Novel Notch regulation in KSHV reactivation

KSHV 重新激活中的新型 Notch 调节

基本信息

批准号：
10053398
负责人：
Vivian Bellofatto
金额：
$ 23.4万
依托单位：
RBHS-NEW JERSEY MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2022-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10053398
关键词：
4-Hydroxy-Tamoxifen Abbreviations Address Alkaline Phosphatase Alleles Animals B-Lymphocytes Binding Binding Sites Biochemical Biological Biological Models CREB3 gene Cell Line Cells ChIP-seq Chronic Lymphocytic Leukemia Complement Factor B Complex DNA DNA Binding DNA Sequence DNA Viruses DNA-Binding Proteins DNA-Protein Interaction Data Differentiation and Growth Disease Drug Targeting Estrogen Receptors Estrogen receptor positive Family Family member Gene Expression Genes Genetic Transcription Genome Goals High-Throughput Nucleotide Sequencing Homeobox Hormones Human Human Herpesvirus 4 Human Herpesvirus 8 Human Pathology Infection Kaposi Sarcoma Light Literature Lymphoma Lytic Measures Modeling Molecular Notch Signaling Pathway Nuclear Nuclear Localization Signal Nuclear Translocation POU domain factors POU2F1 gene Pathogenesis Pathway interactions Phenotype Production Protein Binding Domain Protein Family Protein Isoforms Proteins Publishing Recombinants Regulation Reporter Scientific Advances and Accomplishments Series Signal Pathway Signal Transduction Signal Transduction Pathway Site Specific qualifier value Tetanus Helper Peptide Tetracyclines To specify Trans-Activators Transactivation Transcriptional Activation Domain Up-Regulation VP 16 Valproic Acid Vero Cells Viral Viral Genome Viral Proteins Virus chromatin immunoprecipitation diagnostic biomarker experimental study gain of function loss of function mutant next generation sequencing notch protein novel octamer transcription factor OTF-1 overexpression primary effusion lymphoma programs promoter protein K reactivation from latency response tissue culture transcription factor transcriptome transmission process viral DNA virus host interaction

项目摘要

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.

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了解与卡波西肉瘤相关的 DNA 病毒的持久性和复制至关重要。疱疹病毒 Rta 蛋白对于病毒从潜伏期中出现并复制（裂解 Rta 直接与称为 RBP-Jk 的细胞蛋白相互作用，这也是裂解反应所必需的。 RBP-Jk 通常指定将被细胞 Notch 信号转导激活的基因。通过这种方式，RBP-Jk 充当了 DNA 特异性结合序列的“着陆垫”。激活的 Notch 受体（Notch 胞内结构域 (NICD1)）刺激 RBP-Jk 的 DNA 结合。在病毒重新激活过程中，一种与已建立的规范机制根本不同的机制对于其他 RBP-Jk 激活蛋白，即 NICD1 和 Epstein-Barr 病毒 (EBV) EBNA-2，事实上，NICD1 是。无法刺激病毒完全重新激活，支持启动子特异性机制来控制其最近的数据表明，RBP-Jk 的 DNA 结合受到正向调节。 RBP-Jk 的 DNA 结合调节是一种新的水平。 Notch 通路的调节在文献中一直被低估。该应用的目的是确定在 KSHV 感染中调节 RBP-Jk DNA 结合的基本分子机制细胞，并确定支持病毒重新激活的转录重编程。由启动子决定的生产性和非生产性病毒再激活的基本调节特异性反式激活。因此，我们将实现以下具体目标：目标 1. 确定 KSHV 重新激活期间新型宿主蛋白是否刺激 Jk 与病毒启动子结合。目标 2. 确定特定的 MBP/Jk/DNA 复合物如何编程 Rta 和 Notch 依赖性重新激活。提出了一系列生物化学和分子生物学方法。代表许多实验的基础，并将针对过度表达或细胞蛋白（称为“基序结合蛋白”或 MBP）的功能性缺失会对病毒重新激活产生影响。使用新型、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。该提案将通过下一代测序来检测和测量其直接目标的新版本 Rta。揭示了Notch靶基因如何被指定用于反式激活，并揭示了Notch的新成分信号转导途径。