Multiple EBNA1-binding sites are required to form an EBNA1-dependent enhancer and to activate a minimal replicative origin within oriP of Epstein-Barr virus.
EBNA1 activates the EBV plasmid maintenance sequence oriP by binding to its two essential regions. One region is a family of 30-base-pair (bp) repeats and is activated by EBNA1 to act as a transcriptional enhancer. The other region contains a 65-bp dyad symmetry and lacks enhancer function. To explore the functional differences between the two regions, we determined oriP activities as functions of the number of 30-bp repeats and compared them with activities determined when tandem copies of the dyad symmetry region were used to replace the 30-bp repeats. Three conclusions have been drawn. (i) Activation of the 30-bp repeats by EBNA1 to enhance transcription or to permit plasmid maintenance is a highly cooperative process involving at least six or seven 30-bp repeats for full activity. (ii) Tandem copies of the dyad symmetry region cooperatively enhance transcription but are less effective than 30-bp repeats providing a similar number of EBNA1-binding sites. (iii) Tandem copies of the dyad symmetry region alone cooperatively activate replication, suggesting that the region contains the actual origin of replication. We also report that while rodent-derived cell lines do not support replication of EBV-derived plasmids they do permit EBNA1-dependent enhancer activity. EBV plasmid replication thus requires the interaction of EBNA1 or oriP with a host factor that is not required for enhancement of transcription.
Wysokenski
D A
DA
Department of Human Genetics, Roswell Park Memorial Institute, Buffalo, New York 14263.
Yates
J L
JL
D000956:Antigens, Viral / Q000235:genetics*; D001665:Binding Sites; D015139:Blotting, Southern; D002051:Burkitt Lymphoma; D004261:DNA Replication; D004742:Enhancer Elements, Genetic; D019309:Epstein-Barr Virus Nuclear Antigens; D004854:Herpesvirus 4, Human / Q000235:genetics* / Q000276:immunology / Q000502:physiology; D006801:Humans; D010957:Plasmids; D015183:Restriction Mapping; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured; D014779:Virus Replication
D000956:Antigens, Viral; D019309:Epstein-Barr Virus Nuclear Antigens