用于研究冠状病毒膜融合的单粒子追踪分析
Single particle tracking assay to study coronavirus membrane fusion.
作者信息
Costello Deirdre A, Daniel Susan
机构信息
School of Chemical and Biomolecular Engineering, Cornell University, 120 Olin Hall, Ithaca, NY, 14853, USA.
出版信息
Methods Mol Biol. 2015;1282:183-94. doi: 10.1007/978-1-4939-2438-7_16.
Single particle tracking (SPT) of individual virion fusion with host cell membranes using total internal reflection microscopy (TIRFM) is a powerful technique for quantitatively characterizing virus-host interactions. One significant limitation of this assay to its wider use across many types of enveloped viruses, such as coronavirus, has been incorporating non-lipid receptors (proteins) into the supported lipid bilayers (SLBs) used to monitor membrane fusion. Here, we describe a method for incorporating a proteinaceous viral receptor, feline aminopeptidase N (fAPN), into SLBs using cell blebbing of mammalian cells expressing fAPN in the plasma membrane. This receptor binds feline coronavirus (FECV 1683). We describe how to carry out single particle tracking of FECV fusion in this SLB platform to obtain fusion kinetics.
利用全内反射显微镜（TIRFM）对单个病毒粒子与宿主细胞膜融合进行单粒子追踪（SPT），是定量表征病毒与宿主相互作用的一项强大技术。该检测方法在广泛应用于多种包膜病毒（如冠状病毒）时存在一个重大限制，即如何将非脂质受体（蛋白质）整合到用于监测膜融合的支持脂质双层（SLB）中。在此，我们描述了一种通过在质膜中表达fAPN的哺乳动物细胞进行细胞发泡，将蛋白质类病毒受体猫氨基肽酶N（fAPN）整合到SLB中的方法。该受体可结合猫冠状病毒（FECV 1683）。我们描述了如何在这个SLB平台上对FECV融合进行单粒子追踪以获得融合动力学。
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