MECHANISMS FOR EBV EBNA-2 IMMORTALIZING FUNCTION

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The long-term goal of this research program is to understand the role of EBNA-2 in the establishment and maintenance of latent EBV (Epstein-Barr virus) infection and EBV-driven B cell immortalization. Immortalization of B lymphocytes by EBV requires the viral transcriptional activator protein FBNA-2 and is therefore an attractive target for anti-viral agents. EBNA-2 contains a potent activator domain and targets to promoters through a separate domain by an interaction with the cellular DNA binding protein CBF1/RBPjk (C promoter binding factor 1/recombination signal binding protein). The functional domains characterized so far reside in the carboxy-terminal half of the EBNA-2 protein and are conserved among the EBNA-2 types (from EBV-1 and EBV-2), including the EBNA-2 homologue from herpesvirus papio (HVP). Several questions still remain unsolved. First, genetic studies have indicated that the amino-terminal half of EBNA-2 is also required for immortalization and transactivation. Four of nine conserved regions between the EBNA-2 types reside in the amino-terminal half and are likely to provide insight towards identification of important functions domains. This grant application proposes experiments to test this hypothesis and define additional functional domains in the amino- terminal half of EBNA-2. Second, EBV isolates fall into two types, 1 and 2, that differ in immortalizing efficiency. The biological phenotype can be mapped to the EBNA-2 protein. The mechanism for differences in immortalizing efficiency between type 1 or type 2 EBNA-2 is not known. Third, recent findings have also indicated that participation of cellular factors that bind to EBNA-2 responsive promoters, other than CBF1/RBPjk, are important for EBNA-2 transactivation. One of these proteins called CBF2 (C promoter binding factor 2) is a likely candidate for this effect. Experiments in this proposal are aimed at characterizing and cloning this factor. The specific aims of this proposal focus on examining the mechanism of action of EBNA-2 and are: 1) To identify functional domains in the amino- terminal half of EBNA-2 by mutational analysis of conserved regions 1-4 and testing the mutants for transactivation and immortalization function and seeks to identify direct interactions of conserved regions 1-4 with cellular proteins; 2) To identify fa mechanism for the reduced immortalizing efficiency observed with the type 2 EBV by constructing type 1 and type 2 EBNA-2 chimeras, and 3) To identify and characterize the EBNA-2 enhancer binding protein CBF2 by measuring the effect of CBF2 on EBNA-2 transactivation, examining the effect of spacing between the CBF1 and CBF2 binding sites on EBNA-2 transactivation, defining the CBF2 binding site through mutagenesis, and purification and cloning of CBF2. Completion of these specific aims will give new information about how EBNA-2 functions and its role in EBV transformation, and may provide insight into the general principles of cellular transformation. Knowledge of these biochemical pathways will allow for the rational design of anti-EBV and anti-cancer agents.