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Journal of Virology, July 2001, p. 5796-5811, Vol. 75, No. 13
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.13.5796-5811.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Promoter-Proximal Regulatory Elements Involved in oriP-EBNA1-Independent and -Dependent Activation of the Epstein-Barr Virus C Promoter in B-Lymphoid Cell Lines

Tina Nilsson, Henrik Zetterberg, Yuyan Camilla Wang, and Lars Rymo*

Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, Göteborg University, S-413 45 Göteborg, Sweden

Received 18 December 2000/Accepted 9 April 2001

The identification of the cellular factors that control the transcription regulatory activity of the Epstein-Barr virus C promoter (Cp) is fundamental to the understanding of the molecular mechanisms that control virus latent gene expression. Using transient transfection of reporter plasmids in group I phenotype B-lymphoid cells, we have previously shown that the -248 to -55 region (-248/-55 region) of Cp contains elements that are essential for oriPI-EBNA1-dependent as well as oriPI-EBNA1-independent activation of the promoter. We now establish the importance of this region by a detailed mutational analysis of reporter plasmids carrying Cp regulatory sequences together with or without oriPI. The reporter plasmids were transfected into group I phenotype Rael cells and group III phenotype cbc-Rael cells, and the Cp activity measured was correlated with the binding of candidate transcription factors in electrophoretic mobility shift assays and further assessed in cotransfection experiments. We show that the NF-Y transcription factor interacts with the previously identified CCAAT box in the -71/-63 Cp region (M. T. Puglielli, M. Woisetschlaeger, and S. H. Speck, J. Virol. 70:5758-5768, 1996). We also show that members of the C/EBP transcription factor family interact with a C/EBP consensus sequence in the -119/-112 region of Cp and that this interaction is important for promoter activity. A central finding is the identification of a GC-rich sequence in the -99/-91 Cp region that is essential for oriPI-EBNA1-independent as well as oriPI-EBNA1-dependent activity of the promoter. This region contains overlapping binding sites for Sp1 and Egr-1, and our results suggest that Sp1 is a positive and Egr-1 is a negative regulator of Cp activity. Furthermore, we demonstrate that a reporter plasmid that in addition to oriPI contains only the -111/+76 region of Cp still retains the ability to be activated by EBNA1.

* Corresponding author. Mailing address: Department of Clinical Chemistry and Transfusion Medicine, Sahlgrenska University Hospital, S-413 45 Gothenburg, Sweden. Phone: 46 31 342 40 83. Fax: 46 31 828458. E-mail: lars.rymo{at}clinchem.gu.se.

Journal of Virology, July 2001, p. 5796-5811, Vol. 75, No. 13
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.13.5796-5811.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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