Live-cell analysis of a green fluorescent protein-tagged herpes simplex virus infection
Live-cell analysis of a green fluorescent protein-tagged herpes simplex virus infection
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DOI：
10.1128/jvi.73.5.4110-4119.1999
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发表时间：
1999-05-01
影响因子：
5.4
通讯作者：
O'Hare, P
中科院分区：
文献类型：
--
作者：
Elliott, G;O'Hare, P
单纯疱疹病毒成熟途径的许多阶段尚未被确定。特别是，关于病毒粒子被膜的组装以及随后它被结合到成熟的病毒颗粒中的情况知之甚少。在这里，我们描述了单纯疱疹病毒1型(HSV-1)重组的构建，其中我们用表达绿色荧光蛋白(GFP)-VP22融合蛋白(GFP-22)的基因取代了编码主要被蛋白VP22的基因。我们发现该病毒具有与亲本病毒相同的生长特性，并且新合成的GFP-22早在感染后3h就可以在活细胞中检测到，此外，我们还证明GFP-22与VP22一样有效地掺入HSV-1病毒粒子中，导致荧光显微镜下可以看到颗粒，因此，我们使用时移共聚焦显微镜来监测活细胞感染中的GFP-22，并呈现了GFP-22在病毒整个生命周期中定位的时移动画。这些动画表明，GFP-22在最初表达时存在于弥漫的细胞质位置，但后来演变成颗粒状物质，通过专门的细胞质途径到达细胞外围。通过这种方式，我们首次可视化了疱疹病毒结构成分在活的受感染细胞内的运输。
Many stages of the herpes simplex virus maturation pathway have not yet been defined. In particular, little is known about the assembly of the virion tegument compartment and its subsequent incorporation into maturing virus particles. Here we describe the construction of a herpes simplex virus type 1 (HSV-1) recombinant in which we have replaced the gene encoding a major tegument protein, VP22, with a gene expressing a green fluorescent protein (GFP)-VP22 fusion protein (GFP-22). We show that this virus has growth properties identical to those of the parental virus and that newly synthesized GFP-22 is detectable in live cells as early as 3 h postinfection, Moreover, we show that GFP-22 is incorporated into the HSV-1 virion as efficiently as VP22, resulting in particles which are visible by fluorescence microscopy, Consequently, we have used time lapse confocal microscopy to monitor GFP-22 in live-cell infection, and we present time lapse animations of GFP-22 localization throughout the virus life cycle. These animations demonstrate that GFP-22 is present in a diffuse cytoplasmic location when it is initially expressed but evolves into particulate material which travels through an exclusively cytoplasmic pathway to the cell periphery. In this way, we have for the first time visualized the trafficking of a herpesvirus structural component within live, infected cells.