Epstein-Barr virus (EBV) is a ubiquitous Epstein-Barr virus (EBV) is a ubiquitous human virus infecting greater than 95% of the adult population. Infection with EBV typically leads to infectious mononucleosis in young adults and results in a lifelong infection. EBV is also associated with a number of human cancers which include Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's lymphoma, adult T-cell lymphomas and lymphoproliferative diseases in immunocompromised transplant (PTLD) and HIV positive AIDS patients (ALD). In vitro, EBV can transform human primary B-cells in vitro which results in perpetual proliferation of the primary cells into transformed lymphoblastoid cell lines (LCLs). These nascently infected B lymphocytes express a select set of latent genes one of which is the Epstein-Barr nuclear antigen (EBNA) 3C essential for B cell transformation. The overall goals of this application is to characterize the involvement of EBNA3C with the well known tumor suppressor p53 and to determine the post translational modifications necessary for regulation of p53 which is induced by its interaction with EBNA3C. Thently we have shown that EBNA3C can also interact with the p53 tumor suppressor and this application will focus on developing a model to elucidate the functional significance of this interaction. Thus, similar to HPV, SV40 and BK virus, EBV interacts with Rb and p53 albeit through distinctly different mechanisms. We plan to pursue these studies using an array of molecular genetics, biochemical and molecular biology approaches to develop a comprehensive model for the role of EBNA3C in survival and immortalization of human primary B cells and identify targets for therapeutic approaches in treatment of EBV related diseases. Hypothesis: The essential EBV latent antigen 3C targets and regulates the functions of the major cellular tumor suppressor p53 through post translational modifications for survival and immortalization of human primary B-lymphocytes. In this proposal the specific aims will be to:
Aim I. Investigate the interaction of EBNA3C with the p53 tumor suppressor and to discern the functional significance of this interaction. Tumor viruses like HPV and Polyoma viruses (SV40, BK and JC) can target the tumor suppressor p53 to disrupt its antiproliferative function. EBV has so far been elusive in terms of directly showing the ability of an essential latent antigen to directly bind and alter the function of p53. In this aim we will determine the direct interaction of EBNA3C with p53 by in vitro biochemical assays as well as through association in complex in human cells and EBV transformed cells.
Aim2. We will determine the requirement for phosphorylation of p53 through the regulatory functions of EBNA3C, which can lead stabilization of p53 and suppression of Mdm2 activity which allows for cell proliferation and cell cycle regulation in EBV transformed cells. We will investigate the phosphorylation status of p53 by kinases which include CyclinA/Cdk2, Chk1, Chk2 and ATM which prevents Mdm2 targeted ubiquitination and test the ability of EBNA3C to modulate the function of Mdm2 for p53 stability and function in LCLs.
Aim3. We will determine the targeted acetylation of p53 through the regulatory functions of EBNA3C through recruitment of acetylases important for post-translational modification of p53 which leads to cell proliferation and cell cycle regulation in EBV transformed cells. We will investigate the recruitment of acetylases including p300 and CBP by EBNA3C to determine its ability to modify p53 through acetylation and whether or not phosphorylation is critical for acetylation. Does acetylation of p53 through p300/CBP-EBNA3C allow for interaction of p53 and EBNA3C? What are specific residues important for modulation of p53 function? Specific mutations will be generated at these sites and the mutants tested for functional significance on endogenous promoter activities important for cell survival and proliferation.
Epstein Barr virus nuclear antigen EBNA3C is a latent viral nuclear antigen essential for B cell transformation in vitro. This EBV antigen has been linked to the specific regulation of a number of cellular events which include regulation of transcription, chromatin remodeling, cell proliferation and cell cycle regulation. This proposal will explore a model for the related functions of EBNA3C through its association with two potent tumor suppressors p53 and the retinoblastoma protein in transformation of human B cells.
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