EXPRESSION AND REGULATION OF EBV DNA POLYMERASE

EBV DNA 聚合酶的表达和调控

• 批准号: 3201063
• 负责人: JOSEPH S PAGANO
• 金额: $ 21.22万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-06-23 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3201063
• 关键词: DNA directed DNA polymerase; Epstein Barr virus; gel mobility shift assay; gene deletion mutation; gene expression; genetic mapping; genetic promoter element; genetic regulation; genetic regulatory element; genetic transcription; laboratory rabbit; messenger RNA; nucleic acid sequence; polyadenylate; posttranscriptional RNA processing; site directed mutagenesis; transcription factor; virus antigen

The Epstein-Barr Virus DNA polymerase gene and its regulation are pivotal not only for replication of the virus, but also in the reactivation cascade from the latent infection state. We are the principal laboratory studying both the gene itself and regulation of its expression. Long-term objectives are to work out the genetics of EBV pol function and drug resistance and to study how expression of the EBV pol in its suppressed and activated states contributes to latency and viral reactivation. In work done in the past 2 years we are the first to show 1) that the EBV BALF 5 ORF encodes active core enzyme, shown both in vitro and in E. coli expression systems. 2) We have mapped the mRNA start-site for the gene by four different methods, and we have identified the EBV pol promoter showing that it is TATA-less and yet not constitutively active. 3) We have also demonstrated transactivation of the promoter by EBV immediate-early genes that act synergistically. 4) Finally and most importantly, in mapping the 3'end of the pol mRNA, we have shown that the pol gene lacks a canonical polyadenylation signal. In its place there appears to be a quite novel mechanism with polyadenylation of the pol mRNA accomplished through a cryptic signal that appears to be recognized by a virally encoded or induced replicative function. The proposed work is organized into three parts and builds on each of these areas, focusing in part one on mapping of catalytic domains of the enzyme and mapping of drug-resistance loci as well as study of a long-suspected EBV pol cofactor, the BMRF-1 product, which is thought to be a processivity factor. The second part dissects the EBV pol promoter in order to define cis-acting regulatory elements and the transactivators needed to activate this ordinarily silent gene. The third part is directed at defining the structure and sequence of the new polyadenylation signal contained in the EBV pol mRNA 3'UTR and in defining the exact sequence needed for this new function. Coupled to this work are experiments to show whether an EBV early trans-acting protein, BMLF-1, which we have already shown acts post-transcriptionally perhaps by stabilizing mRNA, targets the pol message, specifically the sequence in the 3'UTR. This last part of the work is designed to define both a hitherto unrecognized mode of polyadenylation of viral messages as well as a virally encoded gene product which may act as a regulatory or processing protein that mediates polyadenylation.

Epstein-Barr病毒DNA聚合酶基因及其调控是关键 不仅用于病毒的复制， 从潜伏感染状态级联而来 我们是主要的实验室 研究基因本身及其表达的调控。 长期目标是研究EBV pol功能的遗传学， 研究EB病毒pol在其耐药性中的表达， 抑制和激活状态有助于潜伏期和病毒 重新激活 在过去2年的工作中，我们首次证明1）EBV BALF 5 ORF编码活性核心酶，在体外和E.杆菌 表达系统。2)我们已经绘制了该基因的mRNA起始位点， 四种不同的方法，我们已经确定了EBV pol启动子 这表明它是TATA较少的，但不是组成型活性的。3)我们 也证实了EBV对启动子的反式激活作用 协同作用的早期基因。4)最后也是最 重要的是，在绘制pol mRNA的3 '末端时，我们已经表明， pol基因缺乏典型的多聚腺苷酸化信号。 在它的位置上 这似乎是一个相当新颖的机制， mRNA通过一个隐藏的信号完成， 通过病毒编码或诱导的复制功能。 拟议的工作分为三个部分，并建立在每一个 这些领域，第一部分侧重于绘制 酶和耐药基因座定位以及研究 长期怀疑的EBV pol辅因子，BMRF-1产物，被认为是 是一个持续性因素。 第二部分剖析了EBV pol启动子 为了确定顺式作用调节元件和反式激活因子， 需要激活这个通常沉默的基因。 第三部分是 旨在定义新的结构和顺序， 多聚腺苷酸化信号包含在EBV pol mRNA 3 'UTR和 定义这个新功能所需的确切序列。 耦合到 这项工作是实验，以显示是否EBV早期反式作用， 我们已经证明，BMLF-1蛋白在转录后发挥作用 也许通过稳定mRNA，瞄准pol信息，特别是 在3 'UTR中的序列。 这最后一部分工作的目的是定义 病毒信息的多聚腺苷酸化是一种迄今未被认识的模式， 以及病毒编码的基因产物，其可以作为调节或 介导多聚腺苷酸化的加工蛋白质。