TRANS-ACTIVATED EXPRESSION OF HSV IMMEDIATE EARLY GENES

在疱疹裂解感染周期的最初阶段 单纯病毒(HSV-1，一种广泛存在于人类的病原体)，转录 在病毒的即刻早期(IE)基因中， 被一种名为VP16的病毒粒子蛋白有效激活。《博大》 这项建议的目的是揭示他的机制的细节 这种反式激活。实现以下目标的初始策略 这一目标是为了识别顺式作用的DNA序列元件 它们对激活作出反应，并表征功能结构域 反式激活蛋白VP16。以前关于IE的工作 编码ICP4蛋白的基因显示存在两个 调节VP16作用的不同顺式反应元件。 然而，VP16并不直接与这些元件结合。相反， VP16的作用显然是由两种宿主细胞蛋白介导的 它们确实与两个顺式元件特别结合。数列 类似于ICP4顺式反应元件，可在 其他IE基因的调控区。一个假设是 在该提案中明确测试的是那些相关序列 也介导VP16的反式激活。假定的反应要素 将使用定点突变技术进行改变。突变质粒 将在瞬时表达分析中进行测试，以确定其能力 回应VP16。一个必然的假设是，宿主蛋白 绑定到ICP4顺式响应元件的元素也将绑定到 其他IE基因的反应元件。这一假设将得到检验。 通过DNase I足迹和凝胶迁移率变化分析 部分纯化的宿主DNA结合蛋白。以前的工作啊 也开始确定VP16激活的功能结构域 蛋白。酸性的羧基叔胺基结构域对 激活，并且很可能是连接到 转录机器。这项提议的一个具体目的是 确定单个氨基酸残基，这些氨基酸残基在 这一功能，使用的是VP16基因的定点突变。 VP16多胺蛋白与TOW宿主细胞相互作用的边界 DNA结合因子也已被确定。这项提议旨在 使用缺失和氨基酸替代突变来获得更多 清楚地定义这些蛋白质的结构域：蛋白质相互作用。 寄主因子的初步鉴定和纯化为 超出了其提议的范围，并正在积极推进 在其他实验室。然而，一旦确定，生化和 将利用免疫学技术直接调查 野生型突变型VP16蛋白与血管内皮细胞的相互作用 关联的宿主因素。

During the initial stages of lytic infectious cycle of herpes simplex virus (HSV-1, a widespread human pathogen), transcription of the viral immediate-early (IE) genes is specifically and potently activated by a virion protein termed VP16. The broad objective of this proposal is to uncover details of he mechanism of this trans-activation. The initial strategy for accomplishing this objective is to discern the cis-acting DNA sequence elements which respond to activation, and to characterize functional domains of the trans-activation proteins VP16. Previous work on the IE gene encoding the ICP4 protein revealed the presence of two distinct cis-response elements which mediate the action of VP16. However, VP16 does not bind directly to these elements. Instead, the action of VP16 is apparently mediated by two host cell proteins which do bind specifically to he two cis elements. Sequences resembling the ICP4 cis-response elements can be found in the regulatory regions of the other IE genes. One hypothesis to be explicitly tested in this proposal is that those related sequences also mediate VP16 trans-activation. Putative response elements will be altered using site-directed mutagenesis. Mutant plasmids will be tested in transient expression assays for th ability to respond to VP16. A corollary hypothesis is that the host proteins which bind to the ICP4 cis-response elements will also bind to the response elemens of other IE genes. This hypothesis will be tested by DNAse I footprinting and gel mobility shift assays using partially purified host DNA binding proteins. Previous work ahs also begun to identify functional domains of the VP16 activating protein. An acidic carboxyl termianl domain is critical for activaton, and is likely to be the physical link to the transcriptional machinery. A specific aim of this proposal is to identify individual amino acid residues which play major roles in this function, using site-directed mutagenesis of the VP16 gene. Boundaries of VP16 doamins which interact with the tow host cell DNA-binding factors have also been identified. This proposal seeks to use deletion and amino acid substitution mutagenesis to more clearly define the domains for these protein:protein interactions. The initial identification and purification of the host factors is beyond the scope of theis proposal, and is being actively pursued in other laboratories. Once identified, however, biochemical and immunological techniques will be employed to directly investigate the interactions of wildtype an mutant VP16 proteins with the associated host factors.