EXPRESSION AND REGULATION OF EBV DNA POLYMERASE

EB病毒DNA聚合酶基因及其调控是关键 不仅用于病毒的复制，而且还用于重新激活 从潜伏感染状态级联而来。我们是首席实验室 研究基因本身及其表达的调控。 长期目标是研究EBV Poll功能的遗传学和 耐药情况及EB病毒POL在其体内的表达 抑制和激活状态导致潜伏期和病毒感染 重新激活。 在过去两年的工作中，我们是第一个表明EBV BALF 5 ORF编码活性核心酶，在体外和在大肠杆菌中均显示 表达系统。2)我们已经通过以下方式绘制了该基因的mrna起始点 四种不同的方法，我们已经鉴定了EBV Poll启动子 表明它没有塔塔，但在构成上并不活跃。3)我们 也证实了EBV对启动子的反式激活 即刻--协同作用的早期基因。4)最后也是最多的 重要的是，在定位polmRNA的3‘端时，我们已经证明了 POL基因缺乏典型的多聚腺苷酸化信号。取而代之的是 这似乎是一种相当新的机制，与POL的聚腺苷酸化 通过一个似乎被识别的隐秘信号完成的mRNA 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，并建立在每个部分的基础上 这些领域，在第一部分集中在绘制催化结构域的 酶与耐药基因定位及耐药菌株的研究 长期以来一直被怀疑的EBV pol辅因子，BMRF-1的产物，被认为是 成为一个过程性因素。第二部分剖析了EBV POL启动子 为了定义顺式作用的调节元件和反式激活剂 需要激活这种通常沉默的基因。第三部分是 旨在定义新的 EB病毒Pol3‘非编码区和非编码区中的多聚腺苷化信号 定义了这个新函数所需的确切顺序。再加上 这项工作是为了证明EBV的早期反式作用 蛋白质，BMLF-1，我们已经证明了它在转录后发挥作用 可能通过稳定mRNA，靶向Poll消息，特别是 3‘端非编码区的序列。这项工作的最后一部分旨在定义 病毒信息的一种迄今未被识别的多聚腺苷酸化模式 以及病毒编码的基因产物，其可作为调节或 介导多聚腺苷酸化的加工蛋白。

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.