Promoter-proximal regulatory elements involved in oriP-EBNA1-independent and -dependent activation of the Epstein-Barr virus C promoter in B-lymphoidcell lines

The identification of the cellular factors that control the transcription r egulatory 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 grou p 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 muta tional analysis of reporter plasmids carrying CD regulatory sequences toget her with or without oriPI. The reporter plasmids were transfected into grou p I phenotype Rael cells and group III phenotype cbc-Rael cells, and the Cp activity measured was correlated with the binding of candidate transcripti on factors in electrophoretic mobility shift assays and further assessed in cotransfection experiments. We show that the NF-Y transcription factor int eracts 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 findin g 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-EBNAl-dependent activity of the promoter. This region contains overlapping binding sites fo r Spl and Egr-1, and our results suggest that Spl is a positive and Egr-1 i s a negative regulator of Cp activity. Furthermore, we demonstrate that a r eporter plasmid that in addition to oriPI contains only the -111/+76 region of Cp still retains the ability to be activated by EBNA1.