Wang Fu-Zhang, Weber Frank, Croce Carlo, Liu Chang-Gong, Liao Xudong, Pellett Philip E
Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
J Virol. 2008 Sep;82(18):9065-74. doi: 10.1128/JVI.00961-08. Epub 2008 Jul 2.
The human genome encodes over 500 microRNAs (miRNAs), small RNAs (19 to 26 nucleotides [nt]) that regulate the expressions of diverse cellular genes. Many cellular processes are altered through a variety of mechanisms by human cytomegalovirus (HCMV) infection. We asked whether HCMV infection leads to changes in the expression of cellular miRNAs and whether HCMV-regulated miRNAs are important for HCMV replication. Levels of most miRNAs did not change markedly during infection, but some were positively or negatively regulated. Patterns of miRNA expression were linked to the time course of infection. Some similarly reregulated miRNAs share identical or similar seed sequences, suggesting coordinated regulation of miRNA species that have shared targets. miRNAs miR-100 and miR-101 were chosen for further analyses based on their reproducible changes in expression after infection and on the basis of having predicted targets in the 3' untranslated regions (3'-UTR) of genes encoding components of the mammalian target of rapamycin (mTOR) pathway, which is important during HCMV infection. Reporter genes that contain the 3'-UTR of mTOR (predicted targets for miR-100 and miR-101) or raptor (a component of the mTOR pathway; predicted site for miR-100) were constructed. Mimics of miR-100 and miR-101 inhibited expression from the mTOR construct, while only miR-100 inhibited the raptor construct. Together, miR-100 and miR-101 reduced mTOR protein levels. While the miR-100 and miR-101 mimics individually modestly inhibited production of infectious progeny, much greater inhibition was achieved with a combination of both (33-fold). Our key finding is that HCMV selectively manipulates the expression of some cellular miRNAs to help its own replication.
人类基因组编码超过500种微小RNA（miRNA），即小RNA（19至26个核苷酸[nt]），它们可调节多种细胞基因的表达。人巨细胞病毒（HCMV）感染通过多种机制改变许多细胞过程。我们询问HCMV感染是否会导致细胞miRNA表达的变化，以及HCMV调节的miRNA对HCMV复制是否重要。大多数miRNA的水平在感染期间没有明显变化，但有些受到正向或负向调节。miRNA表达模式与感染的时间进程相关。一些类似地重新调节的miRNA共享相同或相似的种子序列，表明对具有共同靶标的miRNA种类进行协调调节。基于感染后表达的可重复性变化以及在雷帕霉素哺乳动物靶标（mTOR）途径组成成分编码基因的3'非翻译区（3'-UTR）中有预测靶标，选择了miR-100和miR-101进行进一步分析，mTOR途径在HCMV感染期间很重要。构建了包含mTOR的3'-UTR（miR-100和miR-101的预测靶标）或raptor（mTOR途径的一个组成部分；miR-100的预测位点）的报告基因。miR-100和miR-101的模拟物抑制了mTOR构建体的表达，而只有miR-100抑制了raptor构建体。总之，miR-100和miR-101降低了mTOR蛋白水平。虽然miR-100和miR-101模拟物单独适度抑制了感染性后代的产生，但两者组合可实现更大程度的抑制（33倍）。我们 的关键发现是，HCMV选择性地操纵一些细胞miRNA的表达以帮助其自身复制。
