Paterson T, Everett R D
MRC Virology Unit, Institute of Virology, Glasgow, UK.
Nucleic Acids Res. 1988 Dec 9;16(23):11005-25. doi: 10.1093/nar/16.23.11005.
The immediate-early (IE) protein Vmw175 (ICP4) of HSV-1 is required for the transcription of later classes of viral genes and the repression of IE gene expression. We have previously constructed a panel of plasmid-borne insertion and deletion mutants of the gene encoding Vmw175 and assayed their ability to regulate transcription in transient transfection assays. By this approach we have mapped the regions of the Vmw175 amino acid sequence that are required for transcriptional activation and repression of herpes virus promoters. This paper describes the use of nuclear extracts, made from cells transfected with these mutant plasmids, in gel retardation DNA binding assays in order to define the regions of Vmw175 involved in binding to a specific Vmw175 DNA binding site. The results show that amino acid residues 275-495 (a region which is highly conserved between Vmw175 and the varicella-zoster virus "IE" 140K protein) include structures which are critically required for specific DNA binding, transactivation and repression. This raises the interesting paradox that although the specific DNA sequence recognized by Vmw175 is not commonly found in its target promoters, the protein domain required for recognition of this sequence is required for promoter activation.
单纯疱疹病毒1型（HSV-1）的即刻早期（IE）蛋白Vmw175（ICP4）是后续各类病毒基因转录及IE基因表达抑制所必需的。我们之前构建了一组编码Vmw175基因的质粒携带插入和缺失突变体，并在瞬时转染试验中检测了它们调节转录的能力。通过这种方法，我们绘制了Vmw175氨基酸序列中对于疱疹病毒启动子转录激活和抑制所必需的区域。本文描述了使用从转染了这些突变体质粒的细胞中制备的核提取物进行凝胶阻滞DNA结合试验，以确定Vmw175中与特定Vmw175 DNA结合位点结合的区域。结果表明，氨基酸残基275 - 495（该区域在Vmw175和水痘带状疱疹病毒“IE” 140K蛋白之间高度保守）包含特定DNA结合、反式激活和抑制所必需的结构。这就产生了一个有趣的悖论，即尽管Vmw175识别的特定DNA序列在其靶启动子中并不常见，但识别该序列所需的蛋白质结构域却是启动子激活所必需的。
