Generation of Herpes Viruses for in vivo Observation

用于体内观察的疱疹病毒的产生

• 批准号: 6559762
• 负责人: GERD G MAUL
• 金额: $ 8.21万
• 依托单位: WISTAR INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6559762
• 关键词: biological transport; biotechnology; confocal scanning microscopy; cytomegalovirus; fluorescent dye /probe; genetic techniques; green fluorescent proteins; herpes simplex virus 1; method development; recombinant virus; tissue /cell culture; video microscopy; virus genetics; virus infection mechanism

DESCRIPTION (provided by applicant): The effect of viruses on the host can be observed at different levels of complexity and resolution depending on the technique used. The single-cell observation combined with various labeling techniques has provided great insight into host-virus interactions; however, the observations are made on fixed (dead) infected cells. We propose to develop an in vivo virus genome labeling system that will allow observation of virus genomes in live cells. Visualization of a viral genome in vivo requires tagging its DNA sequence. The construction of a "green" genome is possible by labeling a DNA tag in the viral genome as it enters the nucleus or before packaging with green fluorescent protein (GFP). Cells inducibly producing the GFP-fusion protein to bind to the DNA tag will be generated to assemble a system where upon virus entry into the nucleus or during packaging the viral genome is rendered "green" through very tight binding of the DNA tag and the reporter protein. These genomes can be visualized by confocal microscopy and documented in a time-resolved fashion by time-lapse microscopy. We propose to produce DNA-tagged recombinant herpes simplex virus and mouse cytomegalovirus. To complete the system, we will develop cell lines that inducibly produce GFP-labeled DNA-binding protein, where HSV-1 and MCMV can replicate and which are useful as quiescent ("latent") virus containing cultured cell model systems. Such a new system would recognize single viral genomes directly in the live cells and obviate in situ hybridization. The "green" viral genomes will open up new lines of inquiry into the dynamics of virus entry into the nucleus, the sequence of degradation by endonucleases and/or retention in a nuclease-resistant episome, replication and segregation by multiple observation of single cells or populations during quiescence, affinity immune separation of quiescent viral genomes and identification of viral genomes by immunoelectron microscopy.

描述（由申请人提供）：可以根据所使用的技术观察到病毒对宿主的影响。单细胞观察与各种标记技术相结合，为宿主病毒相互作用提供了充分的见解。但是，观察结果是在固定（死）感染细胞上进行的。我们建议开发一个体内病毒基因组标记系统，该系统将允许观察活细胞中的病毒基因组。体内病毒基因组的可视化需要标记其DNA序列。通过标记病毒基因组中的DNA标签进入细胞核时或用绿色荧光蛋白（GFP）包装之前，可以通过在病毒基因组中标记DNA标签来构造“绿色”基因组。将产生与DNA TAG结合的诱导型细胞，以组装一个系统，该系统在病毒进入细胞核或包装过程中，通过非常紧密的DNA TAG和报告蛋白的结合使病毒基因组在包装过程中变得“绿色”。这些基因组可以通过共聚焦显微镜可视化，并通过延时显微镜以时间分辨的方式进行记录。我们建议产生DNA标记的重组疱疹病毒和小鼠巨细胞病毒。为了完成该系统，我们将开发出可诱导的GFP标记的DNA结合蛋白的细胞系，其中HSV-1和MCMV可以复制并且可作为静态（“潜伏”）病毒有用，其中含有培养的细胞模型系统。这样的新系统将直接在活细胞中识别单个病毒基因组并消除原位杂交。 The "green" viral genomes will open up new lines of inquiry into the dynamics of virus entry into the nucleus, the sequence of degradation by endonucleases and/or retention in a nuclease-resistant episome, replication and segregation by multiple observation of single cells or populations during quiescence, affinity immune separation of quiescent viral genomes and identification of viral genomes by immunoelectron显微镜。