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）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同种型1的细胞蛋白相互作用，其也是裂解再活化所需的。RBP-Jk 1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 1作为活化的Notch受体（Notch胞内结构域（NICD 1））的“着陆垫”。HHV-8 Rta在病毒再活化过程中促进RBP-Jk 1的DNA结合，这一机制与其他RBP-Jk活化蛋白（NICD 1和EB病毒（EBV）EBNA-2）建立的经典机制根本不同。然而，NICD 1诱导24个HHV-8基因的表达，而不刺激完全的病毒再活化，支持启动子特异性机制控制其在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结合受到HHV-8再活化信号的正向和负向调节。RBP-Jk的DNA结合的调节是Notch途径的新水平的调节，其在文献中未被充分认识。本申请的总体目标是确定调节HHV-8感染细胞中RBP-Jk DNA结合的基本分子机制，并确定病毒对Rta或其他Notch途径激活剂的差异转录应答。我们的研究将解释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节。因此，我们将讨论这些具体目标：目标1。确定非典型RBP-Jk亚型是否参与Rta和NICD 1反式激活和HHV-8再激活。目标二。从HHV 8感染的B细胞中鉴定、定量和克隆其他RBP-Jk亚型。提出了一系列生物化学和分子生物学方法。蛋白质-蛋白质和蛋白质- DNA相互作用是许多实验的基础。将在RBP-Jk无效B细胞中使用新型、高度定量的HHV-8报告病毒定量对病毒再活化的影响。该项目的主要部分涉及使用下一代测序对RBP-Jk转录组进行全面组装和定量。这一提议将阐明Notch靶基因是如何被指定为反式激活的，并揭示Notch信号转导途径的新组成部分。

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.