EBV TRANSFORMATION OF B LYMPHOCYTES--EBNA REGULATION

B淋巴细胞的EBV转化--EBNA调节

• 批准号: 2895608
• 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 2002-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2895608
• 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：爱泼斯坦 - 巴尔病毒与伯基特密切相关 淋巴瘤和鼻咽癌，负责 免疫功能低下的个体中的淋巴增生性疾病。 长期 提出的研究的目标是确定 EBV的细胞转换。 当该病毒在体外感染B细胞时， 淋巴细胞变得永生。 EBV W启动子的激活（WP） 是感染期间检测到的第一个病毒转录事件。 WP 指导爱泼斯坦 - 巴尔病毒核抗原（EBNA）的早期表达。 EBNA蛋白参与细胞和病毒基因调节以及 维持慢性EBV潜伏期。 后来感染，有一个开关 使用其他EBNA启动子CP，然后是QP。而CP和QP已经 经过广泛研究，几乎没有有关特定顺式的信息 WP调节的反式成分。 我们的初步研究 证明了W中至少存在三个顺式元素 发起人，因此已选择这些元素进行详细研究 提议。 在第一个特定目的中，特定的细胞转录因子 将表征与WP中关键的顺式元素相互作用。 广泛的启动子诱变，凝胶转移测定法，启动子足迹和 西南凝胶将采用大量约束因素。 第二个特定 AIM将探讨WP CIS元素的功能重要性。 这将是 通过使用表达向量进行的转染研究完成 相关的反式激活器，并使用各种WP缺失和突变 构造。 在第三个目标中，WP的体内足迹和使用 整个EBV突变体和关键顺式元素的破坏将用于 证明WP域的体内意义。 最后，这些研究 将确定作用在trans中作用于WP的因素的体内作用 EBNA启动子切换的时间过程。 从这些中获得的知识 研究最终可能会产生利用EBV基因控制的手段 表达以增强EBV感染的恶性细胞的免疫破坏 伯基特淋巴瘤或霍奇金氏病等灾难性疾病。