Max von Pettenkofer-Institute, Ludwig-Maximilians-University, Munich, Germany.
PLoS Pathog. 2012 Sep;8(9):e1002908. doi: 10.1371/journal.ppat.1002908. Epub 2012 Sep 6.
During viral infections cellular gene expression is subject to rapid alterations induced by both viral and antiviral mechanisms. In this study, we applied metabolic labeling of newly transcribed RNA with 4-thiouridine (4sU-tagging) to dissect the real-time kinetics of cellular and viral transcriptional activity during lytic murine cytomegalovirus (MCMV) infection. Microarray profiling on newly transcribed RNA obtained at different times during the first six hours of MCMV infection revealed discrete functional clusters of cellular genes regulated with distinct kinetics at surprising temporal resolution. Immediately upon virus entry, a cluster of NF-κB- and interferon-regulated genes was induced. Rapid viral counter-regulation of this coincided with a very transient DNA-damage response, followed by a delayed ER-stress response. Rapid counter-regulation of all three clusters indicated the involvement of novel viral regulators targeting these pathways. In addition, down-regulation of two clusters involved in cell-differentiation (rapid repression) and cell-cycle (delayed repression) was observed. Promoter analysis revealed all five clusters to be associated with distinct transcription factors, of which NF-κB and c-Myc were validated to precisely match the respective transcriptional changes observed in newly transcribed RNA. 4sU-tagging also allowed us to study the real-time kinetics of viral gene expression in the absence of any interfering virion-associated-RNA. Both qRT-PCR and next-generation sequencing demonstrated a sharp peak of viral gene expression during the first two hours of infection including transcription of immediate-early, early and even well characterized late genes. Interestingly, this was subject to rapid gene silencing by 5-6 hours post infection. Despite the rapid increase in viral DNA load during viral DNA replication, transcriptional activity of some viral genes remained remarkably constant until late-stage infection, or was subject to further continuous decline. In summary, this study pioneers real-time transcriptional analysis during a lytic herpesvirus infection and highlights numerous novel regulatory aspects of virus-host-cell interaction.
在病毒感染过程中，细胞基因表达受到病毒和抗病毒机制诱导的快速改变。在这项研究中，我们应用了新转录 RNA 的代谢标记 4-硫代尿嘧啶（4sU-标记），以剖析在裂解性鼠巨细胞病毒（MCMV）感染过程中细胞和病毒转录活性的实时动力学。在 MCMV 感染的头 6 小时内的不同时间获得的新转录 RNA 的微阵列分析揭示了受不同动力学以惊人的时间分辨率调节的细胞基因的离散功能簇。病毒进入后，立即诱导了一组 NF-κB 和干扰素调节的基因。这种快速的病毒反调节与非常短暂的 DNA 损伤反应相吻合，随后是延迟的内质网应激反应。这三个簇的快速反调节表明涉及针对这些途径的新型病毒调节剂。此外，还观察到两个参与细胞分化（快速抑制）和细胞周期（延迟抑制）的基因簇下调。启动子分析显示，所有五个簇都与不同的转录因子相关联，其中 NF-κB 和 c-Myc 被验证与新转录 RNA 中观察到的相应转录变化精确匹配。4sU-标记还使我们能够在没有任何干扰病毒相关 RNA 的情况下研究病毒基因表达的实时动力学。qRT-PCR 和下一代测序均表明，在感染的头两个小时内，病毒基因表达出现急剧高峰，包括瞬时早期、早期甚至特征明确的晚期基因的转录。有趣的是，这在感染后 5-6 小时就受到快速基因沉默。尽管在病毒 DNA 复制期间病毒 DNA 负荷迅速增加，但一些病毒基因的转录活性在晚期感染之前仍然保持相当恒定，或者进一步连续下降。总之，本研究开创了裂解性疱疹病毒感染期间的实时转录分析，并突出了病毒-宿主细胞相互作用的许多新的调节方面。
