Fickenscher H, Stamminger T, Rüger R, Fleckenstein B
Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, F.R.G.
J Gen Virol. 1989 Jan;70 ( Pt 1):107-23. doi: 10.1099/0022-1317-70-1-107.
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.
人巨细胞病毒（HCMV）即刻早期（IE）基因1和2转录起始位点上游从核苷酸-530至-120延伸的主要增强子，包含四组重复序列基序，分别由17、18、19或21个碱基对组成。其中一个元件，即19个碱基对的重复序列，是一个对称回文序列，也是其他巨细胞病毒型疱疹病毒IE调控序列的一部分，但不是疱疹病毒组中无关成员的一部分。代表19个碱基对重复单元的合成寡核苷酸在瞬时表达后的共转染实验中强烈降低了IE1/2增强子/启动子的活性。HCMV增强子可以替代猴病毒40（SV40）的72个碱基对重复序列。构建了具有复制能力的SV40/HCMV增强子重组体缺失突变体，其包含一个具有AhaII中心切割位点的单回文19个碱基对重复序列。如果在剩余的单个19个碱基对重复序列中引入缺失，大多数突变病毒在CV-1猴肾细胞中仍具有复制能力。在单个AhaII位点插入两个核苷酸不会显著改变瞬时SV40 T抗原的表达。从单个19个碱基对回文中缺失四个或更多核苷酸会使HCMV增强子/SV40启动子单元对T抗原合成的刺激降低至约50%。更广泛的缺失（28至80个碱基对）不会进一步降低T抗原的表达。所有没有完整19个碱基对重复序列的突变体都包含18个碱基对和/或21个碱基对序列基序。DNase I足迹分析和凝胶阻滞分析表明19个碱基对回文序列存在序列特异性蛋白结合。与增强子活性降低相关的改变的回文序列失去了其大部分蛋白结合特性。因此，19个碱基对重复元件是有助于增强子强度的几个蛋白结合位点之一。然而，19个碱基对序列基序可以完全缺失，从而使活性降低。HCMV IE1/2上游序列似乎是作为反式激活蛋白多个结合位点以模块化方式组成的复合物的增强子的完美模型。
