Sekulovich R E, Leary K, Sandri-Goldin R M
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine 92717.
J Virol. 1988 Dec;62(12):4510-22. doi: 10.1128/JVI.62.12.4510-4522.1988.
The herpes simplex virus type 1 (HSV-1) alpha proteins ICP4, ICP0, and ICP27 are trans-acting proteins which affect HSV-1 gene expression. To investigate potential interactions between these alpha products and to determine the specificity of action of the alpha proteins in combination with each other compared with their activities individually, we performed a series of transient-expression assays. In these assays we used plasmids containing the alpha genes encoding ICP4, ICP0, and ICP27 either singly or in combination as effectors and HSV-1 genes of different kinetic classes and heterologous genes as targets. The HSV-1 targets consisted of promoter-regulatory domains from alpha (ICP0 and ICP27), beta (thymidine kinase and alkaline exonuclease), beta-gamma (glycoprotein D, glycoprotein B, and VP5), and gamma (glycoprotein C) genes, each fused to the chloramphenicol acetyltransferase (CAT) gene. The heterologous target genes consisted of the simian virus 40 early promoter with enhancer and the Rous sarcoma virus long terminal repeat promoter and enhancer each fused to the CAT gene. Target promoter activity was measured by the assay of CAT activity in extracts of transfected cells and by Northern (RNA) blot hybridization of CAT mRNA. The results of these experiments showed that ICP4 activated only HSV-1 target genes, whereas ICP0 activated all of the targets and ICP27 had little effect on any of the targets. ICP4 and ICP0 had a synergistic effect when inducing HSV-1 targets, but they did not have this effect on the heterologous targets pSV2-CAT or pRSV-CAT. In fact, lower levels of CAT activity and CAT mRNA were found in the presence of both effectors than with ICP0 alone. Most interestingly, although the effector plasmid containing the ICP27 gene had little effect on its own, two different and marked effects depending on the target were observed when ICP27 was combined with ICP4 or ICP0 or both. A trans-repression of the induction seen with ICP4 and ICP0 was found when ICP27 was present in the transfections with pSV2-CAT, pRSV-CAT, pICP0-CAT, pICP27-CAT, pTK-CAT, pgD-CAT, pgB-CAT, and pgC-CAT. This resulted in CAT activity levels which were similar to or lower than the basal level of expression of the target genes in the absence of effector plasmids. This trans-repression occurred over a wide range of concentrations of input ICP27 plasmid. In contrast to this repressive effect of ICP27, a trans-activation was seen when ICP4, ICP0, and ICP27 plasmids were combined in transfections with pAE-CAT and pVP5-CAT as targets. This trans-activation also occurred over a 10-fold range of input ICP27 plasmid. These results suggest that ICP27 can facilitate both down
1型单纯疱疹病毒（HSV-1）的α蛋白ICP4、ICP0和ICP27是反式作用蛋白，可影响HSV-1基因表达。为了研究这些α蛋白产物之间的潜在相互作用，并确定α蛋白相互组合时与各自单独活性相比的作用特异性，我们进行了一系列瞬时表达试验。在这些试验中，我们使用了分别含有编码ICP4、ICP0和ICP27的α基因的质粒，单独或组合作为效应物，以及不同动力学类别的HSV-1基因和异源基因作为靶标。HSV-1靶标由来自α（ICP0和ICP27）、β（胸苷激酶和碱性核酸外切酶）、β-γ（糖蛋白D、糖蛋白B和VP5）和γ（糖蛋白C）基因的启动子调控结构域组成，每个结构域都与氯霉素乙酰转移酶（CAT）基因融合。异源靶基因由带有增强子的猴病毒40早期启动子以及劳斯肉瘤病毒长末端重复启动子和增强子组成，每个都与CAT基因融合。通过检测转染细胞提取物中的CAT活性以及对CAT mRNA进行Northern（RNA）印迹杂交来测量靶启动子活性。这些实验结果表明，ICP4仅激活HSV-1靶基因，而ICP0激活所有靶标，ICP27对任何靶标几乎没有影响。ICP4和ICP0在诱导HSV-1靶标时具有协同作用，但它们对异源靶标pSV2-CAT或pRSV-CAT没有这种作用。事实上，在两种效应物存在的情况下，检测到的CAT活性和CAT mRNA水平低于单独使用ICP0时的水平。最有趣的是，尽管含有ICP27基因的效应物质粒本身几乎没有作用，但当ICP27与ICP4或ICP0或两者组合时，根据靶标的不同观察到两种不同且显著的效应。在用pSV2-CAT、pRSV-CAT、pICP0-CAT、pICP27-CAT、pTK-CAT、pgD-CAT、pgB-CAT和pgC-CAT进行转染时，发现ICP27存在时会对ICP4和ICP0诱导的反应产生反式抑制。这导致CAT活性水平与在无效应物质粒时靶基因的基础表达水平相似或更低。这种反式抑制在广泛的输入ICP27质粒浓度范围内都能发生。与ICP27的这种抑制作用相反，当将ICP4、ICP0和ICP27质粒组合用于以pAE-CAT和pVP5-CAT为靶标的转染时，观察到反式激活。这种反式激活也发生在10倍范围的输入ICP27质粒浓度内。这些结果表明，ICP27可以促进两者的下调
