1887

Abstract

The Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA- 2) protein is essential for the immortalization of human primary B cells by EBV. EBNA-2 trans-activates cellular and viral genes like CD23, c, latent membrane protein 1 (LMP1) and terminal protein 1 (TP1). Transactivation of the TP1 promoter and of the promoter has already been investigated in detail and appears to be mediated via protein-protein interactions and not by direct binding of EBNA-2 type A (of EBV type 1) to the DNA. EBNA-2 is able to trans-activate the expression of the LMP gene in several cell lines. Various reports have delineated the cis-acting elements of the LMP promoter through which EBNA-2 mediates transactivation. To determine whether EBNA-2 also trans- activates the LMP promoter by protein-protein interactions, we performed a series of gel retardation assays and competition experiments with LMP promoter fragments of different sizes. We determined that the protein-binding region on the LMP promoter was within a 42 bp fragment encompassing nucleotides −135 to − 176 relative to the LMP transcriptional start site. None of the DNA fragments investigated indicated interaction of EBNA-2 with the DNA via protein- protein interactions. No significant differences between EBNA-2-positive and EBNA-2-negative nuclear extracts could be seen in the gel retardation assay under conditions that clearly showed binding of EBNA-2A to the TP1 promoter. However, analysis of sucrose gradient fractions in the gel retardation assay provided evidence that the LMP promoter-binding proteins form a complex of higher M in EBNA-2-positive cell extracts. These complexes were destroyed by detergent. We deduce from these results that EBNA-2-positive cells might indeed contain specific complexes bound to the LMP promoter which are, however, too labile to be detected in a standard gel retardation assay.

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1994-11-01
2022-05-28
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