Human cytomegalovirus long noncoding RNA4.9 regulates viral DNA replication
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA555104 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9698890.fastq.gz | Fastqsanger.gz | |||
| SRR9698891.fastq.gz | Fastqsanger.gz | |||
| SRR9698892.fastq.gz | Fastqsanger.gz | |||
| SRR9698893.fastq.gz | Fastqsanger.gz | |||
| SRR9698906.fastq.gz | Fastqsanger.gz |
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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: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, which may be important for the initiation of viral DNA replication. Furthermore, targeting the RNA4.9 promoter with CRISPR-Cas9 or genetic relocalization of oriLyt leads to reduced levels of the viral single-stranded DNA-binding protein (ssDBP), suggesting that the levels of ssDBP are coupled to the oriLyt activity. We further identified a similar, oriLyt-embedded, G+C-rich lncRNA in murine cytomegalovirus (MCMV). These results indicate that HCMV RNA4.9 plays an important role in regulating viral DNA replication, that the levels of ssDBP are coupled to the oriLyt activity, and that these regulatory features may be conserved among betaherpesviruses.
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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.
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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
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