EBV GENOME EXPRESSION-LOCALIZATION OF SPECIFIC FUNCTION

EB病毒（EBV）是一种人类疱疹病毒，具有良好的免疫反应性。 与人类肿瘤的相关性EBV始终与 地方性伯基特淋巴瘤，鼻咽癌，移植后 淋巴瘤和AIDS中的原发性中枢神经系统淋巴瘤。 一 混合细胞型霍奇金淋巴瘤、全身性淋巴瘤的比例 艾滋病，鼻T细胞淋巴瘤和未分化胃癌是 也与EBV有关。 共转染-复制试验已经鉴定出六种必需的EBV 复制基因，BALF 5（聚合酶），BMRF 1（聚合持续合成因子）， BALF 2（ssDNA结合蛋白）、BSLF 1（引发酶）、BBLF 4（解旋酶）和 BBLF 2/3（引发酶天冬氨酸蛋白），其产物是 在单纯疱疹病毒中发现的复制蛋白。与HSV（或SV 4 O或 HPV病毒），EBV不编码UL 9样起源结合蛋白， 具有解旋酶活性。EB病毒裂解基因表达受三种基因调控 病毒反式激活因子Zta、Rta和Mta。Zta和Mta都是 在瞬时复制测定中oriLyt复制所需的量。的 本申请寻求：（1）在功能上表征Mta，以便 来区分它的基因调控和复制作用。的 复制基因中的顺式作用序列 对Mta反式激活敏感性将在转染测定中确定 比较了引入不同ORF以及5'和3'端的影响， 非翻译序列将进行Mta的诱变以鉴定 Mta反式激活和复制功能所需的结构域。的 Mta对另一个细胞的细胞内区室化的贡献 将检查复制蛋白。 (2)定义的贡献 Zta到OriLyt复制。Zta激活结构域的区域 将通过诱变来定义复制功能所需的氨基酸序列。 Zta和病毒复制蛋白之间的潜在相互作用将 使用免疫共沉淀试验、GST-亲和试验和 在定向酵母双杂交系统中筛选。ZTA的贡献 将复制蛋白定位到复制前的病灶， 也要检查。(3)为了研究某些顺式作用 赋予TPA反应性的oriLyt元件中的转录信号 将通过DNA酶I足迹法和EMSA来确定oriLyt增强子上的DNA酶活性。 并评价了它们对溶解循环的一般贡献。的 将定位oriLyt启动子转录的序列， 细胞DNA结合负调控因子参与启动子 镇压将被定性。

Epstein-Barr virus (EBV) is a human herpesvirus with a well-established association with human neoplasia EBV is consistently associated with endemic Burkitt's lymphoma, nasopharyngeal carcinoma, post-transplant lymphoma, and primary central nervous system lymphoma in AIDS. A proportion of mixed cellularity Hodgkin's lymphomas, systemic lymphomas in AIDS, nasal T cell lymphomas and undifferentiated gastric carcinomas are also EBV associated. Cotransfection-replication assays have identified six essential EBV replication genes, BALF5 (polymerase), BMRF1 (pol.processivity factor), BALF2(ssDNA binding protein), BSLF1(primase), BBLF4(helicase) and BBLF2/3(primase assoc.protein) whose products are functional homologs of replication proteins found in herpes simplex virus. Unlike HSV (or SV4O or HPV viruses), EBV does not encode a UL9-like origin binding protein that has helicase activity. EBV lytic gene expression is regulated by three viral transactivators, Zta, Rta and Mta. Both Zta and Mta are also required for oriLyt replication in the transient replication assay. The present application seeks: (1) To characterize Mta functionally in order to discriminate between its gene regulation and replication roles. The cis-acting sequences within the replication genes that render them sensitive to Mta transactivation will be determined in transfection assays that compare the effects of introducing different ORF and 5' and 3' untranslated sequences. Mutagenesis of Mta will be undertaken to identify domains required for Mta transactivation and replication functions. The contribution of Mta to the intracellular compartmentalization of the other replication proteins will be examined. (2) To define the contribution of Zta to oriLyt replication. The region of the Zta activation domain required for replication function will be defined by mutagenesis. Potential interactions between Zta and the viral replication proteins will be evaluated using co-immunoprecipitation assays, GST-affinity assays and screening in a directed yeast two-hybrid system. The contribution of Zta to the localization of replication proteins to pre-replicative foci will also be examined. (3) To examine the role of certain cis-acting transcriptional signals in oriLyt Elements that confer TPA-responsiveness on the oriLyt enhancer will be defined by DNase I footprinting and EMSA and their general contribution to lytic cycle permissivity evaluated. The sequences that transcription of the oriLyt promoter will be mapped and the cellular DNA binding negatively regulate factor(s) involved in promoter repression will be characterized.