REGULATION OF EBV ORI-LYT BY CELLULAR AND VIRAL PROTEINS

细胞和病毒蛋白对 EBV Ori-LYT 的调节

• 批准号: 5206918
• 负责人: KENNEY C SHANNON
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5206918
• 关键词: DNA binding protein; DNA directed DNA polymerase; DNA footprinting; DNA replication; DNA replication origin; Epstein Barr virus; epitope mapping; gel mobility shift assay; genetic enhancer element; genetic promoter element; genetic regulation; genetic transcription; host organism interaction; immunoprecipitation; latent virus infection; protein structure function; site directed mutagenesis; tissue /cell culture; transcription factor; tumor suppressor genes; virus DNA; virus genetics; virus infection mechanism; virus protein; virus replication

Epstein-Barr virus (EBV) infection has been associated with the development of several types of human malignancy. The horizontal transmission of EBV from host to host requires activation of the lytic origin of replication, ori-Lyt. In contrast, transformation of cells by EBV is generally associated with latent viral infection and may require that lytic replication be suppressed. Both of the EBV immediate-early transcriptional activators, BZLF1 (Z) and BRLF1 (R), are required for ori-Lyt replication. However, it is not known whether the role of Z and R is limited to transcriptional activation of ori-Lyt or whether one or both of these proteins also plays a more basic role in replication. In addition, the regulation of ori-Lyt replication by cellular factors has not been well studied. In this grant, we propose to study the regulation of ori-Lyt by viral and cellular factors. We hypothesize that the regulation of ori-Lyt replication plays a role in determining the efficiency of productive EBV infection in different cell types. We have recently shown that Z may interact directly with the viral polymerase processivity factor (BMRF1), suggesting a mechanism by which Z could direct the polymerase complex to ori-Lyt. We have shown that Z can also interact directly with p53 and that this interaction may inhibit replication. We have also obtained preliminary data suggesting that three cellular factors (Spl, NF-Y,and zif268) can bind to essential regions of ori-Lyt and potentially regulate its replicative function. We propose the following specific aims: 1) To examine the role of Z and R in ori-Lyt replication, we will compare the effect of deleting Z and R binding sites in ori-Lyt upon transcription versus replication and will attempt to create mutant Z and R proteins which can activate transcription but not support replication. 2) To determine the role of cellular transcription factors in ori-Lyt replication, we will use a variety of techniques to determine which proteins bind to the known essential sites of ori-Lyt in different cell types and will examine the effect of deleting specific transcription factor binding sites upon ori-Lyt replication versus transcription. 3) To examine the effect of p53 upon ori-Lyt replication, we will determine the location of p53 binding sites in ori-Lyt, examine the effect of mutating these sites in different cell types, and determine if mutant Z which no longer interacts with p53 is more (or less) efficient in supporting ori-Lyt replication. 4) We will construct mutations in the intact virus which eliminate the ability of Z to support ori-lyt replication (but still allow transactivation) and determine how these mutations affect the phenotype in vivo.

EB病毒（EBV）感染与发展有关 几种人类恶性肿瘤 EB病毒的水平传播 从宿主到宿主需要激活裂解性复制起点， 奥利-莱特 相比之下，EBV对细胞的转化通常是不可逆的。 与潜伏性病毒感染相关，可能需要溶解 复制被抑制。 EB病毒立即早期转录 ori-Lyt复制需要激活剂BZLF 1（Z）和BRLF 1（R）。 然而，目前尚不清楚Z和R的作用是否仅限于 ori-Lyt的转录激活，或者这些基因之一或两者 蛋白质在复制中也起着更基本的作用。 此外该 细胞因子对ori-Lyt复制的调节作用还不是很好 研究了 在这项资助中，我们计划研究病毒和病毒对ori-Lyt的调控， 细胞因子 我们假设ori-Lyt的调节 复制在决定生产性EBV的效率中起作用， 感染不同类型的细胞。 我们最近发现，Z可能 直接与病毒聚合酶合成因子（BMRF 1）相互作用， 这表明了一种机制，通过这种机制，Z可以指导聚合酶复合物 奥利-莱特 我们已经证明Z也可以直接与p53相互作用， 这种相互作用可以抑制复制。 我们亦得到 初步数据表明，三种细胞因子（Spl，NF-Y，和 zif 268）可以结合ori-Lyt的必需区域并潜在地调节 它的复制功能 我们提出以下具体目标：1）考察Z和R的作用 在ori-Lyt复制中，我们将比较删除Z和R的效果 ori-Lyt在转录与复制时的结合位点， 试图创造突变的Z和R蛋白，可以激活转录 但不支持复制。 2)为了确定细胞的作用， 在ori-Lyt复制的转录因子，我们将使用各种 技术来确定哪些蛋白质结合到已知的必需位点， ori-Lyt在不同的细胞类型，并将检查删除的效果 ori-Lyt复制时特异性转录因子结合位点与 转录。 3)为了检测p53对ori-Lyt复制的影响， 我们将确定ori-Lyt中p53结合位点的位置， 在不同细胞类型中突变这些位点的效果，并确定 不再与p53相互作用的突变体Z在以下方面更有效（或更不有效）： 支持ori-Lyt复制。 4)我们将构建突变， 消除Z支持ori-lyt能力的完整病毒 复制（但仍然允许反式激活），并确定这些 突变影响体内的表型。