IMMEDIATE EARLY GENE REGULATION IN HERPES SIMPLEX VIRUS

这项研究的长期目标是了解基因 对1型单纯疱疹病毒的调控。更具体地说， 提案旨在探索病毒使用的策略，以 自动调节即刻早期基因(编码重要蛋白质 在早期/晚期基因的调控中)和检查顺式作用 网站在直接的早期推广者。当前的工具 生物化学、遗传学和分子生物学将被用于 检测两个序列特异的DNA结合蛋白：1)ICP4 由病毒编码，对病毒复制是必不可少的， 和；2)细胞DNA结合蛋白，识别序列 病毒立即早期增强子/启动子元件中的基序。这些 病毒和细胞编码蛋白将被提纯到均一状态，并 关于它们的DNA结合活性和 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”实验或其他功能分析)， 突变结合结构域中的关键残基以改变特定的 蛋白质/DNA相互作用，然后将突变引入细胞 以评估反应。还计划进行更多的实验 解决ICP4之间蛋白质/蛋白质相互作用的重要性 以及选定病毒启动子内的辅助转录因子 元素。ICP4实验的目的是阐明 ICP4激活和抑制某些基因的机制 其他。细胞实验的直接目标是 DNA结合蛋白决定了这种DNA的重要性 结合蛋白在遗传活性和即刻早期调控中的作用 启动子/增强子区。 这项工作意义重大，与病毒感染循环有关。 因为已知的直接早期基因在 病毒的裂解复制，代表生命中的一个时间点 病毒的一个循环，在这个循环中，人们可以控制 感染。即刻早期基因调控的研究进展 也将有助于设计预防反应的策略 病毒处于潜伏状态。

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.