The long term objective of this project is to understand the mechanisms of gene regulation leading to reactivation of latent Epstein-Barr virus (EBV). Greater than 90% of the population is infected with EBV, and essentially all healthy seropositive individuals intermittently shed virus from mucosal membranes and have latently infected circulating B lymphocytes. EBV has long been thought to be a causal factor in the development of African Burkitt's lymphoma and nasopharyngeal carcinoma. AIDS and other immunosuppressed patients are prone to develop EBV-related B cell lymphomas, and hairy leukoplakia in AIDS patients is likely to be a clinical manifestation of the EBV replicative cycle. There is accumulating evidence that EBV is also associated with CNS lymphomas of the noncompromised. Although EBV is associated with several human cancers, no transforming gene has been identified. Latent cycle gene products have been implicated in immortalization and a lytic cycle transactivator, Zta, has been found to have homology with the cellular Jun oncogene. Furthermore, reactivation of latent virus is required for transmission to susceptible hosts. The specific goals of this research project are to further delineate the functional role of an EBV lytic cycle transcriptional transactivator, Rta. The specific sequences which constitute an Rta response element will be determined by constructing mutated and rearranged versions of the predicted motifs. Footprinting and gel retardation assays will be utilized to determine if Rta binds DNA directly or interacts with DNA through protein- protein interactions. The mechanism of synergism between Rta and the DNA- binding transactivator Zta, will be studied by identifying the required DNA motifs and by protein cross-linking experiments. We postulate that Rta functions by activating enhancers which are scattered throughout the EBV genome, and that Rta is required to activate the lytic cycle. Predicted enhancer elements will be tested for activation by Rta and Zta. Rta expression will be inhibited and the level of reduced Rta expression will be correlated with any decrease in the ability of EBV to reactivate. Because latency and reactivation can be achieved in cell culture, this is an ideal system to study the molecular biology of reactivation from latency. The results of these experiments will enhance our current knowledge of mechanisms of viral reactivation, and are likely to reflect mechanisms of normal eukaryotic gene control.
