Abstract
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.
Highlights
Herpesviruses are large DNA viruses which cause a broad range of disease ranging from the common cold sore to cancer
We first employed 4sU-tagging combined with microarray analysis to study the dynamic changes in transcriptional activity of NIH-3T3 fibroblasts during early murine cytomegalovirus (MCMV) infection
We found virtually all changes in total cellular RNA to be matched by concordant changes in newly transcribed RNA, indicating that alterations in RNA decay rates do not substantially contribute to differential gene expression during early phase of MCMV infection of fibroblasts
Summary
Herpesviruses are large DNA viruses which cause a broad range of disease ranging from the common cold sore to cancer. Cytomegaloviruses (CMV) have co-evolved with their animal and human hosts for millions of years During this time, they have mastered host-cell modulation to facilitate their needs and provide ideal tools to study many fundamental cellular processes. Several highthroughput studies addressed the transcriptional response of the cell to lytic CMV infection by analyzing temporal changes in total RNA levels [3,6,7,8,9,10].
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