Gu B, DeLuca N
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pennsylvania 15261.
J Virol. 1994 Dec;68(12):7953-65. doi: 10.1128/JVI.68.12.7953-7965.1994.
During infection with herpes simplex virus, infected-cell polypeptide 4 (ICP4) activates transcription of most herpes simplex virus genes. In the present study, the mechanism of activation of transcription by ICP4 was investigated by using a reconstituted in vitro system with fractionated and purified general transcription factors, coupled with DNA-binding assays. The templates used in the reactions included regions of the gC and thymidine kinase (tk) promoters in plasmids, and on isolated fragments, allowing for the evaluation of the potential function of naturally occurring and inserted ICP4-binding sites and elements of the core promoter. ICP4 efficiently activated transcription of the gC promoter by facilitating the formation of transcription initiation complexes. ICP4 could not substitute for any of the basal transcription factors. Moreover, TATA-binding protein (TBP) could not substitute for TFIID in activation, suggesting a requirement for TBP-associated factors. Interactions between ICP4 and DNA 3' to the start site was necessary for activation of the gC promoter. The requirement for DNA-protein contacts could be met either by the presence of an ICP4-binding site in the gC leader, by the presence of a site more than 150 nucleotides further downstream, by an inserted site that normally acts to repress transcription, or by the addition of sufficient non-site-containing DNA. The gC TATA box and start site, or initiator element (inr), were individually sufficient for activation by ICP4 and together contributed to optimal activation. In contrast to gC, the tk promoter was poorly activated in the reconstituted system. However, the tk TATA box was efficiently activated when the tk start site region was replaced with the gC inr, suggesting that activation was mediated through the inr and inr-binding proteins. In addition, mutation of the inr core resulted in a gC promoter that was very poorly activated by ICP4. The results of this and previous studies demonstrate that ICP4 activates transcription in a complex manner involving contacts with DNA 3' to the start site, TBP, TFIIB, TBP-associated factors, and possibly proteins functioning at the start site of transcription.
在单纯疱疹病毒感染期间，感染细胞多肽4（ICP4）可激活大多数单纯疱疹病毒基因的转录。在本研究中，通过使用含有分级分离和纯化的通用转录因子的体外重组系统，并结合DNA结合分析，对ICP4激活转录的机制进行了研究。反应中使用的模板包括质粒中gC和胸苷激酶（tk）启动子区域，以及分离的片段，以便评估天然存在和插入的ICP4结合位点及核心启动子元件的潜在功能。ICP4通过促进转录起始复合物的形成有效地激活了gC启动子的转录。ICP4不能替代任何基础转录因子。此外，TATA结合蛋白（TBP）在激活过程中不能替代TFIID，这表明需要TBP相关因子。ICP4与起始位点下游3'端的DNA之间的相互作用对于gC启动子的激活是必要的。DNA-蛋白质接触的要求可以通过gC前导序列中存在ICP4结合位点、下游超过150个核苷酸处存在一个位点、通常起转录抑制作用的插入位点或添加足够的不含位点的DNA来满足。gC TATA框和起始位点或起始子元件（inr）单独就足以被ICP4激活，并且共同促成最佳激活。与gC相反，tk启动子在重组系统中激活较差。然而，当tk起始位点区域被gC inr取代时，tk TATA框被有效激活，这表明激活是通过inr和inr结合蛋白介导的。此外，inr核心的突变导致gC启动子被ICP4激活的能力非常差。本研究及先前研究的结果表明，ICP4以复杂的方式激活转录，涉及与起始位点下游3'端的DNA、TBP、TFIIB、TBP相关因子以及可能在转录起始位点起作用的蛋白质的接触。
