Gribaudo G, Ravaglia S, Gaboli M, Gariglio M, Cavallo R, Landolfo S
Institute of Microbiology, University of Torino, Italy.
Virology. 1995 Aug 1;211(1):251-60. doi: 10.1006/viro.1995.1398.
Transcription of murine cytomegalovirus (MCMV) immediate-early (IE) genes is regulated by the interaction of cellular transcription factors with a strong viral enhancer controlling promoters flanking both sides of the regulatory sequence. We have previously demonstrated that interferon-alpha (IFN-alpha) inhibits MCMV replication by impairing the transcription of IE genes. To define the cis-acting elements and trans-acting factors involved in this inhibition, permissive murine fibroblasts were transferred with DNA constructs containing the chloramphenicol acetyl transferase reporter gene and portions of the IE enhanced. The region spanning -1185 to -259 relative to the IE1-3 promoter was sufficient to allow IFN-alpha-induced inhibition. Since this segment contains several NF-kappa B sites, cells were transfected with a construct containing three copies of NF-kappa B element in front of the homologous minimal IE1-3 promoter. Upon IFN-alpha treatment the reporter gene activity was strongly reduced, indicating that NF-kappa B binding site is sufficient to confer inhibition. The specificity of this inhibition was demonstrated by the lack of a significant effect on the activity of DNA constructs containing either a mutated NF-kappa B trimer or an ATF/CRE trimer. Gel shift assays with NF-kappa B probes revealed that MCMV infection activated NF-kappa B proteins, whereas IFN-alpha treatment significantly reduced their ability to bind NF-kappa B sites. In cotransfection experiments using various NF-kappa B subunit expression vectors and a reporter driven by three copies of an NF-kappa B element, activation of NF-kappa B-dependent transcription was observed with expression of p65 or combinations of p50-p65. Taken as a whole, these results suggest that IFN-alpha inhibits MCMV IE gene enhancer activity by mechanisms that decrease the availability of virus-induced NF-kappa B transcriptionally active in the nuclei of infected cells.
小鼠巨细胞病毒（MCMV）即刻早期（IE）基因的转录受细胞转录因子与一个强大的病毒增强子相互作用的调控，该增强子控制着位于调控序列两侧的启动子。我们之前已经证明，α干扰素（IFN-α）通过损害IE基因的转录来抑制MCMV复制。为了确定参与这种抑制作用的顺式作用元件和反式作用因子，将含有氯霉素乙酰转移酶报告基因和部分IE增强子的DNA构建体转染到允许性小鼠成纤维细胞中。相对于IE1-3启动子，从-1185到-259的区域足以实现IFN-α诱导的抑制作用。由于该片段包含几个核因子κB（NF-κB）位点，因此用在同源最小IE1-3启动子前含有三个NF-κB元件拷贝的构建体转染细胞。经IFN-α处理后，报告基因活性显著降低，表明NF-κB结合位点足以赋予抑制作用。对含有突变的NF-κB三聚体或活化转录因子/环磷腺苷效应元件结合蛋白（ATF/CRE）三聚体的DNA构建体活性没有显著影响，证明了这种抑制作用的特异性。用NF-κB探针进行的凝胶迁移试验表明，MCMV感染激活了NF-κB蛋白，而IFN-α处理显著降低了它们结合NF-κB位点的能力。在使用各种NF-κB亚基表达载体和由三个NF-κB元件拷贝驱动的报告基因的共转染实验中，观察到p65表达或p50-p65组合表达时NF-κB依赖性转录被激活。总体而言，这些结果表明，IFN-α通过降低在受感染细胞核中具有转录活性的病毒诱导的NF-κB的可用性的机制来抑制MCMV IE基因增强子活性。
