FUNCTIONAL INTERACTION BETWEEN THE HUMAN CYTOMEGALOVIRUS 86-KILODALTON IE2 PROTEIN AND THE CELLULAR TRANSCRIPTION FACTOR CREB

The 86-kDa IE2 protein (IE86) of human cytomegalovirus (HCMV) has been described as a promiscuous transactivator of viral, as well as cellul ar, gene expression. Investigation of the mechanism used by IE86 to ac tivate gene expression from the early UL112/113 promoter of HCMV revea led the existence of three binding sites for IE86 located between nucl eotides -290 and -120 relative to the transcriptional start site (H. A rlt, D. Lang, S, Gebert, and T. Stamminger, J. Virol. 68:4117-4125, 19 94). As shown previously, deletion of these target sites resulted in a reduction of IE86-mediated transactivation by approximately 70%. The remaining promoter, however, could still be stimulated about 40-fold, indicating the presence of an additional responsive element within the se sequences. Here, we provide evidence that a binding site for the ce llular transcription factor CREB can also act as a target for IE86 tra nsactivation. By DNase I protection analysis, a binding sequence for C REB could be detected between nucleotides -78 and -56 within the respe ctive promoter region. After in vitro mutagenesis of this CREB-binding site within the context of the entire UL112/113 promoter, a marked re duction in transactivation levels was evident. Moreover, when individu al CREB-binding sites were positioned upstream of a minimal, TATA box- containing UL112/113 promoter, they were able to confer strong IE86 re sponsiveness, whereas a mutated sequence did not exert any effect. In far Western blot and pull-down experiments, a direct interaction of IE 86 with the cellular transcription factor CREB could be observed. The in vivo relevance of this in vitro interaction was confirmed by using various GAL4 fusion proteins in the presence or absence of IE86 which revealed a strong activation only in the presence of both a GAL4-CREB fusion and IE86. This shows that at least one specific member of the A TF/CREB family of transcription factors is involved in mediating trans activation by the HCMV IE86 protein.