title: Real-time transcriptional profiling of cellular and viral gene expression during lytic cytomegalovirus infection.
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.
authors.affiliation: Max von Pettenkofer-Institute, Ludwig-Maximilians-University, Munich, Germany.
authors.forename: Lisa
authors.initials: L
authors.lastname: Marcinowski
authors.forename: Michael
authors.initials: M
authors.lastname: Lidschreiber
authors.forename: Lukas
authors.initials: L
authors.lastname: Windhager
authors.forename: Martina
authors.initials: M
authors.lastname: Rieder
authors.forename: Jens B
authors.initials: JB
authors.lastname: Bosse
authors.forename: Bernd
authors.initials: B
authors.lastname: Rädle
authors.forename: Thomas
authors.initials: T
authors.lastname: Bonfert
authors.forename: Ildiko
authors.initials: I
authors.lastname: Györy
authors.forename: Miranda
authors.initials: M
authors.lastname: de Graaf
authors.forename: Olivia
authors.initials: O
authors.lastname: Prazeres da Costa
authors.forename: Philip
authors.initials: P
authors.lastname: Rosenstiel
authors.forename: Caroline C
authors.initials: CC
authors.lastname: Friedel
authors.forename: Ralf
authors.initials: R
authors.lastname: Zimmer
authors.forename: Zsolt
authors.initials: Z
authors.lastname: Ruzsics
authors.forename: Lars
authors.initials: L
authors.lastname: Dölken
mesh_terms: D000818:Animals; D020869:Gene Expression Profiling / Q000379:methods; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral / Q000235:genetics; D006566:Herpesviridae Infections / Q000235:genetics* / Q000821:virology; D054884:Host-Pathogen Interactions / Q000235:genetics*; D051379:Mice; D046228:Microarray Analysis; D005810:Multigene Family / Q000235:genetics; D018146:Muromegalovirus / Q000235:genetics* / Q000472:pathogenicity; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
chemical_list: D014157:Transcription Factors