EBNA-5, an Epstein-Barr virus-encoded nuclear antigen, binds to the retinoblastoma and p53 proteins.

Abstract

Epstein-Barr virus (EBV) immortalized human lymphoblastoid cell lines express six virally encoded nuclear proteins, designated EBV nuclear antigens 1-6 (EBNA-1-6). We show that the EBNA-5 protein (alternatively designated EBNA-LP) that is required for B-cell transformation can form a molecular complex with the retinoblastoma (RB) and p53 tumor suppressor proteins. Using EBNA-5 deletion mutants, we have found that a 66-amino acid-long peptide, encoded by the W repeat of the EBV genome, is sufficient for binding. Point mutations of RB and p53 that inhibit their complexing with other DNA viral oncoproteins do not affect their binding to EBNA-5. p53 competes with RB for EBNA-5 binding. Our data suggest that the mechanisms involved in EBV transformation may include impairment ofRB and p53 function.Simian virus 40 (SV40), adenoviruses, and human papilloma viruses (HPVs) encode transforming proteins that can form complexes with the two major tumor suppressor proteins, the retinoblastoma (RB) and p53 proteins (1)(2)(3)(4)(5)(6)(7)(8) Herpes viruses are the largest among the DNA tumor viruses. They encode a higher number of transformationassociated proteins than the medium-sized adenoviruses or the small papova viruses, and their transforming strategy must be fundamentally different. It therefore appeared important to investigate whether any of their products interact with RB and p53 as well. We have chosen Epstein-Barr virus (EBV) as the most highly transforming member of the herpes virus family to approach this question.EBV expresses six nuclear proteins in immortalized lymphoblastoid cell lines (LCLs), designated EBV nuclear antigens 1-6 (EBNA-1-6). In a previous study, we have examined the intranuclear distribution of EBNA-1, -2, -3, and -5 and their ability to colocalize with the RB protein, stained with the aRBlCl monoclonal antibody (9). We have found a striking colocalization between EBNA-5, alternatively called EBNA-LP, and RB in the LCL IB4 (10).EBNA-5 is required for B-cell transformation (11 The monoclonal anti-RB antibody aRBlCl was found to stain similar nuclear foci in the LCL IB4. Using computerassisted overlap analysis, we found complete overlapping in the RB and EBNA-5-positive foci (10). They differed from the equally distinct dots of the small nuclear ribonucleoproteinrich splicing islands. Neither EBNA-1, -2, nor -3 showed any colocalization with RB (10, 15).In the present study, we have investigated the ability of EBNA-5 to form complexes with the RB and p53 proteins, using bacterially produced glutathione S-transferase (GST) fusion proteins. We demonstrate that EBNA-5 can bind RB and p53.MATERIALS AND METHODS Cell Lines and Cell Culture. The LCL IARC139, the Burkitt lymphoma (BL) lines BL28, E95A-BL28, Namalwa, and Ramos, the promyelocytic leukemia cell line HL60, and the colon carcinoma cell line SW480 were used in this study. E95A-BL28, a subline of the originally EBV-negative BL28, carries a fragment of EBV DNA encoding EBNA-2 and EBNA-5 but no other members of the EBNA family (16). Cel...

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