Meier J L
Department of Internal Medicine, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
J Virol. 2001 Feb;75(4):1581-93. doi: 10.1128/JVI.75.4.1581-1593.2001.
Inactivity of the human cytomegalovirus (HCMV) major immediate-early regulatory region (MIERR), which is composed of promoter, enhancer, unique region, and modulator, is linked to lack of HCMV replication in latently infected cells and in other nonpermissive cell types, including human embryonal NTera2 carcinoma (NT2) cells. I refined the embryonal NT2 cell model to enable characterization of the unknown mechanistic basis for silencing of HCMV MIERR-dependent transcription and viral replication in nonpermissive cells. These infected NT2 cells contain nonreplicating viral genomes with electrophoretic mobility equivalent to a supercoiled, bacterial artificial chromosome of comparable molecular weight. MIERR-dependent transcription is minimal to negligible. Increasing the availability of positive-acting transcription factors by retinoic acid (RA) treatment after infection is largely insufficient in reactivating the MIERR. In contrast, trichostatin A (TSA), a histone deacetylase inhibitor, reactivates MIERR-dependent transcription. Contrary to prior findings produced from transfected MIERR segments, deletion of the 21-bp repeats and modulator from the MIERR in the viral genome does not relieve MIERR silencing. To demonstrate that MIERR silencing likely results from enhancer inactivity, I examined an HCMV with a heterologous MIERR promoter that is enhancer dependent but exempt from IE2 p86-mediated negative autoregulation. This heterologous promoter, like its neighboring native MIERR promoter, exhibits immediate-early transcriptional kinetics in fibroblasts. In embryonal NT2 cells, the heterologous MIERR promoter is transcriptionally inactive. This silence is relieved by TSA but not by RA. Remarkably, TSA-induced reactivation of MIERR-dependent transcription from quiescent viral genomes is followed by release of infectious virus. I conclude that a mechanism of active repression imposes a block to MIERR-dependent transcription and viral replication in embryonal NT2 cells. Because TSA overcomes the block, viral gene silencing may involve histone deacetylase-based modification of viral chromatin, which might account for the covalently closed circular conformation of quiescent HCMV genomes.
人类巨细胞病毒（HCMV）主要立即早期调控区（MIERR）由启动子、增强子、独特区域和调节子组成，其无活性与潜伏感染细胞及其他非允许细胞类型（包括人类胚胎NTera2癌（NT2）细胞）中HCMV复制的缺乏有关。我优化了胚胎NT2细胞模型，以确定非允许细胞中HCMV MIERR依赖性转录和病毒复制沉默的未知机制基础。这些感染的NT2细胞含有电泳迁移率与分子量相当的超螺旋细菌人工染色体等效的非复制性病毒基因组。MIERR依赖性转录极少甚至可忽略不计。感染后用视黄酸（RA）处理增加正性作用转录因子的可用性，在很大程度上不足以重新激活MIERR。相反，组蛋白脱乙酰酶抑制剂曲古抑菌素A（TSA）可重新激活MIERR依赖性转录。与转染的MIERR片段产生的先前发现相反，从病毒基因组的MIERR中缺失21 bp重复序列和调节子并不能解除MIERR沉默。为证明MIERR沉默可能是由增强子无活性导致的，我研究了一种具有异源MIERR启动子的HCMV，该启动子依赖增强子但不受IE2 p86介导的负性自动调节影响。这个异源启动子与其相邻的天然MIERR启动子一样，在成纤维细胞中表现出立即早期转录动力学。在胚胎NT2细胞中，异源MIERR启动子转录无活性。这种沉默可被TSA解除，但不能被RA解除。值得注意的是，TSA诱导静止病毒基因组中MIERR依赖性转录的重新激活后，会释放出感染性病毒。我得出结论，一种主动抑制机制对胚胎NT2细胞中的MIERR依赖性转录和病毒复制形成了阻碍。由于TSA克服了这种阻碍，病毒基因沉默可能涉及基于组蛋白脱乙酰酶的病毒染色质修饰，这可能解释了静止HCMV基因组的共价闭合环状构象。
