TRANS-ACTIVATED EXPRESSION OF HSV IMMEDIATE EARLY GENES

转录调控是许多生物过程中的关键步骤 在真核细胞中，包括控制细胞生长和分裂， 组织的分化和器官的发育， 细胞外信号和环境条件。转录 监管也是至关重要的时间协调表达 许多真核病毒的遗传程序，包括SV 40， 腺病毒、HIV和疱疹病毒。长期目标是 项目是了解转录的分子机制， activation.单纯疱疹病毒的溶解性感染是一种有用的 模型系统，因为HSV病毒体的组分 （称为VP 16）特异性激活病毒立即- 早期基因VP 16的激活结构域是已知的最有效的， 因此被广泛采用作为研究 转录激活 这个项目已经对建立 转录激活结构域的结构及其相互作用 与转录机制中的假定靶蛋白质结合。一 特别的优势是使用了各种技术， 从遗传学到荧光光谱学来检验特定的假设。的 下一个项目期间的具体目标将继续适用于不同的 方法探讨VP 16激活细胞的机制， 这种蛋白质在病毒体和病毒感染周期中的作用。现场指导， 将使用系统和随机突变方法来鉴定和 表征VP 16激活结构域中的关键氨基酸 它的转录功能。VP 16与 靶蛋白和这些相互作用的功能后果将 使用生化分析进行探索。将引入VP 16突变 进入病毒基因组，以评估转录激活的作用， 在裂解感染期间被VP 16感染。磷酸化和细胞核 VP 16作为病毒体蛋白的定位及其与其它蛋白的相互作用 将涉及病毒体蛋白。最后，具体的假设将 关于转录激活的身份和特征的测试 VP 16同源物的结构域， 兽医的重要性 从该项目中获得的信息将直接关系到 理解RNA聚合酶II通过其他调节因子的转录激活 介导上述广泛生物过程的蛋白质。 该项目还将提供必要的知识，以探索抗病毒 人用和兽用以及HSV开发的策略 载体作为选择性基因治疗的试剂。

The regulation of transcription is a key step in many biological processes in eukaryotic cells, including control of cell growth and division, differentiation of tissues and development of organs, and response to extracellular signals and environmental conditions. Transcriptional regulation is also critical to the temporally-coordinated expression of the genetic programs of many eukaryotic viruses, including SV40, adenoviruses, HIV, and herpesviruses. The long-term objective of this project is to understand the molecular mechanisms of transcriptional activation. Lytic infection by herpes simplex virus represents a useful model system for this objective, in that a component of the HSV virion (termed VP16) specifically activates transcription of the viral immediate- early genes. The activation domain of VP16 is among the most potent known, and thus has been widely adopted as a model for the study of transcriptional activation. This project has already made important contributions to models of the structure of transcriptional activation domains and their interactions with putative target proteins in the transcriptional machinery. A particular strength has been the use of techniques ranging from yeast genetics to fluorescence spectroscopy to test specific hypotheses. The specific aims for the next project period will continue to apply diverse methods to explore the mechanism of activation by VP16, and the role of this protein in the virion and the viral infectious cycle. Site-directed, systematic, and random mutational methods will be used to identify and characterize amino acids in the VP16 activation domain that are critical for its transcriptional function. The physical interactions of VP 16 with target proteins and the functional consequences of those interactions will be explored using biochemical assays. VP16 mutations will be introduced into the viral genome to evaluate the role of transcriptional activation by VP16 during lytic infection. The phosphorylation and nuclear localization of VP16 as a virion protein and its interaction with other virion proteins will be addressed. Finally, specific hypotheses will be tested regarding the identity and character of transcriptional activation domains of VP16 homologs from other alphaherpesviruses of medical and veterinary importance. The information gained from this project will be directly relevant to understanding transcriptional activation of RNA pol II by other regulatory proteins that mediate the wide range of biological processes noted above. This project will also provide knowledge necessary to explore antiviral strategies for human and veterinary use and for exploitation of HSV vectors as agents for selective gene therapy.