Lang D, Gebert S, Arlt H, Stamminger T
Institut für Klinische und Molekulare Virologie, Universität Erlangen-Nürnberg, Germany.
J Virol. 1995 Oct;69(10):6030-7. doi: 10.1128/JVI.69.10.6030-6037.1995.
The 86-kDa IE2 protein (IE86) of human cytomegalovirus (HCMV) has been described as a promiscuous transactivator of viral, as well as cellular, gene expression. Investigation of the mechanism used by IE86 to activate gene expression from the early UL112/113 promoter of HCMV revealed the existence of three binding sites for IE86 located between nucleotides -290 and -120 relative to the transcriptional start site (H. Arlt, D. Lang, S. Gebert, and T. Stamminger, J. Virol. 68:4117-4125, 1994). As shown previously, deletion of these target sites resulted in a reduction of IE86-mediated transactivation by approximately 70%. The remaining promoter, however, could still be stimulated about 40-fold, indicating the presence of an additional responsive element within these sequences. Here, we provide evidence that a binding site for the cellular transcription factor CREB can also act as a target for IE86 transactivation. By DNase I protection analysis, a binding sequence for CREB could be detected between nucleotides -78 and -56 within the respective promoter region. After in vitro mutagenesis of this CREB-binding site within the context of the entire UL112/113 promoter, a marked reduction in transactivation levels was evident. Moreover, when individual CREB-binding sites were positioned upstream of a minimal, TATA box-containing UL112/113 promoter, they were able to confer strong IE86 responsiveness, whereas a mutated sequence did not exert any effect. In far Western blot and pull-down experiments, a direct interaction of IE86 with the cellular transcription factor CREB could be observed. The in vivo relevance of this in vitro interaction was confirmed by using various GAL4 fusion proteins in the presence or absence of IE86 which revealed a strong activation only in the presence of both a GAL4-CREB fusion and IE86. This shows that at least one specific member of the ATF/CREB family of transcription factors is involved in mediating transactivation by the HCMV IE86 protein.
人巨细胞病毒（HCMV）的86-kDa IE2蛋白（IE86）被描述为病毒以及细胞基因表达的多效性反式激活因子。对IE86用于激活HCMV早期UL112/113启动子基因表达的机制进行研究后发现，相对于转录起始位点，在核苷酸-290至-120之间存在三个IE86结合位点（H. Arlt、D. Lang、S. Gebert和T. Stamminger，《病毒学杂志》68:4117-4125，1994年）。如先前所示，删除这些靶位点会导致IE86介导的反式激活降低约70%。然而，剩余的启动子仍可被刺激约40倍，这表明在这些序列中存在一个额外的反应元件。在此，我们提供证据表明，细胞转录因子CREB的结合位点也可作为IE86反式激活的靶点。通过DNase I保护分析，在相应启动子区域内的核苷酸-78至-56之间可检测到CREB的结合序列。在整个UL112/113启动子背景下对该CREB结合位点进行体外诱变后，反式激活水平明显降低。此外，当单个CREB结合位点位于含TATA框的最小UL112/113启动子上游时，它们能够赋予强烈的IE86反应性，而突变序列则没有任何作用。在远Western印迹和下拉实验中，可观察到IE86与细胞转录因子CREB的直接相互作用。通过使用各种GAL4融合蛋白在有或无IE86的情况下进行实验，证实了这种体外相互作用在体内的相关性，结果显示仅在同时存在GAL4-CREB融合蛋白和IE86时才会有强烈的激活。这表明转录因子ATF/CREB家族中至少有一个特定成员参与介导HCMV IE86蛋白的反式激活。
