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.
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). Trans-activation 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 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 trans-activation. 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(r) 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.
Institut für medizinische Mikrobiologie und Hygiene, Universitätskliniken des Saarlandes, Homburg/Saar, Germany.
C
C
Sauder
P
P
Haiss
F A
FA
Grässer
U
U
Zimber-Strobl
N
N
Mueller-Lantzsch
D000956:Antigens, Viral / Q000096:biosynthesis / Q000378:metabolism*; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism; D001483:Base Sequence; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D019309:Epstein-Barr Virus Nuclear Antigens; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D016679:Genome, Viral; D004854:Herpesvirus 4, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D000956:Antigens, Viral; D017931:DNA Primers; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D009838:Oligodeoxyribonucleotides; D015534:Trans-Activators