LSUHSC COBRE: ALPHAHERPESVIRUS REPRESSOR PROTEIN

LSUHSC COBRE：阿尔法疱疹病毒抑制蛋白

• 批准号: 8167464
• 负责人: Seong K Kim
• 金额: $ 20.99万
• 依托单位: LOUISIANA STATE UNIV HSC SHREVEPORT
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8167464
• 关键词: Amino Acids; Biological; Biological Assay; Cells; Computer Retrieval of Information on Scientific Projects Database; DNA Binding Domain; Dominant-Negative Mutation; Dose; Early Promoters; Equid Herpesvirus 1; Equus caballus; Funding; Gene Expression; Gene Expression Regulation; Genes; Grant; Growth; Immediate-Early Genes; Immediate-Early Proteins; Institution; Libraries; Mediating; Modeling; Molecular; Nuclear; Production; Property; Protein Binding; Proteins; Regulator Genes; Repressor Proteins; Research; Research Personnel; Resources; Serine; Source; TATA-Box Binding Protein; Testing; Transcription Factor TFIIB; Transfection; United States National Institutes of Health; Viral; Viral Genes; Virus; base; genetic regulatory protein; human GTF2B protein; mutant; pathogen; programs; promoter; protein function

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Subproject #3: Determine the mechanism by which the EHV-1 IR2 protein inhibits viral gene expression and replication Seong K. Kim Equine herpesvirus 1 (EHV-1) is an important pathogen of equine and a useful model to investigate Alphaherpesvirus gene regulation as its gene program is initiated by expression of a single immediate-early (IE) gene that activates expression of 50 early (E) genes which include E regulatory genes IR2, UL5, EICP0, and IR4. The unique EHV-1 IR2 protein (IR2P) is a 1,165-amino acid truncated form of the immediate-early protein (IEP) and lacks IEP residues 1 to 322 that harbor the trans-activation domain (TAD) essential for trans-activation and viral growth. IEP is multi-functional, and our libraries of IE mutants and 17 IE mutant viruses allowed us to identify and characterize several functional domains essential for EHV-1 replication: trans-activation domain (TAD), the serine-rich tract (SRT), DNA-binding domain (DBD), nuclear localization sequence (NLS), and domains that interact with other EHV-1 proteins or cell proteins, including transcription factors TBP and TFIIB. Transient transfection assays showed that the early regulatory IR2P by itself down-regulated the IE promoter and all early promoters tested and abrogated activation of viral promoters mediated by the IEP and the early regulatory protein UL5P in a dose-dependent manner. The IR2P physically interacted with the general transcription factors TFIIB and TBP. Virus growth assays revealed that the IR2P inhibited virus production by up to 90-fold in equine NBL6 cells. On the basis of these findings, we hypothesize that IR2P functions as a dominant-negative regulator of EHV-1 gene expression by blocking IEP-binding to viral promoter sequences and/or squelching the limited supplies of TFIIB and TBP. Our overall question is how does IR2P inhibit viral gene expression and replication? In this proposal, we are characterizing the biological and molecular properties of IR2P in order to define the mechanism(s) by which IR2P inhibits EHV-1 gene expression and replication.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 子项目#3：确定 EHV-1 IR2 蛋白抑制病毒基因表达和复制的机制 金成K 马疱疹病毒 1 (EHV-1) 是马的重要病原体，也是研究阿尔法疱疹病毒基因调控的有用模型，因为其基因程序是由单个立即早期 (IE) 基因的表达启动，该基因激活 50 早期 (E) 的表达基因，包括E调节基因IR2、UL5、EICP0和IR4。独特的 EHV-1 IR2 蛋白 (IR2P) 是立即早期蛋白 (IEP) 的 1,165 个氨基酸的截短形式，缺乏 IEP 残基 1 至 322，这些残基包含反式激活和反式激活所必需的反式激活结构域 (TAD)。病毒式增长。 IEP 是多功能的，我们的 IE 突变体和 17 种 IE 突变病毒文库使我们能够识别和表征 EHV-1 复制所必需的几个功能域：反式激活域 (TAD)、富含丝氨酸的区域 (SRT)、 DNA 结合结构域 (DBD)、核定位序列 (NLS) 以及与其他 EHV-1 蛋白或细胞蛋白相互作用的结构域，包括转录因子 TBP 和 TFIIB。瞬时转染测定表明，早期调节IR2P本身下调IE启动子，并且所有测试的早期启动子以剂量依赖性方式消除由IEP和早期调节蛋白UL5P介导的病毒启动子的激活。 IR2P 与通用转录因子 TFIIB 和 TBP 发生物理相互作用。病毒生长测定表明，IR2P 在马 NBL6 细胞中抑制病毒产生高达 90 倍。基于这些发现，我们假设 IR2P 通过阻断 IEP 与病毒启动子序列的结合和/或抑制 TFIIB 和 TBP 的有限供应，充当 EHV-1 基因表达的显性负调节因子。我们的总体问题是 IR2P 如何抑制病毒基因表达和复制？在本提案中，我们描述了 IR2P 的生物学和分子特性，以确定 IR2P 抑制 EHV-1 基因表达和复制的机制。