Regulation of a human cytomegalovirus immediate-early gene (US3) by a silencer-enhancer combination.
The US3 open reading frame of human cytomegalovirus (HCMV) is transcribed 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 5R2. Between -340 and -560 bp there are seven copies of a 10-bp dyad repeat, referred to as 7R1. We investigated the roles of these repeats in transcription from the US3 promoter in human foreskin fibroblast or HeLa cells. In transient transfection assays, the region containing 5R2 up-regulated transcription and was responsive to the p65 subunit of NF-kappa B. The DNA region containing 7R1 down-regulated transcription from either the US3 promoter or a heterologous promoter in a position- and orientation-independent manner. Mutational analysis and transient 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 pentanucleotide sequence TGTCG specifically bound cellular proteins, as demonstrated by electrophoretic mobility shift assays and cold-competition electrophoretic mobility shift assays. Two different DNA-protein complexes were detected with DNA probes containing one or two copies of the pentanucleotide. In HCMV-infected cell nuclear extracts, one of the DNA-protein complexes was present in amounts inversely proportional to the amount of US3 transcription. Its formation was affected by dephosphorylation of the DNA-binding protein(s). Transient dephosphorylation of the cellular repressor protein may occur during HCMV infection. Repression of US3 transcription may relate to the number of pentanucleotides and the cellular proteins that bind to it. Twenty-one copies of a TRTCG motif (R = purine) were found clustered upstream of the US3 gene and also in the modulator upstream of the HCMV IE1 and IE2 genes.
Thrower
A R
AR
Program in Genetics, School of Medicine, University of Iowa, Iowa City 52242, USA.
Bullock
G C
GC
Bissell
J E
JE
Stinski
M F
MF
D001483:Base Sequence; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D015342:DNA Probes; D004279:DNA, Viral; D004268:DNA-Binding Proteins / Q000378:metabolism; D004742:Enhancer Elements, Genetic; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D006023:Glycoproteins; D006367:HeLa Cells; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014158:Transcription, Genetic
D015342:DNA Probes; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D006023:Glycoproteins; D017874:Immediate-Early Proteins; D008565:Membrane Proteins; C095111:US3 protein, cytomegalovirus