Sequences in the human cytomegalovirus 2.7-kilobase RNA promoter which mediate its regulation as an early gene.
We have studied the regulation of expression of a major human cytomegalovirus (HCMV) early transcription unit located within the long repeat of the strain AD169 genome. This region specified a 2.7-kilobase RNA which underwent its largest increase in abundance between 8 and 14 h postinfection. To study the regulation of this gene, its promoter was cloned 5' of the gene for chloramphenicol acetyltransferase (CAT) for use in transient expression assays. A construct containing 651 base pairs of upstream sequence and 54 base pairs of leader sequence was transfected into human fibroblast cells, followed by HCMV infection. Analysis of the steady-state levels of RNA expressed from this hybrid gene indicated that it accumulated with the same kinetics as the authentic viral transcript early in the infection. Cotransfection of human fibroblasts with the 2.7-kilobase RNA promoter-CAT construct and plasmids containing different HCMV immediate-early (IE) genes showed that the region of the HCMV genome encoding the transcription units corresponding to IE1 and 2 and the 5' end of IE3 is capable of stimulating promoter activity but not to the same extent as HCMV infection. To define important cis-acting regulatory elements in the promoter, a series of 5' deletion mutants was constructed. Transient expression analysis showed a stepwise reduction in inducible CAT activity, suggesting the presence of multiple regulatory sites. To further characterize the nature of these sites, we used gel mobility shift assays to study DNA-protein interactions occurring within this promoter sequence. With nuclear extracts prepared from HeLa cells as well as from infected and uninfected human foreskin fibroblasts, we found specific binding of a cellular factor to a region of the promoter important in HCMV inducible activity. This region contains a palindromic octamer with homology to the binding site of the cellular factor USF/MLTF.
Klucher
K M
KM
Department of Biology, University of California, San Diego, LaJolla 92093.
Rabert
D K
DK
Spector
D H
DH
D001483:Base Sequence; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000235:genetics*; D015183:Restriction Mapping; D012867:Skin; D014162:Transfection
D004279:DNA, Viral; D012367:RNA, Viral; D015500:Chloramphenicol O-Acetyltransferase