DNA-binding Studies of the Epstein--Barr Virus Nuclear Antigen 2 (EBNA-2): Evidence for Complex Formation by Latent Membrane Protein Gene Promoter-binding Proteins in EBNA-2-positive Cell Lines

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-fgr, latent membrane protein 1 (LMP1) and terminal protein 1 (TP1). Transactivation of the TP1 promoter and of the BamHI C 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 l) 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 transactivates 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 proteinprotein 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 TP 1 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 Mr 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.

PU box-binding transcription factors and a POU domain protein cooperate in the Epstein--Barr virus (EBV) nuclear antigen 2-induced transactivation of the EBV latent membrane protein 1 promoter

“…However, we have not so far been able to demonstrate a direct physical association between EBNA2 and the POU domain protein with EMSA supershift experiments (data not shown). Similar results have been reported by Sauder et al (1994). They identified a protein-binding region in a DNA fragment of the LMP1 promoter that corresponds to the Dc~/Dfl region studied in the present investigation.…”

“…However, analysis by sucrose gradient centrifugation provided evidence that the LMP1 promoter-binding proteins form a complex that sediments with a higher velocity in EBNA2-positive cell extracts. They suggest that EBNA2-positive cells might contain specific complexes bound to the LMP1 promoter that are too labile to be detected by EMSA (Sauder et al, 1994).…”

Epstein-Barr Virus Nuclear Antigen 3C Activates the Latent Membrane Protein 1 Promoter in the Presence of Epstein-Barr Virus Nuclear Antigen 2 through Sequences Encompassing an Spi-1/Spi-B Binding Site

“…Indeed, large amounts of exogenous EBNA-2 are required to detect EBNA-2-J complexes (17,27,46,56). To test this possibility, we added exogenous EBNA-3C, generated by either in vitro translation or baculovirus expression (44), to the binding reactions.…”

The NP9 protein encoded by the human endogenous retrovirus HERV‐K(HML‐2) negatively regulates gene activation of the Epstein‐Barr virus nuclear antigen 2 (EBNA2)

“…The lysate was centrifuged for 20 min at 15,000 rpm and 4 C and the supernatant was used for further experiments or stored at À80 C. Electrophoretic mobility shift assays (EMSAs) were carried out exactly as described. 29,38 The probe used for EMSA is derived from the viral LMP2a (TP1 promoter) and contains two RBPJj-binding sites. For supershift experiments, we employed the EBNA2-specific rat monoclonal antibody (mAb) R3 34 or an appropriate isotype (rat IgG2a) control.…”