胞的融合。IFITM抗性的存在 病毒，如鼠白血病和拉沙病毒，表明限制发生在细胞区室- 具体方式。IFITMs抑制不同病毒融合的机制和逃逸 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。我们制造并验证了功能性荧光标记IFITM蛋白 并首次对单一病毒/IFITM共运输和融合进行成像。初步实验表明， 与IFITM共同运输的细胞被困在半融合阶段，不能形成融合孔，而抗性细胞则不能形成融合孔。 病毒似乎通过缺乏IFITM的内体转运。我们还发现了一个新的角色， 在病毒融合和IFITM限制表型中的磷酸肌醇。我们的工作假设是IFITM 通过以下方式阻断病毒进入：（i）通过改变以下性质在死端半融合阶段捕获病毒融合： 细胞膜;和（ii）有利于扩大的多泡内与腔内小泡的非生产性融合 隔间我们将使用一组经验证的IFITM敏感和耐药的 假病毒以及病毒学、细胞生物学、膜生物物理学和先进成像技术的强大组合 技术.具体而言，我们将：（1）阐明IFITMs与病毒的动态共定位是否是一种有效的方法。 限制的先决条件;（2）确定IFITMs是否通过改变机械性质来抑制病毒融合 细胞膜不利于从半融合到完全融合的转变和/或通过转移病毒 进入非生产性途径;和（3）描绘磷酸肌醇在IFITM限制中的作用 表型。深入了解IFITM介导的病毒限制机制将提供重要线索， 细胞产生有效的抗病毒反应，并提出了预防感染的新策略。

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.