O'Rourke D, O'Hare P
Marie Curie Research Institute, Surrey, England.
J Virol. 1993 Dec;67(12):7201-14. doi: 10.1128/JVI.67.12.7201-7214.1993.
We have examined the cis- and trans-acting factors involved in constitutive transcription of the promoter for the IE110k protein of herpes simplex virus type 1. Our results indicate that while the IE110k gene is activated by Vmw65, it also exhibits very efficient constitutive expression approximating that from the simian virus 40 early enhancer-promoter region. We show that despite the presence of multiple copies of the octamer consensus site which mediate Oct-1 binding and subsequent Vmw65 activation, these upstream sequences have a minor effect on constitutive transcription. By progressive exonuclease digestion and subsequent site-directed mutagenesis of the promoter, we have identified a 15-bp region (termed the EC region), from position -89 to -74, which is required for efficient constitutive expression from the IE110k promoter. We demonstrate that two cellular proteins interact with this region and, by competition and methylation interference analyses, show they have distinct but overlapping sequence requirements for binding. One of these proteins is identified as NF-Y, a CCAAT box-binding factor, which binds an inverted CCAAT box located between positions -71 and -75. The second cellular factor, F2, appears to be novel and binds a region with the sequence CGCGCGGC CAT which overlaps the 3' end of the CCAAT box. The terminal AT of the recognition site for F2 represents, on the opposite strand, the terminal AT of the CCAAT box, and these and adjacent bases are critically required for the binding of both factors. These results together with further competition analysis indicate that these factors bind in a mutually exclusive manner to the EC region. The implications of these results for regulation of expression of the IE110k gene are discussed.
我们研究了单纯疱疹病毒1型IE110k蛋白启动子组成型转录所涉及的顺式和反式作用因子。我们的结果表明，虽然IE110k基因由Vmw65激活，但它也表现出非常高效的组成型表达，接近猿猴病毒40早期增强子-启动子区域的表达水平。我们发现，尽管存在多个介导Oct-1结合及随后Vmw65激活的八聚体共有位点，但这些上游序列对组成型转录的影响较小。通过对启动子进行渐进性核酸外切酶消化及随后的定点诱变，我们确定了一个15bp的区域（称为EC区域），位于-89至-74位，这是IE110k启动子高效组成型表达所必需的。我们证明有两种细胞蛋白与该区域相互作用，通过竞争和甲基化干扰分析表明它们在结合时有不同但重叠的序列要求。其中一种蛋白被鉴定为NF-Y，一种CCAAT盒结合因子，它结合位于-71至-75位之间的反向CCAAT盒。第二种细胞因子F2似乎是新发现的，它结合一个序列为CGCGCGGC CAT的区域，该区域与CCAAT盒的3'端重叠。F2识别位点的末端AT在互补链上代表CCAAT盒的末端AT，这些以及相邻碱基对于两种因子的结合都至关重要。这些结果连同进一步的竞争分析表明，这些因子以互斥的方式结合到EC区域。我们讨论了这些结果对IE110k基因表达调控的意义。
