A HUMAN CYTOMEGALOVIRUS EARLY PROMOTER WITH UPSTREAM NEGATIVE AND POSITIVE CIS-ACTING ELEMENTS - IE2 NEGATES THE EFFECT OF THE NEGATIVE ELEMENT, AND NF-Y BINDS TO THE POSITIVE ELEMENT

The human cytomegalovirus early promoter for the UL4 gene, which codes for an early viral envelope glycoprotein designated gpUL4, requires i mmediate-early viral protein two (IE2) synthesis to be activated (C.-P . Chang, C. L., Malone, and M. F. Stinski, J. Virol. 63:281, 1989). We investigated the cis-acting and trans-acting factors that regulate tr anscription from this UW promoter. In transient transfection assays, t he viral IE2 protein negated the effect of an upstream cis-acting nega tive element and enhanced downstream gene expression. A cis-acting pos itive element contributed to the activity of the viral promoter when a n upstream cis-acting negative element was deleted or when the viral I E2 protein was present. The cellular protein(s) that binds to the cis- acting negative element requires further investigation. The cellular p rotein that binds to the cis-acting positive element was characterized . Two DNA sequence-specific protein complexes were detected with DNA p robes spanning the region containing the cis-acting positive element a nd human cytomegalovirus-infected human fibroblast cell nuclear extrac ts. The more slowly migrating complex was labeled complex A, and the f aster was labeled complex B. Only complex B was detected with mock-inf ected cell nuclear extracts. Competition experiments confirmed the spe cificity of the A and B complexes. The protein bound to the DNA in bot h the complexes contacts a CCAAT box imperfect dyad symmetry (5'CCAATC ACTGG3'). Either CCAAT box within the dyad symmetry could compete for binding the nuclear factor. Mutation of the CCAAT box dyad symmetry re sulted in a decrease of the transcriptional activity from the UL4 prom oter. A cellular transcription factor, antigenically related to nuclea r factor-Y (NF-Y), was found in both complexes A and B. Events associa ted with viral infection caused phosphorylation of protein complex A. Dephosphorylation of the DNA-binding protein converts complex A to com plex B. The effect of phosphorylation of NF-Y is not known.