Development of a novel cell-based assay to monitor the transactivation activity of the HSV-1 protein ICP0.
Development of a novel cell-based assay to monitor the transactivation activity of the HSV-1 protein ICP0.
复制标题
开发一种新的基于细胞的检测方法来监测 HSV-1 蛋白 ICP0 的反式激活活性。
DOI：
10.1016/j.antiviral.2015.04.012
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发表时间：
2015
影响因子：
7.6
通讯作者：
Davido,DavidJ
中科院分区：
文献类型：
--
作者：
Fowler,AngelaM;Shinogle,HeatherE;Davido,DavidJ
单纯疱疹病毒1型（HSV-1）即刻早期磷酸化蛋白感染细胞蛋白0 （ICP0）是一种有效的病毒基因转录激活因子，是有效的病毒复制和潜伏期再激活所必需的。然而，特异性细胞因子在ICP0的反激活子功能中所起的作用在很大程度上是未知的。为了确定这些因素的长期目标，我们开发了一种基于96孔格式的细胞检测方法来测量ICP0的活性。我们用一组HSV-1 GFP报告病毒设计了一个系统，在细胞培养中，ICP0能诱导GFP的表达。在24小时的荧光显微镜下证实了该系统的初步可行性。我们将该实验调整为96孔板格式，用荧光扫描仪定量GFP表达。我们的结果表明，我们建立的基于细胞的检测方法是一种有效的检测ICP0反激活活性的方法。该试验可用于鉴定调节ICP0反激活活性的细胞因子。
The herpes simplex virus type 1 (HSV-1) immediate-early phosphoprotein infected cell protein 0 (ICP0) is a potent transcriptional activator of viral genes and is required for efficient viral replication and reactivation from latency. However, it is largely unknown what role specific cellular factors play in the transactivator function of ICP0. With the long-term goal of identifying these factors, we developed a cell-based assay in a 96-well format to measure this activity of ICP0. We designed a system using a set of HSV-1 GFP reporter viruses in which the expression of GFP is potently induced by ICP0 in cell culture. The initial feasibility of this system was confirmed over a 24-h period by fluorescence microscopy. We adapted this assay to a 96-well plate format, quantifying GFP expression with a fluorescence scanner. Our results indicate that the cell-based assay we developed is a valid and effective method for examining the transactivating activity of ICP0. This assay can be used to identify cellular factors that regulate the transactivating activity of ICP0.