EBNA-3C in B Lymphocyte Transformation by EBV

描述(申请人提供)：本项目将试图确定(S)EB病毒核抗原3C(EBNA-3C) 促进B淋巴细胞转化为淋巴母细胞系(LCLS)。 先前的研究已经证实，缺乏完整EBNA-3C的EBV基因组 不能生长转化B细胞。这个应用程序的中心假设是 EBNA-3C对于建立和维持LCLS和 它通过调节关键细胞的表达来发挥作用 和病毒基因。对EBNA-3C表达的要求 LCLS的维护将使用表达a基因的重组EBV进行测试 羟基他莫昔芬/EBNA-3C融合蛋白。预计，在 在没有羟基他莫昔芬的情况下，这种融合蛋白在 细胞质会导致生长停滞和/或细胞凋亡。在这种情况下， 转EBNA-3C及多个EBNA-3C突变体的能力 补充这一缺陷将被评估。如果相反，EBNA-3C被证明是 对于保持转化来说是必不可少的，EBNA的遗传分析- 将进行3C突变体的能力，以调解建立 LCLS。这些突变体协同共激活的能力 主要的病毒反式激活剂(EBNA-2)也将被评估。使用上面的 信息，EBNA-3C转化能力的转录图谱 突变株将与不能中介转化的突变株进行对比 努力建立EBNA-3C促进B的主要途径 淋巴细胞转化。包括酵母双杂交在内的生化方法 筛查和免疫共沉淀也将被用来发现 细胞蛋白与已识别的EBNA-3C效应域相互作用。

DESCRIPTION (provided by applicant): This project will attempt to define the mechanism(s) by which the Epstein-Barr virus nuclear antigen 3C (EBNA-3C) promotes B lymphocyte transformation into lymphoblastoid cell lines (LCLs). Previous studies have established that EBV genomes lacking an intact EBNA-3C cannot growth transform B cells. The central hypothesis of this application is that EBNA-3C is essential for the establishment and maintenance of LCLs and that it exerts its effect by regulating the expression of critical cellular and viral genes. The requirement for continued EBNA-3C expression in the maintenance of LCLs will be tested using a recombinant EBV expressing a hydroxytamoxifen/EBNA-3C fusion protein. It is anticipated that, in the absence of hydroxytamoxifen, the tethering of this fusion protein in the cytoplasm will lead to growth arrest and/or apoptosis. In this case, the ability of transfected EBNA-3C as well as multiple EBNA-3C mutants to complement this defect will be assessed. If instead, EBNA-3C proves to be dispensable for the maintenance of transformation, a genetic analysis of EBNA- 3C mutants will be conducted for their ability to mediate the establishment of LCLs. The ability of these mutants to synergistically co-activate with the major viral transactivator (EBNA-2) will also be assessed. Using the above information, the transcriptional profile of EBNA-3C transformation competent mutants will be contrasted to mutants unable to mediate transformation in an effort to establish the major pathways through which EBNA-3C promotes B lymphocyte transformation. Biochemical methods including a yeast two hybrid screen and co-immunoprecipitation will also be employed to discover what cellular proteins interact with the identified EBNA-3C effector domains.