Re-specification of the Notch response by the HHV-8 lytic switch protein

描述(由申请人提供)：定义病毒与其宿主之间调节基因特异性反式激活的分子相互作用对于理解DNA病毒的持久性和复制至关重要。人类疱疹病毒-8(HHV-8)ORF50/RTA蛋白是病毒走出潜伏期并进行复制(裂解再激活)所必需的，也是充分的。RTA直接与称为RBP-JK的细胞蛋白相互作用，RBP-JK也是裂解重新激活所必需的。RBP-JK通常通过将序列特异性地与DNA结合来指定将被细胞Notch信号转导途径激活的基因。通过这种方式，RBP-JK作为激活的Notch受体(Notch胞内域(NICD))的“着陆垫”。HHV-8RTA在病毒再激活过程中促进RBP-JK的DNA结合，这一机制与其他RBP-JK激活蛋白的机制根本不同。我们的初步数据表明，在HHV-8感染细胞中，控制RBP-JK依赖活性的基因特异性机制。这项应用的总体目标是确定调控HHV-8感染细胞中RBP-JK依赖反式激活的基本分子机制。我们的研究将阐明启动子特异性反式激活所决定的生产性和非生产性病毒再激活的基本调控。因此，我们将全面鉴定与基本HHV-8启动子结合的蛋白质(目标1)。我们将定义RTA刺激RBP-JK DNA结合所需的分子相互作用(目标2)。我们将确定形成含有RBP-JK的功能性启动子复合体和HHV-8重新激活的分子要求(目标3)。人们提出了一系列的生化和分子生物学方法。对启动子特异的蛋白质-DNA相互作用进行生化筛选是一种主要的方法。启动子-报告、蛋白质-蛋白质和蛋白质-DNA相互作用是许多实验的基础，包括新颖的、高定量的体外和体内分析。这一建议将阐明Notch靶基因是如何被指定的，并揭示Notch信号转导途径的新组成部分。公共卫生相关性：该项目将促进对人类疱疹病毒-8(HHV-8)与人类相互作用致病机制的科学理解。具体地说，这些实验将揭示关键病毒蛋白RTA重新指定目标基因的机制，该机制受到与人类病理相关的细胞信号通路的刺激。独特的药物靶点和诊断标记可能会被揭示。

DESCRIPTION (provided by applicant): Defining the molecular interactions between a virus and its host that regulate gene-specific transactivation has been essential to understanding DNA virus persistence and replication. The Human herpesvirus-8 (HHV-8) ORF50/Rta protein is necessary and sufficient for the virus to emerge from latency and replicate (lytic reactivation). Rta interacts directly with the cellular protein called RBP-Jk, which is also required for lytic reactivation. RBP-Jk normally specifies the genes that will be activated by the cellular Notch signal transduction pathway by binding sequence specifically to DNA. In this fashion, RBP-Jk serves as a "landing pad" for the activated Notch receptor (Notch intracellular domain (NICD)). HHV-8 Rta promotes DNA binding of RBP-Jk during viral reactivation, a mechanism that is fundamentally different from that established for other RBP-Jk-activating proteins. Our preliminary data suggest a gene-specific mechanism for controlling RBP-Jk-dependent activity in HHV-8 infected cells. The overall goal of this application is to define the basic molecular mechanisms that regulate RBP-Jk dependent-transactivation in HHV-8 infected cells. Our studies will shed light on the fundamental regulation of productive and non-productive virus reactivation as determined by promoter-specific transactivation. We will therefore comprehensively identify proteins that bind to essential HHV-8 promoters (Aim 1). We will define the molecular interactions required for Rta stimulation of RBP-Jk DNA binding (Aim 2). We will determine the molecular requirements for forming functional RBP-Jk-containing promoter complexes and HHV-8 reactivation (Aim 3). A series of biochemical and molecular biological approaches are proposed. A biochemical screen for promoter-specific protein-DNA interactions is a major approach. Promoter-reporter, protein-protein and protein-DNA interactions represent the basis for many of the experiments, and include novel, highly quantitative in vitro and in vivo assays. This proposal will shed light on how Notch target genes are specified, and reveal new components of the Notch signal transduction pathway. PUBLIC HEALTH RELEVANCE: This project will advance scientific understanding of the mechanisms by which Human herpesvirus-8 (HHV- 8) interacts with Humans to cause disease. Specifically, the experiments will reveal mechanisms by which the key viral protein Rta re-specifies the target genes stimulated by a cellular signaling pathway that has been associated with Human pathologies. Unique drug targets and diagnostic markers may be revealed.