P53 regulates viral gene expression during infection with human cytomegalovirus
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PROVIDER: PRJNA110027 | ENA |
REPOSITORIES: ENA
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Project description:Human cytomegalovirus (HCMV) is a common cause of morbidity and mortality in immunocompromised and immunosuppressed individuals. During infection, HCMV is known to employ host transcription factors to facilitate viral gene expression. To further understand the previously observed delayed viral replication and protein expression in p53 knockout cells, we conducted microarray analyses on p53+/+ and p53-/- immortalized fibroblast cell lines. At 12 hpi, applying the significance criteria defined above, no genes showed a significant difference in any of the comparisons. At 24 hpi, we found 19 genes to be significantly different in the THF versus p53-/- comparison and 14 genes to be significantly different in the HFF versus p53-/- comparison. Eleven genes were shared between HFF and THF cells when compared to p53-/- cells (UL30, UL54, UL63, UL65, UL69, UL75, UL110, UL122, IRL4, IRS1, TRS1, shown in bold in Table 1). Eight genes were unique in the THF versus p53-/- comparison (UL48, UL68, UL84, UL100, UL106, UL109, UL111, RL5A), and three genes in the HFF versus p53-/- cell comparison (UL14, UL16, US2). When comparing HFF and THF cells, only US2 showed a significant difference. The ratio (HFF versus p53-/- divided by THF versus p53-/-) of the expression levels of the eleven shared genes from the HFF versus p53-/- and THF versus p53-/- comparisons was an averaged 1.2, indicating that, with the exception of US2, these two cell lines behaved similarly with respect to p53-driven viral gene expression. Keywords: time course, virus, cytomegalo, hhv5, infection, p53 Each slide consisted of 4 arrays. Each array consisted of 305 control features, 1265 cellular gene features and 670 viral gene features. The study was performed on 6 slides (HFF-THF, HFF-p53-/-, THF-p53-/- at 12 and 24 hpi). 3 arrays per slide were hybridized with 3 biological replicates of labelled RNA, the 4th array was hybridized with an equimolar mixture of the 3 biological replicate samples. This array was used for control purposes only, and the results were not used for downstream calculations. Each slide was stripped and reprobed two times to provide 3 technical replicates of each sample. As our experiments concentrated on viral genes and their expression, cellular genes were not further analyzed in this study.
2010-05-15 | E-GEOD-13437 | biostudies-arrayexpress
Project description:Human cytomegalovirus (HCMV) is a common cause of morbidity and mortality in immunocompromised and immunosuppressed individuals. During infection, HCMV is known to employ host transcription factors to facilitate viral gene expression. To further understand the previously observed delayed viral replication and protein expression in p53 knockout cells, we conducted microarray analyses on p53+/+ and p53-/- immortalized fibroblast cell lines. At 12 hpi, applying the significance criteria defined above, no genes showed a significant difference in any of the comparisons. At 24 hpi, we found 19 genes to be significantly different in the THF versus p53-/- comparison and 14 genes to be significantly different in the HFF versus p53-/- comparison. Eleven genes were shared between HFF and THF cells when compared to p53-/- cells (UL30, UL54, UL63, UL65, UL69, UL75, UL110, UL122, IRL4, IRS1, TRS1, shown in bold in Table 1). Eight genes were unique in the THF versus p53-/- comparison (UL48, UL68, UL84, UL100, UL106, UL109, UL111, RL5A), and three genes in the HFF versus p53-/- cell comparison (UL14, UL16, US2). When comparing HFF and THF cells, only US2 showed a significant difference. The ratio (HFF versus p53-/- divided by THF versus p53-/-) of the expression levels of the eleven shared genes from the HFF versus p53-/- and THF versus p53-/- comparisons was an averaged 1.2, indicating that, with the exception of US2, these two cell lines behaved similarly with respect to p53-driven viral gene expression. Keywords: time course, virus, cytomegalo, hhv5, infection, p53
2008-12-01 | GSE13437 | GEO
Project description:Human cytomegalovirus long noncoding RNA4.9 regulates viral DNA replication
| PRJNA555104 | ENA
Project description:Viruses are known for their extremely compact genomes composed almost entirely of protein-coding genes. Nonetheless, four long noncoding RNAs (lncRNAs) are encoded by human cytomegalovirus (HCMV). Although these RNAs accumulate to high levels during lytic infection, their functions remain largely unknown. Here, we show that HCMV-encoded lncRNA4.9 localizes to the viral nuclear replication compartment, and that its depletion restricts viral DNA replication and viral growth. RNA4.9 is transcribed from the HCMV origin of replication (oriLyt) and forms an RNA-DNA hybrid (R-loop) through its G+C-rich 5’ end, and this may be important for the initiation of viral DNA replication. Furthermore, interference with RNA4.9 expression drastically reduces the levels of the viral single-stranded DNA-binding protein (ssDBP), and overexpression of ssDBP alleviates the inhibition of viral DNA replication and growth caused by RNA4.9 depletion. We also identified a similar, oriLyt-embedded, G+C-rich lncRNA in murine cytomegalovirus (MCMV). Knockdown of this lncRNA interferes with MCMV ssDBP expression and viral DNA replication. These results indicate that HCMV RNA4.9 plays an important role in regulating viral DNA replication by coupling oriLyt activity to ssDBP levels, and that this novel activity may be conserved in other betaherpesviruses.
