Novel Notch Pathways Govern HHV-8 Reactivation

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转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。人疱疹病毒-8 (HHV-8) Rta蛋白是病毒从潜伏期出现和复制（裂解性再激活）所必需和充分的。Rta直接与称为RBP-Jk异构体1的细胞蛋白相互作用，这也是裂解再激活所必需的。RBP-Jk1通常通过与DNA特异性结合序列指定将被细胞Notch信号转导通路激活的基因。以这种方式，RBP-Jk1作为激活的Notch受体（Notch胞内结构域（NICD1））的“着陆点”。HHV-8 Rta在病毒再激活过程中促进RBP-Jk1的DNA结合，这一机制与其他rbp - jk激活蛋白NICD1和eb病毒EBNA-2的典型机制有着根本的不同。然而，NICD1诱导24个HHV-8基因的表达，而不刺激完全的病毒再激活，支持启动子特异性机制来控制其在HHV-8感染细胞中的活性。最近的数据表明，多种RBP-Jk亚型的DNA结合在响应HHV-8再激活信号时受到正向和负向调节。RBP-Jk的DNA结合调节是Notch通路的一个新的调控水平，在文献中一直被低估。本应用程序的总体目标是确定在HHV-8感染细胞中调节RBP-Jk DNA结合的基本分子机制，并确定病毒对Rta或其他Notch通路激活剂的差异转录反应。我们的研究将解释由启动子特异性转激活决定的产生性和非产生性病毒再激活的基本规则。因此，我们将处理这些具体目标：目标1。确定非典型RBP-Jk亚型是否参与Rta和NICD1的转激活以及HHV-8的再激活。目标2。从hhv8感染的B细胞中鉴定、定量和克隆额外的RBP-Jk亚型。提出了一系列的生化和分子生物学方法。蛋白质-蛋白质和蛋白质- DNA的相互作用是许多实验的基础。对病毒再激活的影响将在RBP-Jk缺失的B细胞中使用一种新的、高定量的HHV-8报告病毒进行定量。该项目的主要部分包括使用下一代测序对RBP-Jk转录组进行全面组装和定量。这一建议将阐明Notch靶基因是如何被指定用于转激活的，并揭示Notch信号转导途径的新成分。

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) 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 isoform 1, which is also required fo lytic reactivation. RBP-Jk1 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-Jk1 serves as a "landing pad" for the activated Notch receptor (Notch intracellular domain (NICD1)). HHV-8 Rta promotes DNA binding of RBP-Jk1 during viral reactivation, a mechanism that is fundamentally different from the canonical mechanism established for other RBP-Jk-activating proteins, NICD1 and Epstein-Barr Virus (EBV) EBNA-2. However, NICD1 induces expression of 24 HHV-8 genes without stimulating complete viral reactivation, supporting a promoter-specific mechanism for controlling its activity in HHV-8 infected cells. Recent data suggest that DNA binding of multiple RBP-Jk isoforms is regulated both positively and negatively in response to HHV-8 reactivation signals. Modulation of DNA binding of RBP-Jk is a novel level of regulation of the Notch pathway that has been underappreciated in the literature. The overall goal of this application is to define the basic molecular mechanisms that regulate RBP-Jk DNA binding in HHV-8 infected cells, and determines the differential transcriptional response of the virus to Rta or to other Notch pathway activators. Our studies will explain the fundamental regulation of productive and non-productive virus reactivation as determined by promoter-specific transactivation. We will therefore address these Specific Aims: Aim 1. To determine whether non-canonical RBP-Jk isoforms participate in Rta and NICD1 transactivation and HHV-8 reactivation. Aim 2. Identify, quantitate, and clone additional RBP-Jk isoforms from HHV8-infected B cells. A series of biochemical and molecular biological approaches are proposed. Protein-protein and protein- DNA interactions represent the basis for many of the experiments. Effects on viral reactivation will be quantitated using a novel, highly quantitative, HHV-8 reporter virus in RBP-Jk null B cells. A major part of the project involves a comprehensive assembly and quantitation of the RBP-Jk transcriptome using next generation sequencing. This proposal will shed light on how Notch target genes are specified for transactivation, and reveal new components of the Notch signal transduction pathway.