Quantitative Studies of Epstein-Barr Virus-Encoded MicroRNAs Provide Novel Insights into Their Regulation
Quantitative Studies of Epstein-Barr Virus-Encoded MicroRNAs Provide Novel Insights into Their Regulation
复制标题
DOI：
10.1128/jvi.01528-10
复制
发表时间：
2011-01-01
影响因子：
5.4
通讯作者：
Bell, Andrew I.
中科院分区：
文献类型：
--
作者：
Amoroso, Richard;Fitzsimmons, Leah;Bell, Andrew I.
EB病毒（Epstein-Barr virus，EBV）编码至少40种微小RNA（microRNAs，miRNAs），这是一类重要的分子，通过转录后机制负调控许多基因的表达。在这里，我们使用实时PCR检测来定量潜伏感染细胞和诱导进入裂解周期的细胞中EBV编码的BHRF 1和BART miRNA的水平。在潜伏期，BHRF 1 miRNAs仅在具有可检测到的Cp和/或Wp启动的EBNA转录物的细胞中观察到，而BART miRNAs在所有形式的潜伏感染中表达。令人惊讶的是，发现不同BART miRNA的水平在细胞系内变化高达50倍。然而，这种变化不能用差异miRNA周转来解释，因为所有EBV miRNA似乎都非常稳定。在进入病毒裂解周期后，miR-BHRF 1 -2和-1-3被快速诱导，与裂解BHRF 1转录物的开始一致，而miR-BHRF 1 -1表达延迟至48小时，并与Cp/Wp起始的EBNA转录物的出现相关。相比之下，尽管BART转录显著增加，但BART miRNA的水平在病毒复制期间相对不变。最后，我们发现，在新鲜感染的原代B细胞培养物中，BHRF 1和BART miRNA相对于BHRF 1和BART转录物的诱导延迟。总之，我们的数据显示BHRF 1和BART转录的变化不一定反映在miRNA水平的改变中，这表明miRNA成熟是调节EBV miRNA稳态水平的关键步骤。
Epstein-Barr virus (EBV) has been shown to encode at least 40 microRNAs (miRNAs), an important class of molecules that negatively regulate the expression of many genes through posttranscriptional mechanisms. Here, we have used real-time PCR assays to quantify the levels of EBV-encoded BHRF1 and BART miRNAs in latently infected cells and in cells induced into the lytic cycle. During latency, BHRF1 miRNAs were seen only in cells with detectable Cp-and/or Wp-initiated EBNA transcripts, while the BART miRNAs were expressed in all forms of latent infection. Surprisingly, levels of different BART miRNAs were found to vary up to 50-fold within a cell line. However, this variation could not be explained by differential miRNA turnover, as all EBV miRNAs appeared to be remarkably stable. Following entry into the virus lytic cycle, miR-BHRF1-2 and -1-3 were rapidly induced, coincident with the onset of lytic BHRF1 transcripts, while miR-BHRF1-1 expression was delayed until 48 h and correlated with the appearance of Cp/Wp-initiated EBNA transcripts. In contrast, levels of BART miRNAs were relatively unchanged during virus replication, despite dramatic increases in BART transcription. Finally, we show that BHRF1 and BART miRNAs were delayed relative to the induction of BHRF1 and BART transcripts in freshly infected primary B cell cultures. In summary, our data show that changes in BHRF1 and BART transcription are not necessarily reflected in altered miRNA levels, suggesting that miRNA maturation is a key step in regulating steady-state levels of EBV miRNAs.