Gu B, Rivera-Gonzalez R, Smith C A, DeLuca N A
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine 15261.
Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9528-32. doi: 10.1073/pnas.90.20.9528.
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.
单纯疱疹病毒1型感染细胞多肽4（HSV-1 ICP4）是一种多功能磷蛋白，对病毒感染至关重要。根据启动子的不同，它既是病毒基因表达的抑制因子，也是激活因子。ICP4可抑制其自身启动子的转录。在本研究中，我们使用来自HeLa细胞核提取物的通用转录因子、重组TATA结合蛋白（TBP）和TFIIB，以及转录激活因子Sp1，对ICP4启动子进行体外转录重建，并研究纯化的ICP4对转录的影响。ICP4能够有效抑制Sp1诱导的来自包含一个或多个Sp1结合位点的ICP4启动子模板的转录。观察到的抑制作用需要跨越转录起始位点的ICP4结合位点。当（i）转录反应中不包含Sp1，（ii）模板不包含Sp1结合位点，以及（iii）反应中使用TBP代替TFIID时，ICP4由于无法抑制转录，因此未抑制基础转录。体外观察结果与单纯疱疹病毒1型基因组在细胞中表达的相同构建体的行为一致。DNase I足迹实验表明，ICP4可以与TBP-TFIIB共同占据ICP4启动子区域，这表明ICP4不一定会阻止这些因子与TATA区域结合。数据表明，本研究中观察到的ICP4的抑制作用是由于ICP4干扰了与Sp1诱导转录相关的相互作用。
