Characteristic of EBV as a human tumor virus is its ability to immortalize B-lymphocytes and establish different degrees of latency. EBV latency products regulate cell-signaling pathways important in cell-cycle progression. Here we propose that cell-signaling molecules regulate the expression of EBV products in a cell-cycle-dependent manner. EBNA-2 is the master regulator of all the promoters used in Type III latency. We show that EBNA-2 is specifically hyperphosphorylated in M phase of the cell cycle, which correlates with suppression of Type III promoter activities. These observations indicate that there may be general inhibition of EBNA-2's transactivation functions in mitosis.
In Aim I we examine regulation of type III latency products during the cell cycle in relation to EBNA-2 phosphorylation status. We have implicated p34/cdc2 kinase in EBNA-2 hyperphosphorylation during M phase and identified a phosphorylation site of EBNA-2 specific for this kinase. We will determine the biological role of this site and also identify other sites of EBNA-2 phosphorylation in M phase through mutational analysis. EBV infection affects important cell-signaling pathways. Recently we discovered that EBV induces the beta-catenin/Tcf pathway, a key cellular oncogenic pathway, in type III latency.
In Aim II we examine how the latent EBV infection state critically alters this pathway and will investigate whether it is essential for immortalization of B-cells by EBV. We will examine if inhibition of this pathway leads to growth arrest and differentiation in EBV-immortalized B-lymphocytes. Finally, in Aim III, based on other discoveries, namely, involvement of both deubiquitination and ubiquitination in beta-catenin stabilization, we will bring together these viral and cellular mechanisms, since they are likely to be involved in EBV oncogenesis. Specifically, we will dissect the mechanism of beta-catenin stabilization in type III latency, focusing on the role of EBNA-2 in deubiquitination, and LMP-1 in ubiquitination. Dysregulation of the balance between these two key proteasomal regulatory processes is likely to be a general phenomenon whereby tumor viruses modulate signaling pathways of host cells.
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