IMMEDIATE EARLY GENE REGULATION IN HERPES SIMPLEX VIRUS

这项研究的长期目标是了解基因 单纯疱疹病毒1型的遗传学研究 更具体而言是 该提案旨在探索病毒使用的策略， 自动调节立即早期基因（其编码重要蛋白质 在早期/晚期基因的调节），并检查顺式作用 立即早期启动子中的位点。 目前的工具 生物化学、遗传学和分子生物学将用于 检测两种序列特异性DNA结合蛋白：1）ICP4， 是由病毒编码的，是病毒复制所必需的， 和; 2）细胞DNA结合蛋白，其识别序列 病毒立即早期增强子/启动子元件中的基序。 这些 病毒和细胞编码的蛋白质将被纯化至同质， 特征在于它们的DNA结合活性， 蛋白质/蛋白质相互作用。 提案的中心主题 是在DNA序列水平上定义蛋白质接触位点 （通过"足迹"实验或其它功能测定）， 突变结合结构域中的关键残基以改变特异性结合结构域， 蛋白质/DNA相互作用，然后将突变引入细胞 来评估反应。 计划进行更多的实验， 解决蛋白质/蛋白质相互作用的重要性， 和选择的病毒启动子内的辅助转录因子 元素 ICP 4实验的目的是阐明 ICP4可以激活某些基因并抑制 他人 细胞实验的直接目标是 DNA结合蛋白是决定这种DNA的重要因素 结合蛋白在遗传活性和调控中的即时早期 启动子/增强子区域。 这项工作是有意义的和相关的病毒传染周期 因为已知直接早期基因在 病毒的裂解性复制代表生命中的一个点 病毒的周期，人们可以对病毒的结果施加控制， 感染 即早基因调控的研究 也将有助于制定战略，以防止反应的 把病毒从潜伏状态转移出来

The long term goal of this research is to understand gene regulation in herpes simplex virus type 1. MOre specifically, the proposal is designed to explore the strategy used by the virus to autoregulate immediate early gene (which encode proteins important in the regulation of early/late genes) and to examine cis-acting sites in the immediate early promoters. Current tools of biochemistry, genetics and molecular biology will be used to examine two sequence specific DNA binding proteins: 1) ICP4 which is encoded by the virus, and is essential for virus replication, and; 2) a cellular DNA binding protein, which recognizes sequence motifs in viral immediate early enhancer/promoter elements. These virus and cell coded proteins will be purified to homogeneity and characterized with regard to their DNA binding activity and protein/protein interactions. The central theme of the proposal is to define at the DNA sequence level, protein contact sites (either by "footprinting" experiments or other functional assays), mutate key residues in the binding domain to alter specific protein/DNA interaction and then introduce the mutation into cells to evaluate the response. Additional experiments are planned which address the importance of protein/protein interaction between ICP4 and ancillary transcription factors within selected viral promoter elements. The objective of the ICP4 experiments is to elucidate mechanisms by which ICP4 can activate some genes and repress others. The immediate objective of experiments with the cellular DNA binding protein is to determine the importance of this DNA binding protein in genetic activity and regulation immediate early promoter/enhancer regions. The work is significant and relevant to the viral infectious cycle because the immediate early genes are known to be essential in the lytic replication of the virus and represent a point in the life cycle of the virus where one can exert control over the outcome of the infection. Studies on the regulation of immediate early genes will also be useful in designing strategies to prevent reaction of the virus from a latent state.