Novel Notch regulation in KSHV reactivation

KSHV 重新激活中的新型 Notch 调节

• 批准号: 10198741
• 负责人: Vivian Bellofatto
• 金额: $ 19.63万
• 依托单位: RBHS-NEW JERSEY MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10198741
• 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; 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; transcriptional reprogramming; transcriptome; transmission process; viral DNA; virus host interaction

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 定义调节基因特异性反式激活的病毒与其宿主之间的分子相互作用 对于理解DNA病毒持久性和复制至关重要。卡波西与肉瘤相关 疱疹RTA蛋白是必需的，足以使病毒从潜伏期和复制中出现（裂解） 重新激活）。 RTA与称为RBP-JK的细胞蛋白直接相互作用，裂解反应也需要 外国。 RBP-JK通常指定将通过细胞Notch信号转移激活的基因 通过结合序列特异性与DNA的途径。以这种方式，RBP-JK充当了 活化的Notch受体（Notch细胞内结构域（NICD1））。 KSHV RTA刺激RBP-JK的DNA结合 在病毒重新激活期间，一种与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。确实，NICD1是 无法刺激完全病毒重新激活，支持启动子特异性机制控制其 在KSHV感染细胞中的活性。最近的数据表明，RBP-JK的DNA结合都受到了积极的调节 并且对KSHV重新激活信号有负面影响。 RBP-JK的DNA结合的调节是一个新水平 在文献中未被低估的缺口途径的调节。总体目标 应用是定义调节KSHV感染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。我们的研究将解释 启动子确定的生产性和非生产性病毒重新激活的基本调节 特定的反式激活。 因此，我们将解决这些具体目标： 目标1。确定新型宿主蛋白是否在KSHV重新激活过程中刺激JK与病毒启动子的结合。 AIM 2。确定特定的MBP/JK/DNA复合物如何RTA和Notch依赖性重新激活。 提出了一系列生化和分子生物学方法。蛋白-DNA相互作用 代表许多实验的基础，将根据过表达或 细胞蛋白的功能缺失（称为“基序结合蛋白”或Mbps）。对病毒重新激活的影响 使用新型的，高度定量的KSHV报告基因病毒进行定量。该项目的主要部分涉及使用 通过下一代测序来检测和测量其直接目标的RTA新颖版本。该提议将 阐明了如何指定档位基因进行反式激活的方式，并揭示了Notch的新组件 信号传输途径。