REGULATION OF EBV ORI-LYT BY CELLULAR AND VIRAL PROTEINS

爱泼斯坦-巴尔病毒(EBV)的感染与该病的发生有关 几种类型的人类恶性肿瘤。EBV的水平传播 从一个主机到另一个主机需要激活复制的裂解起点， 奥里-莱特。相比之下，EBV对细胞的转化通常是 与潜伏的病毒感染有关，并可能需要裂解 抑制复制。EB病毒的即刻-早期转录 Ori-Lyt复制需要激活剂BZLF1(Z)和BRLF1(R)。 然而，尚不清楚Z和R的作用是否仅限于 Ori-Lyt的转录激活或其中之一或两者 蛋白质在复制过程中也扮演着更基本的角色。此外， 细胞因子对Ori-Lyt复制的调控一直不是很好 学习。 在这项拨款中，我们建议研究病毒和病毒对Ori-Lyt的调控 细胞因素。我们假设Ori-Lyt的调节 复制在决定生产性EBV的效率中起着作用 不同细胞类型的感染。我们最近已经证明了Z可以 直接与病毒聚合酶加工因子(BMRF1)相互作用， 提示Z可以通过一种机制将聚合酶复合体引导到 奥里-莱特。我们已经证明Z也可以直接与P53相互作用，并且 这种相互作用可能会抑制复制。我们还获得了 初步数据表明，三种细胞因子(SPL、NF-Y和 Zif268)可以与Ori-Lyt的必需区域结合并潜在地调节 它的复制功能。 我们提出了以下具体目标：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中转录与复制时的结合位点和Will 尝试创造可激活转录的突变Z和R蛋白 但不支持复制。2)确定细胞的作用 在转录因子的Ori-Lyt复制中，我们将使用多种 确定哪些蛋白质与已知的关键部位结合的技术 Ori-Lyt在不同的细胞类型中的表达，并将检查删除 Ori-Lyt复制的特定转录因子结合位点与 抄写。3)为了检测p53对Ori-Lyt复制的影响， 我们将确定P53结合位点在Ori-Lyt中的位置，检查 在不同的细胞类型中突变这些位点的效果，并确定是否 不再与P53相互作用的突变体Z在 支持Ori-Lyt复制。4)我们将在 一种完整的病毒，它消除了Z对组织的支持能力 复制(但仍允许事务激活)，并确定这些 突变会影响体内的表型。

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.