The role of a repetitive palindromic sequence element in the human cytomegalovirus major immediate early enhancer.
The major enhancer, extending from nucleotides -530 to -120 upstream of the transcription initiation site of immediate early (IE) genes 1 and 2 in human cytomegalovirus (HCMV), contains four groups of repeated sequence motifs that consist of 17, 18, 19 or 21 bp, respectively. One of these elements, the 19 bp repeat, is a symmetrical palindrome that is also part of IE regulatory sequences of other cytomegalovirus-type herpesviruses, but not of unrelated members of the herpesvirus group. Synthetic oligonucleotides representing the 19 bp repeat unit strongly reduced the activity of the IE1/2 enhancer/promoter in cotransfection assays after transient expression. The HCMV enhancer can substitute for the 72 bp repeats of simian virus 40 (SV40). Replication-competent deletion mutants of SV40/HCMV enhancer recombinants were constructed that contained a single palindromic 19 bp repeat with a central cleavage site for AhaII. If deletions were introduced into the single remaining 19 bp repeat most of the mutant viruses were still replication-competent in CV-1 monkey kidney cells. Insertion of two nucleotides into the single AhaII site did not significantly alter transient SV40 T antigen expression. Deletion of four nucleotides or more from the single 19 bp palindrome reduced the stimulation of T antigen synthesis by the HCMV enhancer/SV40 promoter unit down to about 50%. More extended deletions (28 to 80 bp) did not further reduce T antigen expression. All mutants without an intact 19 bp repeat contained the 18 bp and/or the 21 bp sequence motif. DNase I footprinting and gel retardation assays indicated sequence-specific protein binding by the 19 bp palindrome. Altered palindromes, correlating with reduced enhancer activity, lost most of their protein-binding properties. Thus, the 19 bp repeat element is one of several protein-binding sites that contribute to enhancer strength. However, the 19 bp sequence motif can be deleted entirely to leave reduced activity. The HCMV IE1/2 upstream sequence appears to be the perfect model of an enhancer as a complex of multiple binding sites for trans-activating proteins in a modular fashion.
Fickenscher
H
H
Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, F.R.G.
Stamminger
T
T
Rüger
R
R
Fleckenstein
B
B
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D003587:Cytomegalovirus / Q000235:genetics* / Q000502:physiology; D004279:DNA, Viral / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D005455:Fluorescent Antibody Technique; D006801:Humans; D017874:Immediate-Early Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D014162:Transfection; D014779:Virus Replication
D000956:Antigens, Viral; D004279:DNA, Viral; D017874:Immediate-Early Proteins; C044582:immediate-early proteins, cytomegalovirus