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Journal of Virology, April 2004, p. 3941-3952, Vol. 78, No. 8
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.8.3941-3952.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification of Epstein-Barr Virus (EBV) Nuclear Antigen 2 (EBNA2) Target Proteins by Proteome Analysis: Activation of EBNA2 in Conditionally Immortalized B Cells Reflects Early Events after Infection of Primary B Cells by EBV

Martin Schlee,1,{dagger}* Tanja Krug,2,{dagger} Olivier Gires,3 Reinhard Zeidler,4 Wolfgang Hammerschmidt,5 Reinhard Mailhammer,1 Gerhard Laux,1 Guido Sauer,1,6 Josip Lovric,1,6 and Georg W. Bornkamm1

Institute of Clinical Molecular Biology and Tumor Genetics,1 Department of Gene Vectors, GSF-National Research Center for EnvironmentHealth,5 Department of Head and Neck Surgery, Ludwig-Maximilians University of Munich,2 Clinical Cooperation Group "Molecular Oncology," GSF-Research Center for Health and Environment, and Department of Head and Neck Surgery, Ludwig-Maximilians University,3 Vaecgene Biotech Inc., D-81377 Munich, Germany,4 Department for Biomolecular Sciences, University of Manchester Institute for Science and Technology, The Mill, M60 1QD Manchester, United Kingdom6

Received 24 June 2003/ Accepted 5 January 2004

The Epstein-Barr virus (EBV) is a ubiquitous B-lymphotropic herpesvirus associated with several malignant tumors, e.g., Burkitt's lymphoma and Hodgkin's disease, and is able to efficiently immortalize primary B lymphocytes in vitro. The growth program of infected B cells is initiated and maintained by the viral transcription factor EBV nuclear antigen 2 (EBNA2), which regulates viral and cellular genes, including the proto-oncogene c-myc. In our study, patterns of protein expression in B cells with and without EBNA2 were analyzed by two-dimensional polyacrylamide gel electrophoresis and mass spectrometry. For this purpose, we used a conditional immortalization system for EBV, a B cell line (EREB2-5) that expresses an estrogen receptor-EBNA2 fusion protein. In order to discriminate downstream targets of c-Myc from c-Myc-independent EBNA2 targets, we used an EREB2-5-derived cell line, P493-6, in which c-Myc is expressed under the control of a tetracycline-regulated promoter. Of 20 identified EBNA2 target proteins, 11 were c-Myc dependent and therefore most probably associated with proliferation, and one of these proteins was a posttranslationally modified protein, i.e., hypusinylated eIF5a. Finally, to estimate the relevance of EBNA2 targets during early EBV infection, we analyzed the proteomes of primary B cells before and after infection with EBV. The protein expression pattern induced upon EBV infection was similar to that following EBNA2 activation. These findings underscore the value of EREB2-5 cells as an appropriate model system for the analysis of early events in the process of EBV-mediated B-cell immortalization.

* Corresponding author. Mailing address: Institute of Clinical Molecular Biology and Tumor Genetics, GSF-National Research Center for Environment and Health, Marchioninistrasse 25, D-81377 Munich, Germany. Phone: 49-89-7099517. Fax: 49-89-7099500. E-mail: schlee{at}gsf.de.

{dagger} M.S. and T.K. contributed equally to this work.

Journal of Virology, April 2004, p. 3941-3952, Vol. 78, No. 8
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.8.3941-3952.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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