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.