一种荧光标记的马尔堡病毒糖蛋白作为研究病毒运输和组装的新工具。
A Fluorescently Labeled Marburg Virus Glycoprotein as a New Tool to Study Viral Transport and Assembly.
机构信息
Institut für Virologie, Philipps-Universität Marburg, Marburg, Germany.
German Center of Infection Research, Partner Site Giessen-Marburg-Langen, Marburg, Germany.
出版信息
J Infect Dis. 2018 Nov 22;218(suppl_5):S318-S326. doi: 10.1093/infdis/jiy424.
The single surface glycoprotein (GP) of filoviruses is indispensable for recognition of its cellular receptor and infection of target cells. To study the intracellular trafficking of GP by using live-cell imaging, the mucin-like domain of Marburg virus (MARV) GP was replaced by the fluorophore mCherry (GP∆MLD_mCherry). Intracellular distribution, surface transport, and recruitment of GP∆MLD_mCherry into virus-like particles were similar to observations for wild-type GP. Using reverse genetics, we generated a recombinant MARV expressing GP∆MLD_mCherry (recMARV MARVGP∆MLD_mCherry). Time-lapse microscopy of recMARV MARVGP∆MLD_mCherry-infected cells revealed that GP∆MLD_mCherry-positive vesicles were transported to the cell surface in a tubulin-dependent manner. Moreover, dual-color live-cell imaging revealed cotransport of GPΔMLD_mCherry and VP40 and their colocalization at the plasma membrane. In this proof-of-concept study we showed that the newly developed GP∆MLD_mCherry is a promising tool to elucidate intracellular trafficking and assembly pathways of MARV.
丝状病毒的单一表面糖蛋白（GP）对于识别其细胞受体和感染靶细胞是不可或缺的。为了通过活细胞成像研究 GP 的细胞内运输，我们用荧光染料 mCherry 取代了马尔堡病毒（MARV）GP 的粘蛋白样结构域（GP∆MLD_mCherry）。GP∆MLD_mCherry 的细胞内分布、表面转运以及募集到病毒样颗粒的情况与野生型 GP 的观察结果相似。通过反向遗传学，我们生成了一株表达 GP∆MLD_mCherry 的重组 MARV（recMARV MARVGP∆MLD_mCherry）。recMARV MARVGP∆MLD_mCherry 感染细胞的延时显微镜观察显示，GP∆MLD_mCherry 阳性囊泡以微管依赖性方式转运至细胞膜表面。此外，双色活细胞成像显示 GPΔMLD_mCherry 和 VP40 的共转运及其在质膜上的共定位。在这项概念验证研究中，我们表明新开发的 GP∆MLD_mCherry 是阐明 MARV 细胞内运输和组装途径的有前途的工具。
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