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  3. Medizin - Open Access LMU - Teil 20/22 Podcast
  4. Real-time transcriptional profiling of cellular and viral gene expression during lytic cytomegalovirus infection.
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.

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