CREB AND CREB-BINDING PROTEINS PLAY AN IMPORTANT ROLE IN THE IE2 86-KILODALTON PROTEIN-MEDIATED TRANSACTIVATION OF THE HUMAN CYTOMEGALOVIRUS 2.2-KILOBASE RNA PROMOTER

The human cytomegalovirus (HCMV) immediate-early region 2 86-kDa prote in (IE2 86) is the major transactivator of the promoter for the 2.2-kb class of early RNAs (open reading frame UL 112-113). Previously, we r eported that a DNA segment on this promoter between nucleotides (nt) - 113 and -59 was critical for activation by IE2 86 in vivo and could be bound by IE2 86 in vitro (R. Schwartz, M. H. Sommer, A. Scully, and D . H. Spector, J. Virol. 68:5613-5622, 1994). With a set of site-specif ic mutations within nt -84 to -61, we have localized the essential cis -acting sequences to nt -72 to -61, which contain an ATF/CREB-binding site. The IE2 86-binding site between nt -113 and -85 is not essential for activation of the promoter by IE2 86 in transient-expression assa ys, but its presence can enhance the level of activation mediated thro ugh the sequences located between nt -84 and -59. Electrophoretic mobi lity shift assays with a segment containing nt -84 to -59 and nuclear extracts from human cells permissive for the HCMV infection revealed a complex band pattern. However, by supershift analysis with specific a ntibodies, we were able to identify CREB as the major ATF/CREB family member in the protein DNA complexes. Further evidence that CREB is a t arget for IE2 86-mediated induction, is provided by the finding that I E2 86 activates the somatostatin promoter to high levels. Although the binding of IE2 86 to nonphosphorylated full-length CREB or Delta CREB is minimal, IE2 86 does form complexes with p300 and the CREB-binding protein (CBP), which in turn bind to CREB and can serve as adaptor pr oteins for CREB function. In addition, the in vivo functional relevanc e of the interaction between IE2 86 and CBP is indicated by the abilit y of IE2 86 to enhance transcriptional activation mediated by a GAL4-C BP fusion protein brought to a promoter by GAL4-binding sites.