Project description: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 from different times during the first six hours of MCMV infection revealed discrete functional networks of cellular genes regulated with distinct kinetics at surprising temporal resolution. Most of these were undetectable in total RNA, either due to low temporal sensitivity of standard gene expression profiling using total RNA, or due to rapid (viral) counter-regulation. Furthermore, metabolic labeling and purification of newly transcribed RNA detailed the real-time kinetics and regulation of viral gene expression in absence of any interfering virion-associated RNA. Both qRT-PCR and next-generation sequencing of newly transcribed RNA demonstrated an unexpected 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 peak was subject to rapid gene silencing (independent of the multiplicity of infection) with similar transcriptional activity only reached late in infection. For some genes, e.g. m152, this resulted in massive down-regulation of transcriptional activity by 6 hours post infection, which was not even reversed late in infection. Our findings thus highlight the importance of transcriptional activity during the first few hours of CMV infection and hint at novel mechanisms governing the kinetics of viral gene expression. In conclusion, studying real-time transcriptional activity during lytic CMV infection provides intriguing new insights into the regulation of cellular and viral gene expression. In total 13 samples were examined: 3 samples of total RNA (mock, 25, 48 hours past MCMV infection), 3 samples pre-existing RNA (mock, 25, 48 hpi), and 7 samples newly-transcribed RNA (mock, 1-2, 5-6, 11-12, 18-19, 24-25, 47-48 hpi)

Project description:Expression data from NIH-3T3 cells left uninfected or infected with MCMV for 2, 4 or 6h on total RNA as well as newly transcribed RNA labeled for 1-2, 3-4, and 5-6hpi. For newly transcribed RNA, the isolated RNA was labeled for 1h and separated from total cellular RNA following Trizol RNA preparation and thiol-specific biotinylation. We used microarrays to analyze the effects of MCMV infection in total and newly transcribed RNA. 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 from different times during the first six hours of MCMV infection revealed discrete functional networks of cellular genes regulated with distinct kinetics at surprising temporal resolution. Most of these were undetectable in total RNA, either due to low temporal sensitivity of standard gene expression profiling using total RNA, or due to rapid (viral) counter-regulation. Furthermore, metabolic labeling and purification of newly transcribed RNA detailed the real-time kinetics and regulation of viral gene expression in absence of any interfering virion-associated RNA. Both qRT-PCR and next-generation sequencing of newly transcribed RNA demonstrated an unexpected 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 peak was subject to rapid gene silencing (independent of the multiplicity of infection) with similar transcriptional activity only reached late in infection. For some genes, e.g. m152, this resulted in massive down-regulation of transcriptional activity by 6 hours post infection, which was not even reversed late in infection. Our findings thus highlight the importance of transcriptional activity during the first few hours of CMV infection and hint at novel mechanisms governing the kinetics of viral gene expression. In conclusion, studying real-time transcriptional activity during lytic CMV infection provides intriguing new insights into the regulation of cellular and viral gene expression. NIH-3T3 cells (5th to 15th passage after thawing) were split from confluent plates 24h before start of the experiment. At the beginning of the experiment about 80% confluency was reached. The experiment was started by applying fresh, prewarmed, CO2-equilibrated medium containing either mock or MCMV particles (MOI 1). The MOI was increased by ~10-fold through centrifugal enhancement (2000g, 30min). Labeling was started 1, 3, or 5hpi after the end of centrifugal enhancement.

Project description:Expression data from NIH-3T3 cells left uninfected or infected with MCMV for 2, 4 or 6h on total RNA as well as newly transcribed RNA labeled for 1-2, 3-4, and 5-6hpi. For newly transcribed RNA, the isolated RNA was labeled for 1h and separated from total cellular RNA following Trizol RNA preparation and thiol-specific biotinylation. We used microarrays to analyze the effects of MCMV infection in total and newly transcribed RNA. 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 from different times during the first six hours of MCMV infection revealed discrete functional networks of cellular genes regulated with distinct kinetics at surprising temporal resolution. Most of these were undetectable in total RNA, either due to low temporal sensitivity of standard gene expression profiling using total RNA, or due to rapid (viral) counter-regulation. Furthermore, metabolic labeling and purification of newly transcribed RNA detailed the real-time kinetics and regulation of viral gene expression in absence of any interfering virion-associated RNA. Both qRT-PCR and next-generation sequencing of newly transcribed RNA demonstrated an unexpected 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 peak was subject to rapid gene silencing (independent of the multiplicity of infection) with similar transcriptional activity only reached late in infection. For some genes, e.g. m152, this resulted in massive down-regulation of transcriptional activity by 6 hours post infection, which was not even reversed late in infection. Our findings thus highlight the importance of transcriptional activity during the first few hours of CMV infection and hint at novel mechanisms governing the kinetics of viral gene expression. In conclusion, studying real-time transcriptional activity during lytic CMV infection provides intriguing new insights into the regulation of cellular and viral gene expression.

Project description:Prenatal Human Cytomegalovirus (HCMV) infection often causes CNS maldevelopment. In a murine model, we detect Murine Cytomegalovirus (MCMV) in brain following intra-peritoneal inoculation at birth. Infected mice show impaired cerebellar development and impaired neurologic function on a beam balance test as adults. Among developmental genes differentially regulated, hindbrain expression of the homeodomain transcription factor HOXa5 was reduced with infection, and fewer HOXa5 expressing neurons were found in vestibular nuclei. Based on the hypothesis that immune activation connects focal viral infection and global CNS maldevelopment, we defined the components of CNS immune response. Flow cytometry showed a large increase in both number and activation of CNS monocytes. Monocytes were found in close association with infected cells by immunohistochemistry (IHC). Oligonucleotide microarrays contained herein identified many differentially expressed genes related to innate immune response. Chemokines, cytokines, cell surface receptors, and proteases are some of the many immunological genes shown to be differentially regulated by MCMV infection. These results together show that MCMV infection induces a complex immune response associated with changes in developmental gene expression and lasting neurologic defecit. Keywords: disease state comparison (virus infection) and competetive hybridization for expression analysis Previous work empirically determined the kinetics of viral spread to and replication in the Balb-C murine CNS. Balb-C mice were inoculated intra-peritoneally at birth, and harvested at 5, 8, 12, and 21 days post inoculation for cerebellar experiments. Controls were mock infected with vehicle (DMEM cell growth media) and harvested at the same timepoints. Each microarray was a two channel MEEBO array, upon which labeled and reverse transcribed cDNA from 3 pooled infected cerebella and three pooled uninfected cerebella competetively hybridized. Three unique biological replicates were performed with day 5 cDNA, 5 unique biological replicates at day 8, 5 unique biological replicates at day 12, and 4 unique biological replicates at day 21. For the analysis, colors channels were seperated and treated as independent hybridizations. However, since the experiments were dual channel, the primary data may be employed to generate ratio based measurements, and each sample is provided a corresponding number that can be used to pair the processed data and derive ratios (mock 3, infected 3). Mice were similarly inoculated and harvested at 5, 15, and 21 days post natal for liver tissue analysis. Inoculation was performed by injection with a microsyringe of 50ul titered virus delivering 500-1000 PFU per animal.

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Real-time transcriptional profiling of cellular and viral gene expression during lytic cytomegalovirus infection

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