EBV GENOME EXPRESSION-LOCALIZATION OF SPECIFIC FUNCTION

EB病毒(Epstein-Barr Virus，EBV)是一种历史悠久的人类疱疹病毒 与人类肿瘤的相关性EBV一直与 地方性Burkitt淋巴瘤、鼻咽癌，移植后 淋巴瘤，以及艾滋病中的原发性中枢神经系统淋巴瘤。一个 混合细胞性霍奇金淋巴瘤、全身性淋巴瘤 艾滋病、鼻T细胞淋巴瘤和未分化胃癌是 也与EB病毒有关。 共转染-复制分析鉴定了六种基本的EBV 复制基因，BALF5(聚合酶)，BMRF1(极化加工性因子)， BALF2(单链DNA结合蛋白)、BSLF1(启动酶)、BBLF4(解旋酶)和 BBLF2/3(Primase assoc.Protein)，其产物是 在单纯疱疹病毒中发现的复制蛋白。与HSV(或SV4O或 HPV病毒)，EBV不编码UL9样的起源结合蛋白， 具有解旋酶活性。EBV裂解基因表达受三种基因调控 病毒反式激活剂、ZTA、RTA和MTA。Zta和MTA也是 在瞬时复制试验中，oriLyt复制所需的。这个 本申请寻求：(1)有序地表征MTA的功能 来区分它的基因调控和复制作用。这个 构成它们的复制基因内的顺式作用序列 将在转染法中确定对MTA反式激活的敏感性 比较了引入不同ORF以及5‘和3’的效果 未翻译的序列。将对MTA进行诱变以确定 MTA转录激活和复制功能所需的域。这个 MTA对另一种细胞内区隔作用的贡献 将对复制蛋白进行检测。(2)界定……的贡献 Zta来定向Lyt复制。Zta激活结构域的区域 复制功能所需的基因将通过突变来定义。 Zta和病毒复制蛋白之间的潜在相互作用将 使用免疫共沉淀分析、GST亲和分析和 定向酵母双杂交系统的筛选。Zta的贡献 将复制蛋白定位到复制前的焦点将 也要接受检查。(3)研究某些顺位代理的角色 OriLyt元件中赋予TPA反应性的转录信号 在oriLyt增强子上将由DNase I足迹和EMSA定义 并估算了它们对裂解循环透过率的总体贡献。这个 OriLyt启动子的转录将被映射的序列和 细胞DNA结合负性调节因子(S)参与启动子 镇压将成为其特征。

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.