Re-specification of the Notch response by the HHV-8 lytic switch protein

HHV-8 裂解开关蛋白对 Notch 反应的重新特异性

• 批准号: 7765515
• 负责人: DAVID M LUKAC
• 金额: $ 38.61万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-02-15 至 2014-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7765515
• 关键词: Affinity Chromatography; Amino Acids; Animal Viruses; Architecture; Binding; Binding Proteins; Binding Sites; Biochemical; Biological; Biological Assay; Biological Models; Cells; Complex; DNA; DNA Binding; DNA Sequence; DNA Viruses; DNA-Binding Proteins; DNA-Protein Interaction; Data; Diagnostic; Disease; Drug Delivery Systems; EMSA; Elements; Enhancers; Gene Expression Profile; Gene Targeting; Genes; Genome; Goals; Human; Human Herpesvirus 4; Human Herpesvirus 8; Human Pathology; Hybridization Array; Hybrids; In Vitro; Infection; Light; Lytic; Mass Spectrum Analysis; Modeling; Molecular; Mutation; Notch Signaling Pathway; Oligonucleotide Microarrays; Pathway interactions; Protein Binding; Proteins; Regulation; Reporter; Repressor Proteins; Role; Scientific Advances and Accomplishments; Screening procedure; Series; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Specific qualifier value; Transactivation; Transcriptional Activation; Viral; Viral Proteins; Virus; Yeasts; base; cell growth; chromatin immunoprecipitation; genome-wide; in vivo; mutant; notch protein; novel; promoter; public health relevance; research study; response; tissue culture; transcription factor; virus host interaction

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) ORF50/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 reactivation. 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 (NICD)). HHV-8 Rta promotes DNA binding of RBP-Jk during viral reactivation, a mechanism that is fundamentally different from that established for other RBP-Jk-activating proteins. Our preliminary data suggest a gene-specific mechanism for controlling RBP-Jk-dependent activity in HHV-8 infected cells. The overall goal of this application is to define the basic molecular mechanisms that regulate RBP-Jk dependent-transactivation in HHV-8 infected cells. Our studies will shed light on the fundamental regulation of productive and non-productive virus reactivation as determined by promoter-specific transactivation. We will therefore comprehensively identify proteins that bind to essential HHV-8 promoters (Aim 1). We will define the molecular interactions required for Rta stimulation of RBP-Jk DNA binding (Aim 2). We will determine the molecular requirements for forming functional RBP-Jk-containing promoter complexes and HHV-8 reactivation (Aim 3). A series of biochemical and molecular biological approaches are proposed. A biochemical screen for promoter-specific protein-DNA interactions is a major approach. Promoter-reporter, protein-protein and protein-DNA interactions represent the basis for many of the experiments, and include novel, highly quantitative in vitro and in vivo assays. This proposal will shed light on how Notch target genes are specified, and reveal new components of the Notch signal transduction pathway. PUBLIC HEALTH RELEVANCE: This project will advance scientific understanding of the mechanisms by which Human herpesvirus-8 (HHV- 8) interacts with Humans to cause disease. Specifically, the experiments will reveal mechanisms by which the key viral protein Rta re-specifies the target genes stimulated by a cellular signaling pathway that has been associated with Human pathologies. Unique drug targets and diagnostic markers may be revealed.

描述（由申请人提供）：定义病毒与其宿主之间的分子相互作用来调节基因特异性反式激活对于理解DNA病毒持久性和复制至关重要。人类疱疹病毒8（HHV-8）ORF50/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，这也是裂解重新激活所必需的。 RBP-JK通常指定通过特异性结合与DNA结合的细胞缺口信号转导途径将激活的基因。以这种方式，RBP-JK充当激活的Notch受体（Notch细胞内结构域（NICD））的“着陆垫”。 HHV-8 RTA在病毒重生过程中促进RBP-JK的DNA结合，该机制与其他RBP-JK-JK激活蛋白的机制根本不同。 我们的初步数据提出了一种基因特异性机制，用于控制HHV-8感染细胞中RBP-JK依赖性活性。该应用的总体目标是定义调节HHV-8感染细胞中RBP-JK依赖性转移的基本分子机制。我们的研究将阐明由启动子特异性反式激活确定的生产性和非生产性病毒重新激活的基本调节。因此，我们将全面识别与必不可少的HHV-8启动子结合的蛋白质（AIM 1）。我们将定义RTA刺激RBP-JK DNA结合所需的分子相互作用（AIM 2）。我们将确定形成功能性RBP-JK启动子络合物和HHV-8重新激活的分子需求（AIM 3）。 提出了一系列生化和分子生物学方法。启动子特异性蛋白DNA相互作用的生化筛选是一种主要方法。启动子重孢子，蛋白质 - 蛋白质和蛋白-DNA相互作用代表了许多实验的基础，包括新型的，高度定量的体外和体内测定。该建议将阐明如何指定Notch靶基因，并揭示Notch信号转导途径的新组件。公共卫生相关性：该项目将进一步了解人类疱疹病毒8（HHV-8）与人类相互作用以引起疾病的机制。具体而言，这些实验将揭示关键病毒蛋白RTA重新指定的机制，通过与人类病理相关的细胞信号传导途径刺激的靶基因。可以揭示独特的药物靶标和诊断标记。