EBV TRANSFORMATION OF B LYMPHOCYTES--EBNA REGULATION

B淋巴细胞的EBV转化--EBNA调节

• 批准号: 6376277
• 负责人: DAVID E GUTSCH
• 金额: $ 10.68万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-06-17 至 2001-06-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6376277
• 关键词: B lymphocyte; DNA footprinting; Epstein Barr virus; cell transformation; cytotoxic T lymphocyte; gel mobility shift assay; gene expression; genetic promoter element; genetic regulatory element; human genetic material tag; human tissue; proliferating cell nuclear antigen; southern blotting; transcription factor; transfection; transfection /expression vector; virus genetics; virus protein

DESCRIPTION: Epstein-Barr virus is strongly associated with Burkitt lymphoma and nasopharyngeal carcinoma and is responsible for lymphoproliferative disease in immunocompromised individuals. The long-term objectives of the studies proposed are to determine the mechanisms of cellular transformation by EBV. When this virus infects B cells in vitro, the lymphocytes become immortalized. Activation of the EBV W promoter (Wp) is the first viral transcriptional event detected during infection. Wp directs the early expression of Epstein-Barr virus nuclear antigens (EBNA). The EBNA proteins are involved in cellular and viral gene regulation and the maintenance of chronic EBV latency. Later in infection, there is a switch to usage of the other EBNA promoters, Cp, then Qp. While Cp and Qp have been studied extensively, almost no information exists regarding the specific cis and trans constituents of Wp regulation. Our preliminary studies have demonstrated the presence of at least three cis elements within the W promoter, so these elements have been selected for detailed study in this proposal. In the first specific aim, the particular cellular transcription factors which interact with critical cis elements in Wp will be characterized. Extensive promoter mutagenesis, gel shift assays, promoter footprinting and southwestern gels of bound factors will be employed. The second specific aim will explore the functional importance of Wp cis elements. This will be accomplished with transfection studies using expression vectors for pertinent transactivators and using various Wp deletional and mutational constructs. In the third aim, in vivo footprinting of Wp, and the use of whole EBV mutants with disruption of key cis elements will be used to demonstrate the in vivo significance of Wp domains. Finally, these studies will ascertain the in vivo role of factors acting upon Wp in trans during the time course of EBNA promoter switching. Knowledge gained from these studies might ultimately produce means of exploiting the control of EBV gene expression to augment immune destruction of EBV-infected malignant cells in such catastrophic diseases as Burkitt lymphoma or Hodgkin's disease.

描述：EB病毒与伯基特密切相关 淋巴瘤和鼻咽癌，并负责 免疫功能低下个体的淋巴组织增生性疾病。 长期 建议的研究目标是确定 EBV的细胞转化。 当这种病毒在体外感染B细胞时， 淋巴细胞就会永生化。 EBV W启动子（Wp）的激活 是感染期间检测到的第一个病毒转录事件。 Wp 指导EB病毒核抗原（EBNA）的早期表达。 EBNA蛋白参与细胞和病毒基因调控， 维持慢性EBV潜伏期。 在感染后期， 使用其他EBNA启动子Cp，然后是Qp。虽然Cp和Qp已经被 经过广泛的研究，几乎没有关于特定独联体的信息。 和Wp调节的反式成分。 我们的初步研究表明 证明了W中存在至少三个顺式元件 启动子，因此这些元件已被选择用于详细的研究， 提议 在第一个具体目标中，特定的细胞转录因子 其与WP中的关键顺式元件相互作用将被表征。 广泛的启动子诱变，凝胶迁移分析，启动子足迹和 将使用结合因子的西南凝胶。 第二特定 目的是探索Wp顺式元件的功能重要性。 这将是 通过使用表达载体的转染研究完成， 相关的反式激活因子，并使用各种Wp缺失和突变 结构。 在第三个目标中，Wp的体内足迹，以及Wp的使用。 具有关键顺式元件破坏的完整EBV突变体将用于 证明了Wp结构域的体内意义。 最后，这些研究 将确定在体内作用于Wp反式的因素， EBNA启动子转换的时间过程。 知识来自这些 研究可能最终产生利用EBV基因控制的方法， 表达以增强EBV感染的恶性细胞的免疫破坏， 像伯基特淋巴瘤或霍奇金病这样的灾难性疾病。