This project will attempt to define the mechanism(s) by which the Epstein-Barr virus nuclear antigen 3C (EBNA-3C) promotes B lymphocyte transformation into lymphoblastoid cell lines (LCLs). Previous studies have established that EBV genomes lacking an intact EBNA-3C cannot growth transform B cells. The central hypothesis of this application is that EBNA-3C is essential for the establishment and maintenance of LCLs and that it exerts its effect by regulating the expression of critical cellular and viral genes. The requirement for continued EBNA-3C expression in the maintenance of LCLs will be tested using a recombinant EBV expressing a hydroxytamoxifen/EBNA-3C fusion protein. It is anticipated that, in the absence of hydroxytamoxifen, the tethering of this fusion protein in the cytoplasm will lead to growth arrest and/or apoptosis. In this case, the ability of transfected EBNA-3C as well as multiple EBNA-3C mutants to complement this defect will be assessed. If instead, EBNA-3C proves to be dispensable for the maintenance of transformation, a genetic analysis of EBNA- 3C mutants will be conducted for their ability to mediate the establishment of LCLs. The ability of these mutants to synergistically co-activate with the major viral transactivator (EBNA-2) will also be assessed. Using the above information, the transcriptional profile of EBNA-3C transformation competent mutants will be contrasted to mutants unable to mediate transformation in an effort to establish the major pathways through which EBNA-3C promotes B lymphocyte transformation. Biochemical methods including a yeast two hybrid screen and co-immunoprecipitation will also be employed to discover what cellular proteins interact with the identified EBNA-3C effector domains.
| Maruo, Seiji; Johannsen, Eric; Illanes, Diego et al. (2005) Epstein-Barr virus nuclear protein 3A domains essential for growth of lymphoblasts: transcriptional regulation through RBP-Jkappa/CBF1 is critical. J Virol 79:10171-9 |
| Johannsen, Eric; Luftig, Micah; Chase, Michael R et al. (2004) Proteins of purified Epstein-Barr virus. Proc Natl Acad Sci U S A 101:16286-91 |
| Rosendorff, Adam; Illanes, Diego; David, Gregory et al. (2004) EBNA3C coactivation with EBNA2 requires a SUMO homology domain. J Virol 78:367-77 |
| Cooper, Andrew; Johannsen, Eric; Maruo, Seiji et al. (2003) EBNA3A association with RBP-Jkappa down-regulates c-myc and Epstein-Barr virus-transformed lymphoblast growth. J Virol 77:999-1010 |
| Maruo, Seiji; Johannsen, Eric; Illanes, Diego et al. (2003) Epstein-Barr Virus nuclear protein EBNA3A is critical for maintaining lymphoblastoid cell line growth. J Virol 77:10437-47 |
