Arlt H, Lang D, Gebert S, Stamminger T
Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, Erlangen, Germany.
J Virol. 1994 Jul;68(7):4117-25. doi: 10.1128/JVI.68.7.4117-4125.1994.
The 86-kDa IE2 protein (IE86) of human cytomegalovirus (HCMV) can act as both an activator and a repressor of gene expression. The mechanisms for both of these functions are not well defined. It has recently been demonstrated that this protein has sequence-specific DNA binding properties: it interacts directly with a target sequence that is located between the TATA box and the cap site of its own promoter. This sequence, termed the CRS (cis repression signal) element, is required for negative autoregulation of the IE1/IE2 enhancer/promoter by IE2. We demonstrate now that binding of this protein to DNA is not confined to this site but occurs also within an early promoter of HCMV that has previously been shown to be strongly IE2 responsive. By DNase I protection analysis using a purified, procaryotically expressed IE2 protein, we could identify three binding sites within the region of -290 to -120 of the UL112 promoter of HCMV. Competition in DNase I protection experiments as well as gel retardation experiments showed that the identified binding sites are specific and have high affinity. Deletion of IE2 binding sites from this promoter reduced the level of transactivation; however, the remaining promoter could still be stimulated about 40-fold. Constructs in which IE2 binding sites were fused directly to the TATA box of the UL112 promoter did not reveal a significant contribution of these sequences to transactivation. However, if an IE2 binding site was reinserted upstream of nucleotide -117 of the UL112 promoter, an increase in transactivation by IE2 was obvious, whereas a mutated sequence could not mediate this effect. This finding suggests that DNA-bound IE2 can contribute to transactivation but seems to require the presence of additional transcription factors. Moreover, a comparison of the detected IE2 binding sites could not detect a strong homology, suggesting that this protein may be able to interact with a broad spectrum of different target sequences.
人巨细胞病毒（HCMV）的86-kDa IE2蛋白（IE86）既可以作为基因表达的激活剂，也可以作为抑制剂。这两种功能的机制尚未完全明确。最近有研究表明，该蛋白具有序列特异性DNA结合特性：它直接与位于自身启动子的TATA盒和帽位点之间的靶序列相互作用。这个序列被称为CRS（顺式抑制信号）元件，是IE2对IE1/IE2增强子/启动子进行负向自动调节所必需的。我们现在证明，该蛋白与DNA的结合并不局限于这个位点，在HCMV的一个早期启动子中也会发生结合，该启动子先前已被证明对IE2有强烈反应。通过使用纯化的、原核表达的IE2蛋白进行DNase I保护分析，我们可以在HCMV UL112启动子的-290至-120区域内鉴定出三个结合位点。DNase I保护实验以及凝胶阻滞实验中的竞争表明，所鉴定的结合位点具有特异性且亲和力高。从该启动子中删除IE2结合位点会降低反式激活水平；然而，剩余的启动子仍可被刺激约40倍。将IE2结合位点直接与UL112启动子的TATA盒融合的构建体并未显示这些序列对反式激活有显著贡献。但是，如果在UL112启动子的核苷酸-117上游重新插入一个IE2结合位点，IE2的反式激活会明显增加，而突变序列则无法介导这种效应。这一发现表明，与DNA结合的IE2可以促进反式激活，但似乎需要其他转录因子的存在。此外，对检测到的IE2结合位点进行比较未发现强烈的同源性，这表明该蛋白可能能够与广泛的不同靶序列相互作用。
