FUNCTIONS OF EPSTEIN-BARR VIRUS NUCLEAR ANTIGEN 1

EB病毒是一种疱疹病毒， 人类的疾病，并可以通过感染使人类B细胞永生， 体外 对于在转化细胞中表达的一种病毒蛋白， 抗原EBNA 1，至少一种功能是已知的。 EBNA 1是必需的反式 EBV的质粒维持元件oriP的活性。 一 oriP的一个重要组成部分，一个30 bp重复序列的家族， EBNA 1的存在，成为一个强大的转录增强子。 目的 其中一项工作是确定EBNA 1如何激活DNA复制。 和转录，并评估其可能的作用， 增长转化 一个主要目标是从人类细胞中纯化足够量的EBNA 1 以进行生物化学研究。 重组动物病毒载体 感染性腺病毒载体或EBV/SV 40质粒载体，将用于 在培养的细胞中过量产生EBNA 1。 这将使蛋白质 纯化至接近均匀的毫克量。 蛋白质将 生物化学特征，并将努力证明其 体外复制和转录的激活。 最终目标 将确定EBNA 1与哪些细胞成分相互作用， 促进这两项活动。 突变将被引入整个 EBNA 1基因，试图将这两种活性分开， 通过定义蛋白质的区域来补充生物化学研究 这对他们的活动至关重要。 还将进行实验 用培养细胞中的质粒来确定 携带oriP的质粒受活性EBNA 1的量或受 限制DNA在每个细胞周期中复制一轮的机制。 EBNA 1在生长转化中的可能作用将通过以下方法进行评估： 测试EBNA 1基因改变细胞生长特性的能力， 未建立的啮齿动物和人类细胞培养。

Epstein-Barr virus is a herpes virus that causes lymphoproliferative disorders in people and can immortalize human B-cells by infection in vitro. For one viral protein expressed in transformed cells, nuclear antigen EBNA1, at least one function is known. EBNA1 is required in trans for activity of the plasmid-maintenance element of EBV, oriP. One essential component of oriP, a family of 30-bp repeats, is activated in the presence of EBNA1 to become a strong transcriptional enhancer. The purpose of the proposed work is to determine how EBNA1 activates DNA replication and transcription and to evaluate its possible role in growth-transformation. A major goal is to purify sufficient quantities of EBNA1 from human cells to permit biochemical studies. Recombinant animal virus vectors, an infectious adenovirus vector or an EBV/SV40 plasmid vector, will be used to overproduce EBNA1 in cultured cells. This should allow the protein to be purified to near homogeneity in milligram quantities. The protein will be characterized biochemically, and efforts will be made to demonstrate its activation of replication and transcription in vitro. The eventual goal will be to determine with what cellular components EBNA1 interacts to stimulate these two activities. Mutations will be introduced throughout the EBNA1 gene in an attempt to separate the two activities and to complement the biochemical studies by defining the regions of the protein that are essential to its activities. Experiments will also be performed with plasmids in cultured cells to determine whether replication of oriP-carrying plasmids is limited by the amount of active EBNA1 or by a mechanism that limits DNA to one round of replication per cell cycle. A possible role for EBNA1 in growth-transformation will be evaluated by testing the capacity of the EBNA1 gene to alter the growth properties of nonestablished rodent and human cells in culture.