Transcriptional regulation of the latent membrane protein 1 gene by Epstein-Barr virus nuclear antigen 2

Abstract: The Epstein-Barr virus (EBV) is a ubiquitous human herpes virus, which infects B-cells. This leads to a life-long latent infection, which in most cases is non-pathogenic. However, EBV is etiologically associated with several human malignancies, including endemic Burkitt's lymphoma, nasopharyngeal carcinoma and post-transplant lymphoma. This association is probably linked to the ability of the virus to immortalise B-cells in vitro and to induce B-cell proliferation. Mutagenesis of the viral genome has defined a subset of four nuclear proteins (EBNA1, 2, 3, and 6) and one membrane protein (LMP1) required for the immortalisation process. The aim of the present study was to increase our knowledge about the different mechanisms by which the expression of the LMP1 gene is regulated in B-cells focussing on the role played by the virally encoded transactivating factor EBNA2. The importance of an ATF/CRE site and an Sp site in the proximal part of the LMP1 regulatory sequence (LRS) was established. Mutations of the ATF/CRE and Sp sites decreased both EBNA2-dependent and -independent LMP1 promoter activity in transient transfection experiments. Both cAMP and okadaic acid, an inhibitor of protein phosphatase 1 (PP1) and 2A (PP2A), activated the promoter in an ATF/CRE-dependent fashion. Immunoaffinity experiments showed that EBNA2 co-purified with a PP1-like protein from an EBV-immortalised B-cell line and that a recombinant EBNA2 fusion protein specifically bound and inhibited a PP1-like activity in B-cell extracts. Thus, we conclude that the ATF/CRE and Sp site is important in transactivation of the LMP1 promoter and that the ability of EBNA2 to block the effect exerted by protein phosphatases on a certain transcription factor can be part of the mechanism by which the LMP1 promoter is activated.Electrophoretic mobility shift assays showed that both the positive Sp1 and the negative Sp3 transcription factor bound to the Sp site. However, overexpression of the Sp1 transcription factor did not add to the activity induced by EBNA2. The heterodimeric transcription factor complexes ATF1/CREB1 and c-Jun/ATF2 bound to the ATF1/CRE site in LRS. The binding of ATF1/CREB1 activated the LMP1 promoter in the absence of EBNA2. The ATF2 and c-Jun transcription factors, on the other hand, did not activate the LMP1 promoter in the absence of EBNA2 but did so in the presence of EBNA2. The activating effect was abolished if the phosphate-accepting amino acid residues Thr69 and Thr71 in ATF2 were mutated. Furthermore, EBNA2 could interact with the c-Jun/ATF2 heterodimer. We conclude that EBNA2 is targeted to the promoter by these factors to transactivate the LMP1 promoter.Histone acetylation at the LMP1 promoter was studied in two different B-cell lines. Both EBNA2 and the histone deacetylase inhibitor Trichostatin A (TSA) rapidly increased the level of histone acetylation at the LMP1 promoter in resting ER/EB2-5 B-cells. However, only EBNA2 induced LMP1 transcription. This was in contrast to the situation in proliferating P3HR1 B-cells, in which TSA both increased histone acetylation at and activated transcription from the LMP1 promoter. Mutational analysis of LRS showed that the ATF/CRE element was an important mediator of the TSA effect. Our results also suggest that both EBNA2 and TSA require the presence of phosphorylated ATF2 to activate the LMP1 promoter.