Department of Microbiology and Immunology, Center for Molecular and Tumor Virology, Louisiana State University Health Sciences Center, Shreveport, LA 71130-3932, USA.
Virus Res. 2012 Apr;165(1):52-60. doi: 10.1016/j.virusres.2012.01.005. Epub 2012 Jan 14.
The immediate-early protein (IEP), the major regulatory protein encoded by the IE gene of equine herpesvirus 1 (EHV-1), plays a crucial role as both transcription activator and repressor during a productive lytic infection. To investigate the mechanism by which the EHV-1 IEP inhibits its own promoter, IE promoter-luciferase reporter plasmids containing wild-type and mutant IEP-binding site (IEBS) were constructed and used for luciferase reporter assays. The IEP inhibited transcription from its own promoter in the presence of a consensus IEBS (5'-ATCGT-3') located near the transcription initiation site but did not inhibit when the consensus sequence was deleted. To determine whether the distance between the TATA box and the IEBS affects transcriptional repression, the IEBS was displaced from the original site by the insertion of synthetic DNA sequences. Luciferase reporter assays revealed that the IEP is able to repress its own promoter when the IEBS is located within 26-bp from the TATA box. We also found that the proper orientation and position of the IEBS were required for the repression by the IEP. Interestingly, the level of repression was significantly reduced when a consensus TATA sequence was deleted from the promoter region, indicating that the IEP efficiently inhibits its own promoter in a TATA box-dependent manner. Taken together, these results suggest that the EHV-1 IEP delicately modulates autoregulation of its gene through the consensus IEBS that is near the transcription initiation site and the TATA box.
立即早期蛋白（IEP）是马疱疹病毒 1（EHV-1）IE 基因编码的主要调节蛋白，在产生活性裂解感染过程中作为转录激活子和转录阻遏物发挥关键作用。为了研究 EHV-1 IEP 抑制其自身启动子的机制，构建了包含野生型和突变型 IEP 结合位点（IEBS）的 IE 启动子-荧光素酶报告质粒，并用于荧光素酶报告基因检测。IEP 在存在位于转录起始位点附近的共识 IEBS（5'-ATCGT-3'）的情况下抑制自身启动子的转录，但当删除共识序列时则不抑制。为了确定 TATA 盒和 IEBS 之间的距离是否影响转录抑制，通过插入合成 DNA 序列将 IEBS 从原始位置置换。荧光素酶报告基因检测显示，当 IEBS 位于 TATA 盒 26 个碱基内时，IEP 能够抑制自身启动子。我们还发现，IEBS 的适当取向和位置对于 IEP 的抑制作用是必需的。有趣的是，当从启动子区域删除共识 TATA 序列时，抑制水平显著降低，表明 IEP 以 TATA 盒依赖性方式有效地抑制自身启动子。总之，这些结果表明，EHV-1 IEP 通过位于转录起始位点附近的共识 IEBS 和 TATA 盒精细地调节其基因的自身调节。
