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，包括控制细胞的生长和分裂， 组织分化和器官发育，以及对 胞外信号和环境条件。转录的 监管也是时间上协调表达的关键 许多真核病毒的遗传程序，包括SV40， 腺病毒、艾滋病毒和疱疹病毒。这样做的长期目标是 该项目是为了了解转录的分子机制 激活。单纯疱疹病毒的溶血性感染代表着一种有用的 这一目标的模型系统，因为HSV病毒粒子的一个组件 (称为VP16)特异性地激活病毒即时转录- 早期基因。VP16的激活结构域是已知的最有效的结构域之一， 因此被广泛地用作研究的模型。 转录激活。 该项目已经为模型的建立做出了重要贡献 转录激活结构域的结构及其相互作用 在转录机制中带有假定的目标蛋白。一个 尤其是使用了从酵母到酵母的各种技术 从遗传学到荧光光谱学来检验特定的假说。这个 下一个项目期的具体目标将继续适用于不同的 方法探讨VP16的激活机制及其作用机制。 这种蛋白质在病毒粒子和病毒感染循环中。以站点为导向， 将使用系统的和随机的突变方法来识别和 确定VP16激活区中关键氨基酸的特征 因为它的转录功能。VP16与VP16的物理相互作用 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。VP16突变将被引入 进入病毒基因组以评估转录激活的作用 在裂解感染过程中被VP16感染。磷酸化与核 VP16作为病毒粒子蛋白的定位及其与其他病毒粒子的相互作用 病毒粒子蛋白将被解决。最后，具体的假设将是 关于转录激活的身份和特征的测试 其他甲型疱疹病毒VP16同源物的结构域 兽医的重要性。 从这个项目中获得的信息将直接与 通过其他调节机制了解RNA PolII的转录激活 调节上述各种生物过程的蛋白质。 该项目还将提供探索抗病毒药物所需的知识。 人类和兽医使用以及开发单纯疱疹病毒的战略 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.