TRANSCRIPTIONAL ACTIVATION BY HERPESVIRUS VP16 PROTEIN

转录调控是许多生物学过程中的关键步骤， 真核细胞中的过程，包括细胞生长的控制， 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 转录调控，我们研究了单纯疱疹病毒VP16蛋白 1型病毒（HSV-1），其已被广泛采用作为 真核转录激活。 本研究职业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激活中的氨基添加物， 这些结构域对于其转录功能至关重要。我们有 已经确定了一些氨基酸的功能至关重要， VP16的亚结构域（残基413 - 456）。我们最近的研究表明 残基450 - 490构成独立的激活结构域， 利用了不同的氨基酸模式我们将在现场- 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鉴定和进一步表征 VP16两个不同亚结构域的关键氨基酸 转录激活因子2.我们将定义 VP16激活结构域及其各种推定的靶蛋白。 生物化学和遗传学方法已被用于鉴定推定的 转录激活因子的靶蛋白，包括基础 转录因子TFIID（TATA结合蛋白TBP和TFIID） 相关的TAF蛋白）、THIIB和TFIIH;衔接蛋白，如ADA 2 和ADA3;甚至已知在复制中起作用的蛋白质（例如， RPA）。我们将通过使用 我们收集了VP16在生物化学、生物物理学和遗传学方面的突变， 测定。3.我们将探讨的二级和三级结构的 VP16激活结构域。先前的报告和未发表的结果显示， VP16的激活结构域在溶液中大部分是非结构化的。 使用荧光和NMR光谱的初步结果表明， 在假定的靶分子存在时， proteins. VPI 6的结构将使用这些和相关的 方法，与研究人员合作， 在这些领域的声誉。4.我们将描述转录 来自相关疱疹病毒的VP16同源物的激活结构域。病毒 与HSV-1相关的病毒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同源物中假定的转录激活结构域 从这些病毒。ORF10基因产物的初步观察 水痘带状疱疹病毒，与杰弗里科恩和 同事们已经验证了这种方法。我们将把这种方法应用于 识别和表征其他同源物的激活结构域。 5.我们将评估VP16在转录激活过程中的作用。 裂解性感染过程。VP16的转录激活作用已经被证实。 使用VP16基因和蛋白质 从它们在病毒基因组和病毒体中的生物学位点分离， 分别我们现在已经构建了携带 VP16激活结构域的部分或全部的缺失突变。我们将 研究这些突变对病毒生长的影响， 培养，并在病毒IE基因的转录激活过程中， 在培养物中裂解感染。

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.