Epstein-Barr virus (EBV) infection has been associated with lymphoid and epithelial cell malignancies. The viral genes expressed during latent infection (in particular the EBNA 2 and LMP 1 proteins) are sufficient to immortalize cells in vitro, and are the only gene products expressed in EBV-associated Burkitt's lymphomas. However, other EBV-associated tumors (including nasopharyngeal carcinoma, lymphomas in immunocompromised patients, and Hodgkins's Disease) commonly contain malignant cells expressing the EBV immediate-early protein, BZLF1 (Z). Therefore, EBV proteins (such as Z) expressed during productive infection could potentially enhance tumor development initiated by the latent (transforming) viral gene products. We have recently shown that the EBV Z protein (a transcriptional activator which regulates the switch from latent to lytic viral infection) can interact directly with wild-type p53 through direct protein-protein interactions. The interaction between Z and p53 appears to play a role in regulating lytic viral replication. We find that p53 binds directly to an essential region of the lytic viral origin (ori-Lyt), and that over- expression of wild-type p53 inhibits the ability of Z to disrupt viral latency. In addition, we have shown that Z inhibits the ability of wild- type p53 to activate promoters containing upstream p53 binding sites. Therefore, expression of Z (like the SV40 T antigen, the papilloma virus E6 protein, and the adenovirus EIB protein) may inactivate p53 function nd contribute to the malignant phenotype in EBV-associated tumors. In this grant, we propose to further study the interaction between Z and 53. We hypothesize that th e interaction between Z and p53 may contribute to cellular transformation. In the first specific aim, we will further investigate the interaction betweeen Z and p53 by determining if Z can affect the protein stability, nuclear localization, conformation, DNA binding ability, or phosphorylation status of p53. In the second specific aim, we will map the domains of Z and p53 required for direct interaction and attempt to create Z mutants which no longer interact with p53 (but still retain transactivator and replicative functions). In our third specific aim, we will determine if Z can transform cell lines in vitro either by itself, or in combination with a variety of known oncogenes (such as myc, ras, and E1A), and whether Z can inhibit P53-MEDIATED apoptosis. If so, we will determine if the same domains of Z required for direct interaction with p53 are also required for inhibition of p53 tumor suppressor function and p53-mediated apoptosis. In the fourth specific aim, we will recombine mutant forms of Z no longer able to interact with p53 (created in specific aim #2) back into the intact virus and determine whether the mutant virus is les tumorigenic in SCID mice. These studies should illuminate the function of the Z/p53 interaction in EBV infection, in addition to providing valuable information regarding the possible role of this interaction in EBV-associated transformation.
| Mauser, Amy; Saito, Shin'ichi; Appella, Ettore et al. (2002) The Epstein-Barr virus immediate-early protein BZLF1 regulates p53 function through multiple mechanisms. J Virol 76:12503-12 |
