EBV GENOME EXPRESSION-LOCALIZATION OF SPECIFIC FUNCTION

EBV基因组表达-特定功能的定位

• 批准号: 2683421
• 负责人: S DIANE HAYWARD
• 金额: $ 24.49万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-09-30 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2683421
• 关键词: DNA footprinting; Epstein Barr virus; gel mobility shift assay; gene expression; genetic enhancer element; genetic mapping; genetic promoter element; genetic regulation; immunoprecipitation; laboratory rabbit; transcription factor; virus DNA; virus genetics; virus replication; yeast two hybrid system

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.

EB 病毒 (EBV) 是一种人类疱疹病毒，具有公认的 与人类肿瘤的关联 EBV 始终与 地方性伯基特淋巴瘤、鼻咽癌、移植后 淋巴瘤和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、系统性淋巴瘤的比例 艾滋病、鼻T细胞淋巴瘤和未分化胃癌 也与 EBV 相关。 共转染复制测定已鉴定出六种必需的 EBV 复制基因、BALF5（聚合酶）、BMRF1（聚合持续因子）、 BALF2​​（ssDNA结合蛋白）、BSLF1（引物酶）、BBLF4（解旋酶）和 BBLF2/3（引物酶相关蛋白），其产物是以下功能的同系物 单纯疱疹病毒中发现的复制蛋白。与 HSV（或 SV4O 或 HPV 病毒），EBV 不编码类似 UL9 的起源结合蛋白 具有解旋酶活性。 EBV裂解基因表达受三个因素调控 病毒反式激活因子、Zta、Rta 和 Mta。 Zta 和 Mta 也是 瞬时复制测定中 oriLyt 复制所需的。这 本申请寻求： (1) 功能性地表征 Mta，以便 区分其基因调控和复制作用。这 复制基因内的顺式作用序列使它们 对 Mta 反式激活的敏感性将在转染测定中确定 比较引入不同 ORF 以及 5' 和 3' 的效果 未翻译的序列。将进行 Mta 诱变以确定 Mta 反式激活和复制功能所需的域。这 Mta 对其他细胞内区室化的贡献 将检查复制蛋白。 (2) 定义贡献 Zta 到 oriLyt 复制。 Zta激活域区域 复制功能所需的将通过诱变来定义。 Zta 和病毒复制蛋白之间的潜在相互作用将 使用免疫共沉淀测定、GST 亲和力测定和 在定向酵母双杂交系统中进行筛选。 Zta的贡献 复制蛋白在复制前焦点的定位 也予以检查。 (3) 检验某些顺式作用的作用 oriLyt 元件中赋予 TPA 响应性的转录信号 oriLyt 增强子上的信息将由 DNase I 足迹和 EMSA 定义 并评估了它们对溶解循环介电常数的一般贡献。这 oriLyt 启动子的转录将被定位的序列和 启动子参与的细胞 DNA 结合负调控因子 压制将具有特征。