Prösch Susanna, Wuttke Regine, Krüger Detlev H, Volk Hans-Dieter
Institute of Virology, University Hospital Charité, Humboldt University, D-10098 Berlin, Germany.
Biol Chem. 2002 Oct;383(10):1601-9. doi: 10.1515/BC.2002.181.
From clinical studies the proinflammatory cytokine TNFalpha was proposed to play a key role in human cytomegalovirus (HCMV) reactivation from latency. In vitro experiments confirmed that TNFalpha stimulates the activity of the HCMV IE1/2 enhancer/promoter, which controls immediate early protein IE1 and IE2 gene expression via activation of the transcription factor NF-kappaB and its binding to putative binding sites in the IE1/2 enhancer. NF-kappaB was also proposed to be involved in IE1-mediated autostimulation of this promoter. The IE1/2 enhancer of HCMV contains four putative NF-kappaB binding sites which differ in their distance to the transcription start site as well as in their sequence. Construction and testing of a series of promoter mutants demonstrated that NF-kappaB is essential for both TNFalpha and IE1 stimulation. Furthermore, we were able to show that although all four NF-kappaB sites bind NF-kappaB with similar affinity in vitro, the contribution to TNFalpha and IE1 stimulation differs in correlation with the distance to the transcription start site and the sequence. Site 1 and 3 play the most dominant role and site 2 an intermediate, while site 4, which is conserved in sequence but far distant from the transcription start site, had no influence on NF-kappaB-mediated regulation of the IE1/2 promoter. Specific inhibition of NF-kappaB signalling by co-expression of a dominant-negative IkappaB variant reduced TNFalpha stimulation of the IE1/2 enhancer/promoter by up to 80%. From this data, inhibitors of NF-kappaB activation are suggested to be an alternative therapeutical strategy to interfere with HCMV (re)activation in undifferentiated monocyte/granulocyte progenitor cells in patients with a high risk of inflammation-related HCMV (re)activation.
临床研究表明，促炎细胞因子肿瘤坏死因子α（TNFα）在人类巨细胞病毒（HCMV）潜伏状态的重新激活中起关键作用。体外实验证实，TNFα可刺激HCMV IE1/2增强子/启动子的活性，该增强子/启动子通过激活转录因子核因子κB（NF-κB）及其与IE1/2增强子中假定结合位点的结合来控制即刻早期蛋白IE1和IE2基因的表达。也有人提出NF-κB参与IE1介导的该启动子的自身刺激。HCMV的IE1/2增强子包含四个假定的NF-κB结合位点，它们与转录起始位点的距离以及序列各不相同。一系列启动子突变体的构建和测试表明，NF-κB对于TNFα和IE1的刺激均至关重要。此外，我们能够证明，尽管所有四个NF-κB位点在体外以相似的亲和力结合NF-κB，但它们对TNFα和IE1刺激的贡献与到转录起始位点的距离和序列相关。位点1和3起最主要的作用，位点2起中间作用，而位点4虽然序列保守但距离转录起始位点很远，对NF-κB介导的IE1/2启动子调控没有影响。通过共表达显性负性IκB变体对NF-κB信号进行特异性抑制，可使TNFα对IE1/2增强子/启动子的刺激降低多达80%。基于这些数据，对于有炎症相关HCMV重新激活高风险的患者，NF-κB激活抑制剂被认为是一种干扰未分化单核细胞/粒细胞祖细胞中HCMV重新激活的替代治疗策略。