2020-03-03 | GSE134423 | GEO
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)
2013-02-14 | E-GEOD-35833 | biostudies-arrayexpress
Project description:Viral disruption of innate immune signaling is a critical determinant of productive infection. The Human Cytomegalovirus (HCMV) UL26 protein prevents anti-viral gene expression during infection, yet the mechanisms involved are unclear. We used TurboID-driven proximity proteomics to identify putative UL26 interacting proteins during infection to address this issue. We find that UL26 forms a complex with several immuno-regulatory proteins, including several STAT family members and various PIAS proteins, a family of E3 SUMO ligases. Our results indicate that UL26 prevents STAT phosphorylation during infection and antagonizes transcriptional activation induced by either interferon α (IFNA) or tumor necrosis factor α (TNFα). Additionally, we find that the inactivation of PIAS1 sensitizes cells to inflammatory stimulation, resulting in an anti-viral transcriptional environment similar to ΔUL26 infection. Further, PIAS1 is important for HCMV cell-to-cell spread, which depends on the presence of UL26, suggesting that the UL26-PIAS1 interaction is vital for modulating intrinsic anti-viral defense.
2024-04-27 | GSE264589 | GEO
Project description:Small RNA deep sequencing analysis was conducted on primary human fibroblasts infected with human cytomegalovirus (HCMV). HCMV-encoded miRNAs accumulated to ~20% of the total smRNA population at late stages of infection, and our analysis led to improvements in viral miRNA annotations and identification of novel HCMV miRNAs. Through crosslinking and immunoprecipitation of Argonaute-bound RNAs from infected cells, followed by high-throughput sequencing (Ago CLIP-seq), we obtained direct evidence for incorporation of all HCMV miRNAs into the endogenous host silencing machinery. Additionally, significant upregulation was observed during infection for a host miRNA cluster containing miR-96, miR-182 and miR-183. We also identified novel non-miRNA forms of virus-derived smRNAs, revealing greater complexity within the smRNA population during HCMV infection. High-throughput profiling of smRNAs, Ago1-, and Ago2-associated miRNAs from HCMV-infected fibroblast cells. Wild-type HCMV Towne (Genbank FJ616285.1) was used for these studies.
2011-11-10 | E-GEOD-33584 | biostudies-arrayexpress
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.
2013-02-14 | GSE35833 | GEO
Project description:We characterized the role of the conserved murine cytomegalovirus (MCMV) gene M79. Using a recombinant MCMV virus carrying a tagged M79 coding sequence, we showed that M79 encoded a protein (pM79) which was expressed at late times of infection and localized to nuclear viral replication compartments. M79 transcription was largely dependent on viral DNA synthesis but was markedly stimulated by pM79, suggesting a positive feedback loop. To investigate its role, we created the recombinant virus SMin79, in which the M79 coding sequence was disrupted by an 88-nt insertion. We subsequently repaired the mutation to generate marker-rescued virus SMrev79. While SMrev79 grew efficiently in fibroblasts, SMin79 failed to produce infectious progeny but was rescued by pM79 expression in trans. During SMin79 infection, representative viral immediate early and early gene products, as well as viral DNA, accumulated efficiently. Formation of viral replication compartments also appeared normal. Pulsed field gel electrophoresis analysis indicated that the overall structure of replicating viral DNA was indistinguishable in cells infected with SMin79 compared to that with wild type virus. However, the accumulation of viral products for late gene M55 was severely compromised. Viral oligonucleotide tiled array analysis revealed that the accumulation of many late transcripts, defined by their sensitivity to viral DNA synthesis inhibitor phosphonoacetic acid, was markedly reduced by pM79 mutation. This study extends our previous work to suggest that cytomegaloviruses use a conserved mechanism to promote transcription at late stages of infection, and that pM79 regulates expression of a subset of viral DNA synthesis-dependent transcripts. This study consists of 4 unreplicated samples. RNA from Mock-infected, WT infected, WT infected in the presence of PAA, and a mutant M79 version of MCMV.
2013-07-31 | E-GEOD-47586 | biostudies-arrayexpress
Project description:Small RNA deep sequencing analysis was conducted on primary human fibroblasts infected with human cytomegalovirus (HCMV). HCMV-encoded miRNAs accumulated to ~20% of the total smRNA population at late stages of infection, and our analysis led to improvements in viral miRNA annotations and identification of novel HCMV miRNAs. Through crosslinking and immunoprecipitation of Argonaute-bound RNAs from infected cells, followed by high-throughput sequencing (Ago CLIP-seq), we obtained direct evidence for incorporation of all HCMV miRNAs into the endogenous host silencing machinery. Additionally, significant upregulation was observed during infection for a host miRNA cluster containing miR-96, miR-182 and miR-183. We also identified novel non-miRNA forms of virus-derived smRNAs, revealing greater complexity within the smRNA population during HCMV infection.
2011-11-10 | GSE33584 | GEO