EBNA-3C in B Lymphocyte Transformation by EBV

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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.