REGULATION OF REPLICATION AND LATENCY BY EBV EBNAS

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Epstein-Barr virus (EBV), a human herpesvirus, establishes a latent infection in B lymphocytes. These cells are immortalized by the virus and become capable of continuous proliferation in culture. EBV has traditionally been associated with two malignant diseases, Burkitt's lymphoma and nasopharyngeal carcinoma. Recently, awareness of EBVs potential to cause lymphoproliferative disease has been heightened by data more clearly associating EBV with a subset of Hodgkin's lymphomas and by the development of EBV associated lymphomas in organ transplant recipients and AIDS patients. Only a limited number of EBV genes are expressed in latently infected B cells. The research described in this proposal focuses on the role of two of them, EBNA-l and EBNA-2, in the establishment and maintenance of latency and in the establishment of the immortalized B cell growth phenotype. EBNA-l is functionally pleiotropic, contributing to the regulation of its own expression and being the sole viral protein required for replication via the latency origin, ori-P. EBNA-ls function is mediated through direct DNA binding. However the mechanisms by which EBNA-l activates transcription and ori-P replication remain unresolved. It is proposed (l) To further characterize the DNA recognition and dimerization domains that are required for EBNA-l binding by mutational analysis. (2) To determine the mechanism of EBNA-l transactivation by identifying a transcriptional activation domain(s). (3) To examine the contribution to ori-P replication of EBNA-l induced structural changes and determine the role in replication and immortalization of EBNA-l interaction with cell proteins. Assessment of EBNA-ls contribution (if any) to R cell immortalization is pertinent since the EBNA-l/ori-P combination is being touted as a non-integrating vector system for human gene therapy. EBNA-2 is essential for EBV induced immortalization. EBNA-2 up-regulates transcription of both the EBNA and LMP families of latency genes and also alters expression of specific B cell genes. It is proposed to (4) Characterize the domains of EBNA-2 required for transactivation and immortalization including the domain for interaction with the cellular targeting protein, CBF1; the domain for interaction with the cell transcription complex, and the domains of presently unknown function that contain evolutionarily conserved amino acid motifs. In the final specific aim (5) the cell CBF1 protein that targets EBNA-2 to responsive promoters will be characterized and the role of CBF1 in the uninfected cell examined.