Sun Shuzhen, Yan Jingjun, Xia Chao, Lin Yuanyuan, Jiang Xiaorui, Liu Haojing, Ren Huanping, Yan Junwei, Lin Jusheng, He Xingxing
Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China; Department of Gastroenterology, the First Affiliated Hospital, Zhengzhou University, Zhengzhou, China.
Liver Int. 2014 Nov;34(10):1532-42. doi: 10.1111/liv.12419. Epub 2013 Dec 23.
Study on viruses has greatly benefited from visualization of viruses tagged with green fluorescent protein (GFP) in living cells. But GFP tag, as a large inserted fragment, is not suitable for labelling Hepatitis B virus (HBV) that is a compact virion with limited internal space.
To visualize HBV in living cells, we constructed several recombinant HBV fluorescently labelled with biarsenical dye to track the behaviour of HBV in the cytoplasm of infected cells.
By mutagenesis, a smaller size tetracysteine (TC) tag (C-C-P-G-C-C) that could be bound with a biarsenical fluorescent dye was genetically inserted at different cell epitopes of HBV core protein expressed in transfected cells.
Confocal microscopy and transmission electron microscopy (TEM) observations showed that TC-tagged core proteins bound with biarsenical dye could specifically fluoresce in cells and be incorporated into nucleocapsid to form fluorescent virions. The recombinant fluorescent HBV virions retained their infectivity as wild-type ones. Moreover, tracking of fluorescent HBV particles in living cells reveals microtubule-dependent motility of the intracellular particles.
To the best of our knowledge, this is the first time to generate fluorescent HBV virions with biarsenical labelling and to visualize their trafficking in living cells. The fluorescent HBV may become one highly valuable tool for further studying detailed dynamic processes of HBV life cycle and interaction of HBV with host in live-imaging approach.
对病毒的研究极大地受益于在活细胞中对标记有绿色荧光蛋白（GFP）的病毒进行可视化观察。但是，GFP标签作为一个大的插入片段，并不适合标记乙型肝炎病毒（HBV），因为HBV是一种内部空间有限的致密病毒粒子。
为了在活细胞中对HBV进行可视化观察，我们构建了几种用双砷染料荧光标记的重组HBV，以追踪HBV在受感染细胞胞质中的行为。
通过诱变，将一个能与双砷荧光染料结合的较小尺寸的四半胱氨酸（TC）标签（C-C-P-G-C-C）基因插入到转染细胞中表达的HBV核心蛋白的不同细胞表位。
共聚焦显微镜和透射电子显微镜（TEM）观察表明，与双砷染料结合的TC标签化核心蛋白能在细胞中特异性发出荧光，并被整合到核衣壳中形成荧光病毒粒子。重组荧光HBV病毒粒子保留了与野生型病毒粒子相同的感染力。此外，对活细胞中荧光HBV颗粒的追踪揭示了细胞内颗粒依赖微管的运动。
据我们所知，这是首次产生用双砷标记的荧光HBV病毒粒子并在活细胞中可视化其运输。荧光HBV可能成为进一步以活体成像方法研究HBV生命周期详细动态过程以及HBV与宿主相互作用的一个极有价值的工具。
