Rivera-Gonzalez R, Imbalzano A N, Gu B, Deluca N A
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pennsylvania 15261.
Virology. 1994 Aug 1;202(2):550-64. doi: 10.1006/viro.1994.1377.
The herpes simplex virus (HSV) type 1 immediate-early protein ICP4 represses transcription of its own gene and possibly that of other immediate-early genes. In the present study, we analyzed the role that an ICP4 binding site present in two HSV-1 promoters plays in the level and kinetics of expression during HSV-1 infection. Wild-type and mutant forms of the IE-3 (ICP4) promoter and the latency associated promoter (LAP) were fused to the thymidine kinase (tk) coding sequences and transferred to the genome of an ICP4-deficient virus. Promoter mutants were constructed to assess the effect of the ICP4 binding site in the presence and absence of defined binding sites for cellular and other viral factors in the promoters. The activities of the promoter constructs were inferred from the level of tk mRNA seen following viral infection in the absence of ICP4, in Vero cells and in the presence of ICP4 in ICP4 expressing E5 cells. Kinetics of expression and the dependence on DNA synthesis for expression were examined following infection of E5 cells. In the presence of the ICP4 binding site in LAP and in IE3 promoters lacking TAATGARAT motifs, expression was maximal late after infection and was greatly reduced when DNA synthesis was inhibited. When the ICP4 binding site was removed, both LAP and the IE3 construct retaining an Sp1 site were more abundantly expressed and exhibited kinetics of expression indistinguishable from that of the tk promoter. In vitro, ICP4 repressed LAP transcription mediated by the general transcription factors and upstream activating proteins. Deletion of the ICP4 binding site not only relieved repression, but in the presence of USF activity, ICP4 further induced LAP transcription. The results of these experiments suggest a role for the repressor activity of ICP4 in the temporal regulation of HSV-1 transcription.
单纯疱疹病毒1型（HSV-1）的立即早期蛋白ICP4可抑制其自身基因以及其他可能的立即早期基因的转录。在本研究中，我们分析了HSV-1两个启动子中存在的ICP4结合位点在HSV-1感染期间的表达水平和动力学中所起的作用。将IE-3（ICP4）启动子和潜伏相关启动子（LAP）的野生型和突变型形式与胸苷激酶（tk）编码序列融合，并转移至ICP4缺陷型病毒的基因组中。构建启动子突变体以评估在启动子中存在和不存在细胞及其他病毒因子的特定结合位点时ICP4结合位点的作用。从在Vero细胞中无ICP4的病毒感染后以及在表达ICP4的E5细胞中有ICP4存在时所观察到的tk mRNA水平推断启动子构建体的活性。在感染E5细胞后，检测表达动力学以及表达对DNA合成的依赖性。在LAP以及缺乏TAATGARAT基序的IE3启动子中存在ICP4结合位点时，感染后期表达最高，而当DNA合成受到抑制时表达大幅降低。当去除ICP4结合位点时，保留Sp1位点的LAP和IE3构建体均表达更为丰富，并且表现出与tk启动子无法区分的表达动力学。在体外，ICP4抑制由通用转录因子和上游激活蛋白介导的LAP转录。删除ICP4结合位点不仅解除了抑制作用，而且在存在USF活性的情况下，ICP4进一步诱导LAP转录。这些实验结果表明ICP4的阻遏活性在HSV-1转录的时间调控中发挥作用。
