The Epstein-Barr virus nuclear antigen 2 interacts with an EBNA2 responsive cis-element of the terminal protein 1 gene promoter.
The Epstein-Barr virus protein EBNA2 acts as a transcriptional activator of cellular and viral genes and plays a crucial role in the immortalization of human primary B-cells by EBV. We have shown previously that EBNA2 transactivates the promoters of the latent membrane antigens LMP, TP1 and TP2. The promoter of the TP1 gene was chosen as a model system to study the molecular mechanism of EBNA2 mediated transactivation. To identify an EBNA2 dependent cis-acting element, various TP1 promoter-reporter gene constructs were transfected in the absence and presence of an EBNA2 expression vector into the established B-cell line BL41-P3HR1. We were able to delineate an 81 bp EBNA2 responsive region between -258 and -177 relative to the TP1 RNA start site. The element worked in either orientation and could mediate EBNA2 dependent transactivation on a heterologous promoter. Electrophoretic mobility shift assays revealed three specific protein-DNA complexes formed with sequences of the EBNA2 responsive element. Two of these were not cell type specific, but the third was detected only in EBNA2 positive cell extracts. Gel-shift analysis in the presence of EBNA2 specific monoclonal antibodies revealed that EBNA2 is a component of the third complex. Thus, these experiments demonstrate that EBNA2 interacts with an EBNA2 responsive cis-element of the TP1 promoter.
Zimber-Strobl
U
U
Institut für Klinische Molekularbiologie und Tumorgenetik, GSF, München, Germany.
Kremmer
E
E
Grässer
F
F
Marschall
G
G
Laux
G
G
Bornkamm
G W
GW
D000954:Antigens, Surface / Q000235:genetics*; D000956:Antigens, Viral / Q000378:metabolism*; D001402:B-Lymphocytes; D001483:Base Sequence; D002460:Cell Line; D002472:Cell Transformation, Viral / Q000235:genetics*; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019309:Epstein-Barr Virus Nuclear Antigens; D005814:Genes, Viral; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000276:immunology; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015183:Restriction Mapping; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection; D014763:Viral Matrix Proteins / Q000235:genetics*
D000954:Antigens, Surface; D000956:Antigens, Viral; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D009838:Oligodeoxyribonucleotides; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014763:Viral Matrix Proteins; D015500:Chloramphenicol O-Acetyltransferase