Herpes simplex virus infected cell polypeptide 4 preferentially represses Sp1-activated over basal transcription from its own promoter.

Herpes simplex virus type 1 infected cell polypeptide 4 (HSV-1 ICP4) is a multifunctional phosphoprotein that is essential for viral infection. It is both a repressor and an activator of viral gene expression depending upon the promoter. ICP4 represses transcription from its own promoter. In the present study, we used general transcription factors from HeLa cell nuclear extracts, recombinant TATA binding protein (TBP) and TFIIB, and the transcriptional activator Sp1 to reconstitute in vitro transcription for the ICP4 promoter and to examine the effects of purified ICP4 on transcription. ICP4 was able to effectively repress Sp1-induced transcription from ICP4 promoter templates that contain one or multiple Sp1 binding sites. The observed inhibition required the ICP4 binding site that spans the transcription initiation site. ICP4 did not inhibit basal transcription as inferred by its inability to inhibit transcription when (i) Sp1 was not included in transcription reactions, (ii) the templates contained no Sp1 binding sites, and (iii) TBP was used in place of TFIID in the reactions. The in vitro observations were consistent with the behavior of the same constructs expressed in cells from the herpes simplex virus type 1 genome. DNase I footprinting experiments revealed that ICP4 could co-occupy the ICP4 promoter region with TBP-TFIIB, indicating that ICP4 does not necessarily exclude these factors from binding to the TATA region. The data suggest that the repressive effects of ICP4 observed in this study result from ICP4 interfering with the interactions contributing to Sp1-induced transcription. IMAGES:1型单纯疱疹病毒感染细胞多肽4（HSV-1 ICP4）是一种多功能磷酸化蛋白，对病毒感染至关重要。其可根据启动子类型的不同，同时作为病毒基因表达的阻遏物与激活物。ICP4可阻遏自身启动子的转录。 本研究中，我们以HeLa细胞核提取物中的通用转录因子、重组TATA结合蛋白（TATA binding protein, TBP）及转录因子IIB（TFIIB），辅以转录激活因子Sp1，重建了ICP4启动子的体外转录体系，并探究纯化ICP4对转录的调控作用。ICP4可有效阻遏携带单个或多个Sp1结合位点的ICP4启动子模板中Sp1诱导的转录。该抑制效应依赖于跨越转录起始位点的ICP4结合位点。当转录反应体系未添加Sp1、模板不含Sp1结合位点，或反应中以TBP替代转录因子IID（TFIID）时，ICP4均无法抑制转录，由此可推断ICP4不会抑制基础转录。上述体外实验结果与1型单纯疱疹病毒基因组中相同构建体在细胞内的表达特性一致。DNase I足迹实验结果显示，ICP4可与TBP-TFIIB复合物共同结合ICP4启动子区域，这表明ICP4并非必然会阻断这些因子与TATA区域的结合。本研究数据表明，本次实验中观测到的ICP4阻遏效应，源于ICP4干扰了参与Sp1诱导转录的相互作用过程。图像：