Identification of sequence requirements and trans-acting functions necessary for regulated expression of a human cytomegalovirus early gene.
We analyzed the regulation of expression of a human cytomegalovirus (HCMV) early transcription unit encoded by EcoRI fragments R and d (map units, 0.682 to 0.713), located within the long unique segment of the genome. This region specified a 2.2-kilobase class of spliced transcripts which encode several related proteins. To define important upstream regulatory elements of this gene, we generated hybrid plasmids in which 5'-promoter sequences were fused to the Escherichia coli chloramphenicol acetyltransferase (CAT) gene and tested these hybrid genes in transient expression assays in human fibroblast cells. The stimulation of CAT activity in HCMV-infected cells was found to reflect an induction of correctly initiated hybrid mRNA, which was dependent on the de novo synthesis of some virally induced factor(s). A time course experiment showed the correct early kinetics of CAT expression. Analysis of a series of 5'-promoter deletion plasmids, ending between -323 and -7 base pairs relative to the transcription start site, showed a stepwise reduction in inducible CAT activity, suggesting that this HCMV early promoter consists of multiple elements. One of these elements resembles the binding site of a previously identified cellular "transcription" factor. We also examined the role of specific virus-encoded factors in the transactivation of this promoter. Cotransfection of human fibroblasts with the 2.2-kilobase RNA promoter-CAT construct and plasmids containing different immediate-early genes showed that expression of CAT from this promoter was stimulated by the region of the HCMV genome encoding the immediate-early 1 and 2 gene products.
Staprans
S I
SI
Department of Biology, University of California, San Diego, La Jolla 92093.
Rabert
D K
DK
Spector
D H
DH
D000123:Acetyltransferases / Q000235:genetics; D000956:Antigens, Viral / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase; D003587:Cytomegalovirus / Q000235:genetics*; D004720:Endonucleases; D005347:Fibroblasts; D005786:Gene Expression Regulation; D005814:Genes, Viral; D006801:Humans; D017874:Immediate-Early Proteins; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000032:analysis; D012091:Repetitive Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic; D014162:Transfection
D000956:Antigens, Viral; D017874:Immediate-Early Proteins; D012367:RNA, Viral; D014157:Transcription Factors; C044582:immediate-early proteins, cytomegalovirus; D000123:Acetyltransferases; D015500:Chloramphenicol O-Acetyltransferase; D004720:Endonucleases; D015719:Single-Strand Specific DNA and RNA Endonucleases