TRANSCRIPTIONAL ACTIVATION BY HERPESVIRUS VP16 PROTEIN

转录调控是许多生物学中的关键步骤 真核细胞中的过程，包括控制细胞生长和 组织的分裂、分化和器官的发育 对细胞外信号的反应。转录调控也是 对许多真核生物的遗传程序的表达至关重要 病毒，包括众所周知的SV40、腺病毒和 疱疹病毒。在我们努力理解如何启动 转录受调控，我们研究了单纯疱疹病毒的VP16蛋白 1型病毒(HSV-1)，已被广泛采用为 真核转录激活。 本研究支持的项目的长期目标 发展奖是对分子细节的理解，通过 VPI6通过宿主RNA激活病毒IE基因的转录 在本项目期间，我们将通过以下方式实现这一目标 五个具体目标。1.我们将确定VP16激活中的氨基酸添加 对其转录功能至关重要的结构域。我们有 已经确定了一些对肌动蛋白功能至关重要的氨基酸 VP16的一个亚区(残基413-456)。我们最近已经证明， 残基450-490构成一个独立的激活结构域， 利用了一种截然不同的氨基酸模式。我们将雇用工地- 定向诱变、丙氨酸扫描诱变和随机突变 利用遗传选择进行诱变以鉴定和进一步鉴定 VP16两个不同亚区的关键氨基酸 转录激活剂。2.我们将定义 VP16激活区及其各种可能的靶蛋白。 生化和遗传学方法已被用来鉴定推定的 转录激活剂的靶蛋白，包括碱基 转录因子TFIID(TATA结合蛋白，TBP和AN 相关的TAF蛋白)、THIIB和TFIIH；接头蛋白，如ADA2 和ADA3；甚至是已知它们在复制中的作用的蛋白质(例如， RPA)。我们将通过采用以下方法来描述这些交互 我们收集的VP16在生化、生物物理和遗传方面的突变 化验。3.我们会探讨该委员会的二级及三级架构 VP16激活域。之前的报告和未发布的结果显示 VPl6的激活结构域在溶液中基本上是非结构化的。 荧光和核磁共振光谱的初步结果表明 在假定目标存在的情况下，结构域可能变得更加有序 蛋白质。VPI6的结构将使用这些和相关的 方法，与研究人员合作，区分 在这些领域的声誉。4.我们将描述转录的 相关疱疹病毒VP16同源物的激活结构域。病毒 与HSV-1相关的是人类和农业上的重要病原体 重要的动物。我们使用了一种新的蛋白质序列分析方法 鉴定VP16同源物中可能的转录激活结构域 从这些病毒中。ORF10基因产物的初步观察 与杰弗里·科恩和杰弗里·科恩合作 同事们，已经验证了这种方法。我们将把此方法应用于 鉴定和鉴定其他同系物的活化域。 5.我们将评估VP16在转录激活过程中的作用 裂解感染过程。VP16的转录激活已经被 初步研究使用VP16基因和蛋白质 是从它们在病毒基因组和病毒粒子中的生物学位置被翻译出来的， 分别进行了分析。我们现在已经构建了携带有 部分或全部VP16激活域的缺失突变。我们会 检查这些突变对病毒生长的影响 培养，以及病毒IE基因在转录过程中的激活 培养物中的裂解性感染。

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. Transcriptional regulation is also critical to the expression of the genetic programs of many eukaryotic viruses, including the well-known examples of SV40, adenoviruses, and herpesviruses. In our effort to understand how the initiation of transcription is regulated, we study the VP16 protein of herpes simplex virus type 1 (HSV-1), which has been widely adopted as a paradigm for eukaryotic transcriptional activation. The long-term objective for the project supported by this Research Career Development Award is an understanding of the molecular details by which VPI6 activates transcription of the viral IE genes by the host RNA polymerase II. In this project period, we will pursue that objective with five specific aims. 1. We will identify amino adds in the VP16 activation domain that are critical for its transcriptional function. We have already identified a number of amino acids critical to the function of a subdomain of VP16 (residues 413-456). We have recently shown that residues 450-490 constitute an independent activation domain that utilizes a much different pattern of amino acids. We will employ site- directed mutagenesis, alanine-scanning mutagenesis, and random mutagenesis with genetic selection to identify and further characterize the critical amino acids for the two distinct subdomains of the VP16 transcriptional activator. 2. We will define the interactions between the VP16 activation domain and its various putative target proteins. Biochemical and genetic methods have been used to identify putative target proteins for transcriptional activators, including the basal transcription factors TFIID (both the TATA-binding protein, TBP, and an associated TAF protein), THIIB, and TFIIH; adaptor proteins, such as ADA2 and ADA3; and even proteins known for their role in replication (e.g., RPA). We will characterize a number of these interactions by employing our collection of VP16 mutations in biochemical, biophysical, and genetic assays. 3. We will explore the secondary and tertiary structure of the VP16 activation domain. Previous reports and unpublished results show that the activation domain of VPl6 is largely unstructured in solution. Preliminary results using fluorescence and NMR spectroscopy suggest that the domain may become more ordered in the presence of putative target proteins. The structure of VPI6 will be pursued using these and related methods, in collaboration with investigators having distinguished reputations in these fields. 4. We will characterize the transcriptional activation domains of VP16 homologs from related herpesviruses. Viruses related to HSV-1 are significant pathogens of humans and agriculturally important animals. We have used a novel protein sequence analysis method to identify putative transcriptional activation domains in VP16 homologs from these viruses. Preliminary observations on the ORF10 gene product of varicella-zoster virus, in collaboration with Jeffrey Cohen and colleagues, have validated this approach. We will apply this method to identify and characterize the activation domains of additional homologs. 5. We will evaluate the role of transcriptional activation by VP16 during the lytic infection process. Transcriptional activation by VP16 has been primarily investigated using methods in which the VP16 gene and protein are explanted from their biological sites in the viral genome and virion, respectively. We have now constructed recombinant viruses bearing deletion mutations of part or all of the VP16 activation domain. We will examine the effects of these mutations on the growth of the virus in culture, and upon the transcriptional activation of viral IE genes during lytic infection in culture.