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病毒持久性和复制至关重要。人类疱疹病毒-8（HHV-8）ORF 50/Rta蛋白是病毒从潜伏期出现和复制（裂解再活化）所必需和充分的。Rta直接与称为RBP-Jk的细胞蛋白相互作用，这也是裂解再活化所需的。RBP-Jk通常通过特异性结合DNA的序列指定将被细胞Notch信号转导途径激活的基因。以这种方式，RBP-Jk充当活化的Notch受体（Notch胞内结构域（NICD））的“着陆垫”。HHV-8 Rta在病毒再活化过程中促进RBP-Jk的DNA结合，这一机制与其他RBP-Jk活化蛋白的机制根本不同。 我们的初步数据表明，在HHV-8感染的细胞中控制RBP-Jk依赖性活性的基因特异性机制。本申请的总体目标是确定在HHV-8感染的细胞中调节RBP-Jk依赖性反式激活的基本分子机制。我们的研究将阐明由启动子特异性反式激活决定的生产性和非生产性病毒再激活的基本调控。因此，我们将全面地鉴定与必需的HHV-8启动子结合的蛋白质（目的1）。我们将定义Rta刺激RBP-Jk DNA结合所需的分子相互作用（目的2）。我们将确定形成功能性RBP-Jk启动子复合物和HHV-8再活化的分子要求（目的3）。 提出了一系列生物化学和分子生物学方法。启动子特异性蛋白质-DNA相互作用的生物化学筛选是一种主要方法。启动子-报告子、蛋白质-蛋白质和蛋白质-DNA相互作用代表了许多实验的基础，并且包括新颖的、高度定量的体外和体内测定。这一提议将阐明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）与人类相互作用导致疾病的机制的科学理解。具体而言，这些实验将揭示关键病毒蛋白Rta重新指定由与人类病理学相关的细胞信号传导途径刺激的靶基因的机制。独特的药物靶点和诊断标志物可能会被发现。

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.