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.
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.
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