LSUHSC COBRE: ALPHAHERPESVIRUS REPRESSOR PROTEIN

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 子项目3：确定EHV-1IR2蛋白抑制病毒基因表达和复制的机制 金胜坤 马疱疹病毒1型(EHV-1)是一种重要的马病原，是研究甲型疱疹病毒基因调控的有用模型，其基因程序由单个即刻早期(IE)基因启动，激活50个早期(E)基因的表达，包括E调节基因IR2、UL5、EICP0和IR4。独特的EHV-1IR2蛋白(IR2P)是1,487个氨基酸(AA)即刻早期蛋白(IEP)的截短形式，缺乏IEP残基1-322，该残基含有反式激活和病毒生长所必需的反式激活结构域(TAD)和富含丝氨酸的链(SRT)。IEP是多功能的，我们的IE突变体库和17个IE突变病毒库使我们能够识别和表征EHV-1复制所必需的几个功能结构域：反式激活结构域(TAD)、富丝氨酸链(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以及EAP蛋白。瞬时转染实验表明，早期调节IR2P单独下调IE启动子和所有早期启动子，并以剂量依赖的方式抑制IEP和早期调节蛋白UL5P介导的病毒启动子的激活。IR2P与一般转录因子TFIIB和TBP发生物理相互作用。病毒生长分析表明，IR2P可抑制马NBL6细胞中病毒的产生，最高可达90倍。在这些发现的基础上，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的有限供应来发挥EHV-1基因表达的显性-负性调节作用。我们的总体问题是IR2P是如何抑制病毒基因表达和复制的？在这项研究中，我们对IR2P的生物学和分子特性进行了研究，以确定IR2P抑制EHV-1基因表达和复制的机制(S)。

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 truncated form of the 1,487 amino acid (aa) immediate-early protein (IEP) and lacks IEP residues 1 to 322 that harbor the trans-activation domain (TAD) and serine-rich tract (SRT) 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 and the EAP protein. 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.