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.

Murine Cytomegalovirus Protein pM79 Is a Key Regulator for Viral Late Transcription

“…sion, and 61 have been found to be expressed at elevated levels at late stages of MCMV infection (i.e., late genes) (45,46). Therefore, transcripts of a large set (76%) of annotated ORFs sensitive to PAA are also known to be highly expressed at late times, consistent with the notion that most viral DNA synthesis-dependent transcripts are derived from late genes.…”

“…b The mean intensity of a transcriptional region was (Y) or was not (N) reduced by at least 3-fold in array analysis. c Transcription of an ORF is considered to be late kinetics (L) when it is elevated by at least 50% from 12 to 24 hpi, as described by Marcinowski and coworkers (46), or absent at 6.5 hpi but detectable at 24 hpi, as described by Lacaze and coworkers (45). Blank entries, early kinetics.…”

25-Hydroxycholesterol Activates the Integrated Stress Response to Reprogram Transcription and Translation in Macrophages

“…2I). MCMV, like human cytomegalovirus, stimulates multiple stress response pathways during infection that likely account for the 25OHC-independent elevations in Chop mRNA (35,36). Thus, macrophage production of 25OHC during MCMV infection contributes to, but is not solely responsible for, stress gene activation during MCMV infection of macrophages.…”

Murine Cytomegalovirus Virion-Associated Protein M45 Mediates Rapid NF-κB Activation after Infection

“…Also like HCMV, MCMV induces a transient phase of NF-B activation within the first few hours of infection (29,30). However, the viral proteins and cellular signaling pathways involved have not been defined.…”