) The objective of the proposed research is to define the direct contribution of the Epstein-Barr virus (EBV) EBNA-1 protein to EBV-associated lymphoma. Although EBNA-1 indirectly contributes to the tumorigenic potential of EBV through its viral genome maintenance function, several observations support a direct role of EBNA-1 in tumorigenesis. Specifically: i) EBNA-1 is a transcriptional regulator with the potential to target cellular gene expression, a mechanism that would enable EBNA-1 to influence cellular processes important to tumorigenesis; ii) loss of EBNA-1 expression in Akata Burkitt lymphoma (BL) cells is associated with lower tumorigenic potential; and iii) actively expressed EBNA-1 transgenes predispose mice to B-cell tumors. Using BL as a model system, the applicant will address the contribution of EBNA-1 to tumorigenesis focusing on the two defining features of this tumor: EBV infection in which EBNA-1 is the only known viral protein expressed and the deregulated expression of the c-myc proto-oncogene. To fulfill the objective, the applicant proposes three specific aims.
Aim 1 : Determine the tumorigenic potential of EBNA-1 in an EBV-negative BL-cell background; to do this, EBNA-1 will be stably expressed in EBV-negative Akata and other BL cells, and the tumorigenic potential of these cells relative to their EBV-positive and EBV-negative counterparts will be assayed.
Aim 2 : Define direct contributions of EBNA-1 to the oncogenic potential of EBV; specifically, the applicant will determine the role of EBNA-1 in EBV-mediated deregulation of c-myc expression and the ability of EBNA-1 to inhibit c-myc-induced apoptosis. Additionally, he will assess the potential of EBNA-1 to complement c-myc function in co-transformation assays.
Aim 3 : Identify EBNA-1 responsive cellular genes and evaluate their contribution to tumorigenic potential; using an inducible EBNA-1 expression system and representational difference analysis, the applicant will identify cellular genes that are activated or repressed by EBNA-1 and will then delineate the functions of the proteins encoded by these genes with respect to tumorigenesis.
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