TRANSCRIPTIONAL ACTIVATION BY HERPESVIRUS VP16 PROTEIN

疱疹病毒 VP16 蛋白的转录激活

• 批准号: 2671365
• 负责人: Steven J. Triezenberg
• 金额: $ 7.51万
• 依托单位: MICHIGAN STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-30 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2671365
• 关键词: Herpesviridae; X ray crystallography; alanine; chemical binding; eukaryote; fluorescence spectrometry; genetic transcription; molecular cloning; mutant; nuclear magnetic resonance spectroscopy; point mutation; protein structure; site directed mutagenesis; transcription factor; virus genetics; virus infection mechanism; virus protein; yeasts

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.

转录调控是许多生物学的关键步骤 真核细胞中的过程，包括细胞生长的控制和 组织的分裂、分化和器官的发育，以及 对细胞外信号的反应。转录调控也 对许多真核生物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。在我们努力了解如何启动 转录受到调控，我们研究了单纯疱疹病毒的VP16蛋白 1 型病毒 (HSV-1)，已被广泛采用作为范例 真核转录激活。 本研究生涯支持的项目的长期目标 发展奖是对分子细节的理解 VPI6 通过宿主 RNA 激活病毒 IE 基因的转录 聚合酶II。在本项目期间，我们将努力实现这一目标 五个具体目标。 1. 我们将鉴定VP16激活中的氨基 对其转录功能至关重要的结构域。我们有 已经鉴定出许多对功能至关重要的氨基酸 VP16 的一个子结构域（残基 413-456）。我们最近表明 残基 450-490 构成独立的激活结构域 利用非常不同的氨基酸模式。我们将雇用现场- 定向诱变、丙氨酸扫描诱变和随机诱变 通过遗传选择进行诱变以识别和进一步表征 VP16 两个不同子结构域的关键氨基酸 转录激活剂。 2. 我们将定义之间的相互作用 VP16 激活结构域及其各种假定的靶蛋白。 生化和遗传学方法已被用来鉴定假定的 转录激活因子的靶蛋白，包括基础蛋白 转录因子 TFIID（TATA 结合蛋白、TBP 和 相关 TAF 蛋白）、THIIB 和 TFIIH；接头蛋白，例如 ADA2 和 ADA3；甚至是以其在复制中的作用而闻名的蛋白质（例如， 机器人流程自动化）。我们将通过使用 我们收集的 VP16 生化、生物物理和遗传突变 化验。 3. 我们将探索其二级和三级结构 VP16激活域。之前的报告和未发表的结果显示 VPl6 的激活域在溶液中很大程度上是非结构化的。 使用荧光和核磁共振光谱的初步结果表明 在存在假定目标的情况下，域可能会变得更加有序 蛋白质。 VPI6的结构将利用这些和相关的 方法，与有区分的研究人员合作 在这些领域享有盛誉。 4. 我们将表征转录 来自相关疱疹病毒的 VP16 同源物的激活域。病毒 与 HSV-1 相关的病毒是人类和农业的重要病原体 重要的动物。我们使用了一种新颖的蛋白质序列分析方法 鉴定 VP16 同源物中假定的转录激活域 来自这些病毒。 ORF10基因产物的初步观察 与 Jeffrey Cohen 合作研究水痘带状疱疹病毒 同事们已经验证了这种方法。我们将把这个方法应用到 识别并表征其他同源物的激活域。 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 主要使用 VP16 基因和蛋白质的方法进行研究 从病毒基因组和病毒体中的生物位点移出， 分别。我们现在已经构建了带有 部分或全部 VP16 激活结构域的缺失突变。我们将 检查这些突变对病毒生长的影响 培养，并在病毒 IE 基因转录激活期间 培养物中的溶解性感染。