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.
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.
Schlee
Martin
M
Institute of Clinical Molecular Biology and Tumor Genetics, GSF-National Research Center for EnvironmentHealth, Munich, Germany. schlee@gsf.de
Krug
Tanja
T
Gires
Olivier
O
Zeidler
Reinhard
R
Hammerschmidt
Wolfgang
W
Mailhammer
Reinhard
R
Laux
Gerhard
G
Sauer
Guido
G
Lovric
Josip
J
Bornkamm
Georg W
GW
D001402:B-Lymphocytes / Q000166:cytology / Q000821:virology*; D001483:Base Sequence; D002472:Cell Transformation, Viral; D004279:DNA, Viral / Q000235:genetics; D020031:Epstein-Barr Virus Infections / Q000235:genetics / Q000378:metabolism / Q000821:virology; D019309:Epstein-Barr Virus Nuclear Antigens / Q000235:genetics / Q000502:physiology*; D015870:Gene Expression; D016259:Genes, myc; D004854:Herpesvirus 4, Human / Q000472:pathogenicity / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D014176:Protein Biosynthesis; D020543:Proteome; D000090063:Proto-Oncogene Mas; D014764:Viral Proteins
D004279:DNA, Viral; C402641:EBNA-2 protein, Human herpesvirus 4; D019309:Epstein-Barr Virus Nuclear Antigens; C000715470:MAS1 protein, human; D020543:Proteome; D000090063:Proto-Oncogene Mas; D014764:Viral Proteins