Epstein-Barr virus (EBV) is etiologically associatedwith many malignant diseases, including nasopharyngeal and gastric cancer, and Burkitt's, Hodgkin's and non-Hodgkin's lymphoma. EBV remains atent in EBV-associated tumors and in normal B cells. Reactivation is required for the virus to spread between cells and among people. Reactivation from latency into the lytic cycle may be oncolytic. Lytic cycle products may also possess direct oncogenic effects. Our lab discovered and has intensively studied the EBV BZLF1 gene product, ZEBRA, which mediates the switch between latency and the lytic cycle. The global objective of work supported by CA12055 is to understandZEBRA's mechanism of action and regulation of its expression. In this application for an extensionof the MERIT award, we describe significant published and preliminary data of the past 4 years to accomplish this objective. We discoveredZEBRA point mutants which arrest the EBV lytic cycle at distinct phases of immediate-early, early and late gene expression. Each class of ZEBRA mutant elicits unique nuclear reorganization. We mapped the in vivo phosphorylation sites on ZEBRA and showed that phosphorylation of ZEBRA is required for its activity as a temporally regulated represser of viral late genes and for its activity in binding and activating the origin of lytic DMAreplication. We identified phosphoacceptor site mutants which specifically impaired ZEBRA's capacity to activate DMAreplication, but did not affect ZEBRA's ability to act as a transcriptional activator. We showed that induction of BZLF1 expression by histone deacetylase inhibitors and other inducing stimuli is mediated by newly synthesized cellular proteins. We found that a DMA methyltransferase inhibitor induces BZLF1 expression by a novel mechanism without detectably demethylating the promoter of this gene. We developed methods to separate lymphoma cells in which EBV is latent or lytic. Using this method we have shown that primary EBV infection is associated with an IgA response to early lytic antigens, such as ZEBRA. The proposed studies, utilizing tools of cell biology, biochemistry, molecular genetics, immunology and epidemiology, build on these seminal observations. The experiments address both basic questions of control of eukaryotic gene expression that are relevant to cancer and specific unresolved issues about molecular pathogenesis of an important human cancer virus.
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