Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN 38163, USA.
Methods. 2011 Oct;55(2):127-36. doi: 10.1016/j.ymeth.2011.09.002. Epub 2011 Sep 10.
Viruses exploit cellular machinery to gain entry and initiate their replication cycle within host cells. The development of methods to visualize virus entry in live cells has provided new insights to the cellular processes involved in virus entry and the intracellular locations where viral payloads are deposited. The use of fluorescently labeled virus and high-resolution microscopy is currently the method of choice to study virus entry in live cells. While fluorescent protein fusions (e.g. viral proteins fused to GFP) have been used, the labeling of viral proteins that contain a small tetracysteine (tc) tag with biarsenical fluorescent compounds (e.g. FlAsH, ReAsH, Lumio-x) offers several advantages over conventional xFP-fusion constructs. This article describes methods for generating fluorescently labeled viruses encoding tc-tagged proteins that are suitable for the study of virus entry in live cells by fluorescence microscopy. Critical parameters required to quantify fluorescence signals from the labeled, tc-tagged proteins in individual virus particles during the entry process and the subsequent fate of the labeled viral proteins after virus uncoating are also described.
病毒利用细胞机制进入宿主细胞并启动其复制周期。开发用于在活细胞中可视化病毒进入的方法，为病毒进入涉及的细胞过程以及病毒有效负载沉积的细胞内位置提供了新的见解。目前，使用荧光标记病毒和高分辨率显微镜是研究活细胞中病毒进入的首选方法。虽然已经使用了荧光蛋白融合体（例如与 GFP 融合的病毒蛋白），但用双砷荧光化合物（例如 FlAsH、ReAsH、Lumio-x）标记含有小四半胱氨酸（tc）标签的病毒蛋白与传统的 xFP 融合构建体相比具有几个优势。本文描述了生成荧光标记病毒的方法，这些病毒编码 tc 标记蛋白，适合通过荧光显微镜研究活细胞中的病毒进入。还描述了在进入过程中量化标记的 tc 标记蛋白在单个病毒颗粒中的荧光信号所需的关键参数，以及标记的病毒蛋白在病毒脱壳后的后续命运。
