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

Abstract

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.Epstein-Barr virus (EBV) is a ubiquitous B-lymphotropic herpesvirus that after a usually asymptomatic infection is carried by more than 90% of adults for the rest of their lives. When the primary infection is postponed to late childhood or adolescence, EBV can cause infectious mononucleosis. The virus has been linked to the development of several malignant tumors, including Burkitt's lymphoma, Hodgkin's disease, lymphoproliferative disease in immunosuppressed individuals, certain forms of T-cell lymphoma, and some lymphoepithelial tumors, such as nasopharyngeal carcinoma and a proportion of gastric cancers (reviewed in reference 4). Infection of primary B cells with EBV in vitro causes the activation of the cells and drives them into continuous proliferation, a process termed immortalization. Cell lines established by infection of primary human B cells with EBV are called lymphoblastoid cell lines (LCLs).The first viral genes expressed in EBV-infected B cells are EBNA2 and EBNA-LP, two nuclear antigens that act as transcription activators in concert with each other and regulate the expression of viral as well as cellular genes involved in the initiation and maintenance of cell proliferation. EBNA2 in conjunction with EBNA-LP upregulates the expression of the viral membrane latent genes LMP1 and LMP2 and regulates the BamHI-C promoter driving the expression of all nuclear antigens (reviewed in refe...

Different Patterns of Epstein-Barr Virus Latency in Endemic Burkitt Lymphoma (BL) Lead to Distinct Variants within the BL-Associated Gene Expression Signature

“…Not surprisingly, there are parallels with the expression profiles reported for conventional LCLs and also that seen in a model B cell line when switched from a c-myc-driven (BL-like) to an EBV-driven (LCLlike) growth program (47). Many of these LCL-associated changes have been assigned to the effects of two major effectors of the EBV-induced transformation process, namely, EBNA2 as a transcriptional activator (48,54,55) and LMP1 as an activator of the NF-B pathway (50,56). It is therefore worth noting that, as the present study moves on to look for more subtle changes distinguishing between latency I and Wp-restricted BL lines, these two forms of infection lack both EBNA2 and LMP1.…”

Cellular Target Genes of Epstein-Barr Virus Nuclear Antigen 2

“…This expression pattern of EBV transcription, accompanied by a specific cellular gene expression pattern, is called latency III. Important primary targets of EBNA-2 in LCLs are the proto-oncogene c-myc and the viral LMP1 gene, which contribute to the initiation of the secondary target gene cascade (6,12,24,47,57,60). The mechanism by which EBNA-2 activates viral target genes has been intensively studied.…”

Changes in the Nasopharyngeal Carcinoma Nuclear Proteome Induced by the EBNA1 Protein of Epstein-Barr Virus Reveal Potential Roles for EBNA1 in Metastasis and Oxidative Stress Responses

“…2-D DiGE technology is a powerful and quantitative tool to address proteomic changes in complex samples. This and related 2-D gel techniques have been used previously to provide insight into cellular changes induced by viral infection or by specific viral proteins (1,2,7,9,14,42,53). Using this approach, we have identified 19 cellular proteins whose abundance is altered in the presence of EBNA1 in NPC cells.…”