Novel Notch regulation in KSHV reactivation

项目描述/摘要 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。卡波西肉瘤相关的 疱疹病毒Rta蛋白是病毒从潜伏期出现和复制（裂解）所必需和充分的 再活化）。Rta直接与称为RBP-Jk的细胞蛋白相互作用，RBP-Jk也是裂解反应所必需的。 vation。RBP-Jk通常指定将被细胞Notch信号转导激活的基因 通过特异性结合序列到DNA的途径。以这种方式，RBP-Jk充当用于该系统的“着陆垫”。 活化的Notch受体（Notch胞内结构域（NICD 1））。KSHV Rta刺激RBP-Jk的DNA结合 在病毒再活化过程中，一种与建立的典型机制根本不同的机制， 其他RBP-Jk激活蛋白，即NICD 1和EB病毒（EBV）EBNA-2。事实上，NICD 1 不能刺激完全的病毒再活化，支持启动子特异性机制来控制其 KSHV感染细胞的活性。最近的数据表明，RBP-Jk的DNA结合受到正向调节， 而对KSHV再激活信号的反应为阴性。RBP-Jk对DNA结合的调节是一个新的水平 Notch信号通路的调节在文献中被低估了。这个项目的总体目标是 应用是确定在KSHV感染中调节RBP-Jk DNA结合的基本分子机制， 细胞，并确定支持病毒再活化的转录重编程。我们的研究将解释 由启动子决定的生产性和非生产性病毒再活化的基本调节- 特异性反式激活 因此，我们将致力于实现这些具体目标： 目标1.确定在KSHV再活化过程中，新的宿主蛋白是否刺激Jk与病毒启动子结合。 目标2.确定特定MBP/Jk/DNA复合物如何编程Rta和Notch依赖性再激活。 提出了一系列生物化学和分子生物学方法。蛋白质-DNA相互作用 代表了许多实验的基础，并将响应于过表达或 细胞蛋白（称为“基序结合蛋白”或MBP）的功能缺失。对病毒再活化的影响将 使用新的、高度定量的KSHV报告病毒进行定量。该项目的一个主要部分涉及使用 通过下一代测序检测和测量其直接靶点的新型Rta。这项建议会 阐明了Notch靶基因是如何被指定为反式激活的，并揭示了Notch的新组成部分。 信号转导途径

Project Description/Abstract 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 Kaposi’s sarcoma-associated Herpesivrus 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 reacti- vation. 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 (NICD1)). KSHV Rta stimulates DNA binding of RBP-Jk during viral reactivation, a mechanism that is fundamentally different from the canonical mechanism established for other RBP-Jk-activating proteins, namely NICD1 and Epstein-Barr Virus (EBV) EBNA-2. Indeed, NICD1 is unable to stimulate complete viral reactivation, supporting a promoter-specific mechanism for controlling its activity in KSHV infected cells. Recent data suggest that DNA binding of RBP-Jk is regulated both positively and negatively in response to KSHV 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 KSHV infected cells, and determine the transcriptional reprogramming that supports viral reactivation. 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. Determine if novel host proteins stimulate Jk binding to viral promoters during KSHV reactivation. Aim 2. Determine how specific MBP/Jk/DNA complexes program Rta and Notch-dependent reactivation. A series of biochemical and molecular biological approaches are proposed. Protein-DNA interactions represent the basis for many of the experiments, and will be evaluated in response to overexpression or functional deletion of cellular proteins (termed ‘motif binding proteins’, or MBPs). Effects on viral reactivation will be quantitated using a novel, highly quantitative, KSHV reporter virus. A major part of the project involves using a novel version of Rta to detect and measure its direct targets by 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.