LSUHSC COBRE: ALPHAHERPESVIRUS REPRESSOR PROTEIN

LSUHSC COBRE：阿尔法疱疹病毒抑制蛋白

• 批准号: 8359694
• 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: 2011
• 资助国家: 美国
• 起止时间: 2011-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8359694
• 关键词: Amino Acids; Biological; Biological Assay; Cells; Centers of Research Excellence; 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; Libraries; Mediating; Modeling; Molecular; National Center for Research Resources; Nuclear; Principal Investigator; Production; Property; Protein Binding; Proteins; Regulator Genes; Repressor Proteins; Research; Research Infrastructure; Resources; Serine; Source; TATA-Box Binding Protein; Testing; Transcription Factor TFIIB; Transfection; United States National Institutes of Health; Viral; Viral Genes; Virus; base; cost; genetic regulatory protein; human GTF2B protein; mutant; pathogen; programs; promoter; protein function; tumor; virology

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. 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.

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其他NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 子项目#3：确定EHV-1 IR 2蛋白抑制病毒基因表达和复制的机制 成金 马疱疹病毒1型（Equine herpesvirus 1，EHV-1）是马的重要病原体，是研究α疱疹病毒基因调控的重要模型，其基因程序由一个立即早期（immediate-early，IE）基因启动，该基因激活50个早期（early，E）基因的表达，包括E调控基因IR 2、UL 5、EICP 0和IR 4。独特的EHV-1 IR 2蛋白（IR 2 P）是立即早期蛋白（IEP）的1，165个氨基酸截短形式，缺乏IEP残基1至322，这些残基包含反式激活和病毒生长所必需的反式激活结构域。 IEP是多功能的，我们的IE突变体和17个IE突变病毒库使我们能够鉴定和表征EHV-1复制所必需的几个功能结构域：反式激活结构域（trans-activation domain，EHV-1）、富含丝氨酸的区域（serine-rich tract，SRT）、DNA结合结构域（DNA binding domain，DBD）、核定位序列（nuclear localization sequence，NLS）以及与其他EHV-1蛋白或细胞蛋白（包括转录因子TBP和TFIIB）相互作用的结构域。瞬时转染试验表明，早期调节IR 2 P本身下调IE启动子和所有早期启动子测试和废除激活的病毒启动子介导的IEP和早期调节蛋白UL 5 P在剂量依赖性的方式。IR 2 P与一般转录因子TFIIB和TBP物理相互作用。病毒生长测定显示，IR 2 P在马NBL 6细胞中抑制病毒产生高达90倍。基于这些发现，我们推测IR 2 P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，作为EHV-1基因表达的显性负调控因子发挥作用。我们的总体问题是IR 2 P如何抑制病毒基因表达和复制？在这项提案中，我们正在表征IR 2 P的生物学和分子特性，以确定IR 2 P抑制EHV-1基因表达和复制的机制。