REGULATION OF A HUMAN CYTOMEGALOVIRUS IMMEDIATE-EARLY GENE (US3) BY ASILENCER-ENHANCER COMBINATION

The US3 open reading frame of human cytomegalovirus (HCMV) is transcri bed at immediate-early (IE) times after infection, Upstream of the US3 promoter, between -84 and -259 bp relative to the transcription start site, there are five copies of an 18-bp repeat, referred to as 5R(2). Between -340 and -560 bp there are seven copies of a 10-bp dyad repea t, referred to as 7R(1). We investigated the roles of these repeats in transcription from the US3 promoter in human foreskin fibroblast or H eLa cells. In transient transfection assays, the region containing 5R( 2) up-regulated transcription and was responsive to the p65 subunit of NF-kappa B. The DNA region containing 7R(1) down-regulated transcript ion from either the US3 promoter or a heterologous promoter in a posit ion and orientation-independent manner. Mutational analysis and transi ent transfections indicated that DNA containing the 10-bp dyad or one- half of the dyad was sufficient to cause repression of downstream gene expression, DNA probes containing one or more copies of the pentanucl eotide sequence TGTCG specifically bound cellular proteins, as demonst rated by electrophoretic mobility shift assays and cold-competition el ectrophoretic mobility shift assays. Two different DNA-protein complex es were detected with DNA probes containing one or two copies of the p entanucleotide, In HCMV-infected cell nuclear extracts, one of the DNA -protein complexes was present In amounts inversely proportional to th e amount of US3 transcription, Its formation was affected by dephospho rylation of the DNA-binding protein(s), Transient dephosphorylation of the cellular repressor protein may occur during HCMV infection, Repre ssion of US3 transcription mag relate to the number of pentanucleotide s and the Cellular proteins that bind to it, Twenty one copies of a TR TCG motif (R = purine) were found clustered upstream of the US3 gene a nd also in the modulator upstream of the HCMV IE1 and IE2 genes.