Etienne Lyns, Joshi Poorval, Dingle Laura, Huang Eugene, Grzesik Peter, Desai Prashant J
Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
J Virol Methods. 2017 Mar;241:46-51. doi: 10.1016/j.jviromet.2016.12.012. Epub 2016 Dec 21.
Our laboratory was one of the first to engineer a live fluorescent tag, enhanced green fluorescent protein (eGFP), that marked the capsid of herpes simplex virus type 1 (HSV-1) and subsequently maturing virus as the particle made its way to the cell surface. In the present study we sought to increase the repertoire of colors available as fusion to the small capsid protein, VP26, so that they can be used alone or in conjunction with other fluorescent tags (fused to other HSV proteins) to follow the virus as it enters and replicates within the cell. We have now generated viruses expressing VP26 fusions with Cerulean, Venus, mOrange, tdTomato, mCherry, and Dronpa3 fluorescent proteins. These fusions were made in a repaired UL35 gene (VP26) background. These fusions do not affect the replication properties of the virus expressing the fusion polypeptide and the fusion tag was stably associated with intranuclear capsids and mature virions. Of note we could not isolate viruses expressing fusions with fluorescent proteins that have a tendency to dimerize.
我们的实验室是最早设计出一种活荧光标记物——增强型绿色荧光蛋白（eGFP）的实验室之一，该标记物可标记1型单纯疱疹病毒（HSV-1）的衣壳，随后随着病毒颗粒向细胞表面移动，标记成熟的病毒。在本研究中，我们试图增加可与小衣壳蛋白VP26融合的可用颜色种类，以便它们可以单独使用或与其他荧光标记物（与其他HSV蛋白融合）结合使用，来追踪病毒进入细胞并在细胞内复制的过程。我们现已构建出表达与蓝色荧光蛋白、维纳斯荧光蛋白、橙色荧光蛋白、串联二聚体红色荧光蛋白、单体红色荧光蛋白和绿色荧光蛋白Dronpa3融合的VP26的病毒。这些融合体是在修复后的UL35基因（VP26）背景下构建的。这些融合体不影响表达融合多肽的病毒的复制特性，并且融合标签与核内衣壳和成熟病毒粒子稳定相关。值得注意的是，我们无法分离出表达与有二聚化倾向的荧光蛋白融合的病毒。
