Correction: Comprehensive Analysis of Human Cytomegalovirus MicroRNA Expression during Lytic and Quiescent Infection.
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ABSTRACT:
SUBMITTER: Shen ZZ
PROVIDER: S-EPMC7147750 | biostudies-literature | 2020
REPOSITORIES: biostudies-literature
Publications
Correction: Comprehensive Analysis of Human Cytomegalovirus MicroRNA Expression during Lytic and Quiescent Infection.
Shen Zhang-Zhou ZZ Pan Xing X Miao Ling-Feng LF Ye Han-Qing HQ Chavanas Stéphane S Davrinche Christian C McVoy Michael M Luo Min-Hua MH
PloS one 20200410 4
[This corrects the article DOI: 10.1371/journal.pone.0088531.]. ...[more]
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Project description:BackgroundHuman cytomegalovirus (HCMV) encodes microRNAs (miRNAs) that function as post-transcriptional regulators of gene expression during lytic infection in permissive cells. Some miRNAs have been shown to suppress virus replication, which could help HCMV to establish or maintain latent infection. However, HCMV miRNA expression has not been comprehensively examined and compared using cell culture systems representing permissive (lytic) and semi-permissive vs. non-permissive (latent-like) infection.MethodsViral miRNAs levels and expression kinetics during HCMV infection were determined by miRNA-specific stem-loop RT-PCR. HCMV infected THP-1 (non-permissive), differentiated THP-1 (d-THP-1, semi-permissive) and human embryo lung fibroblasts (HELs, fully-permissive) were examined. The impact of selected miRNAs on HCMV infection (gene expression, genome replication and virus release) was determined by Western blotting, RT-PCR, qPCR, and plaque assay.ResultsAbundant expression of 15 HCMV miRNAs was observed during lytic infection in HELs; highest peak inductions (11- to 1502-fold) occurred at 48 hpi. In d-THP-1s, fourteen mRNAs were detected with moderate induction (3- to 288-fold), but kinetics of expression was generally delayed for 24 h relative to HELs. In contrast, only three miRNAs were induced to low levels (3- to 4-fold) during quiescent infection in THP-1s. Interestingly, miR-UL70-3p was poorly induced in HEL (1.5-fold), moderately in THP-1s (4-fold), and strongly (58-fold) in d-THP-1s, suggesting a potentially specific role for miR-UL70-3p in THP-1s and d-THP-1s. MiR-US33, -UL22A and -UL70 were further evaluated for their impact on HCMV replication in HELs. Ectopic expression of miR-UL22A and miR-UL70 did not affect HCMV replication in HELs, whereas miR-US33 inhibited HCMV replication and reduced levels of HCMV US29 mRNA, confirming that US29 is a target of miR-US33.ConclusionsViral miRNA expression kinetics differs between permissive, semi-permissive and quiescent infections, and miR-US33 down-regulates HCMV replication. These results suggest that miR-US33 may function to impair entry into lytic replication and hence promote establishment of latency.
| S-EPMC3922878 | biostudies-literature
Project description:MicroRNAs (miRNAs) are expressed in a wide variety of organisms, ranging from plants to animals, and are key posttranscriptional regulators of gene expression. Virally encoded miRNAs are unique in that they could potentially target both viral and host genes. Indeed, we have previously demonstrated that a human cytomegalovirus (HCMV)-encoded miRNA, miR-UL112, downregulates the expression of a host immune gene, MICB. Remarkably, it was shown that the same miRNA also downregulates immediate-early viral genes and that its ectopic expression resulted in reduced viral replication and viral titers. The targets for most of the viral miRNAs, and hence their functions, are still unknown. Here we demonstrate that miR-UL112 also targets the UL114 gene, and we present evidence that the reduction of UL114 by miR-UL112 reduces its activity as uracil DNA glycosylase but only minimally affects virus growth. In addition, we show that two additional HCMV-encoded miRNAs, miR-US25-1 and miR-US25-2, reduce the viral replication and DNA synthesis not only of HCMV but also of other viruses, suggesting that these two miRNAs target cellular genes that are essential for virus growth. Thus, we suggest that in addition to miR-UL112, two additional HCMV miRNAs control the life cycle of the virus.
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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 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-?B- 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-?B 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.
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Project description:Expression of the human cytomegalovirus (HCMV) IE1 and IE2 proteins is critical for the establishment of lytic infection and reactivation from viral latency. Defining the mechanisms controlling IE1 and IE2 expression is therefore important for understanding how HCMV regulates its replicative cycle. Here we identify several novel transcripts encoding full-length IE1 and IE2 proteins during HCMV lytic replication. Two of the alternative major immediate early (MIE) transcripts initiate in the first intron, intron A, of the previously defined MIE transcript, while others extend the 5' untranslated region. Each of the MIE transcripts associates with polyribosomes in infected cells and therefore contributes to IE1 and IE2 protein levels. Surprisingly, deletion of the core promoter region of the major immediate early promoter (MIEP) from a plasmid containing the MIE genomic locus did not completely abrogate IE1 and IE2 expression. Instead, deletion of the MIEP core promoter resulted in increased expression of alternative MIE transcripts, suggesting that the MIEP suppresses the activity of the alternative MIE promoters. While the canonical MIE mRNA was the most abundant transcript at immediate early times, the novel MIE transcripts accumulated to levels equivalent to that of the known MIE transcript later in infection. Using two HCMV recombinants, we found that sequences in intron A of the previously defined MIE transcript are required for efficient IE1 and IE2 expression and viral replication. Together, our results identify new regulatory sequences controlling IE1 and IE2 expression and suggest that multiple transcription units act in concert to regulate IE1 and IE2 expression during lytic infection.The HCMV IE1 and IE2 proteins are critical regulators of HCMV replication, both during primary infection and reactivation from viral latency. This study expands our understanding of the sequences controlling IE1 and IE2 expression by defining novel transcriptional units controlling the expression of full-length IE1 and IE2 proteins. Our results suggest that alternative promoters may allow for IE1 and IE2 expression when MIEP activity is limiting, as occurs in latently infected cells.
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Project description:Human cytomegalovirus (HCMV) microRNAs (miRNAs) significantly rewire host signaling pathways to support the viral lifecycle and regulate host cell responses. Here we show that SMAD3 expression is regulated by HCMV miR-UL22A and contributes to the IRF7-mediated induction of type I IFNs and IFN-stimulated genes (ISGs) in human fibroblasts. Addition of exogenous TGFβ interferes with the replication of a miR-UL22A mutant virus in a SMAD3-dependent manner in wild type fibroblasts, but not in cells lacking IRF7, indicating that downregulation of SMAD3 expression to limit IFN induction is important for efficient lytic replication. These findings uncover a novel interplay between SMAD3 and innate immunity during HCMV infection and highlight the role of viral miRNAs in modulating these responses.
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Project description:The human cytomegalovirus (HCMV) clinical strain Toledo and the attenuated strain AD169 exhibit a striking difference in pathogenic potential and cell tropism. The virulent Toledo genome contains a 15-kb segment, which is present in all virulent strains but is absent from the AD169 genome. The pathogenic differences between the 2 strains are thought to be associated with this additional genome segment. Cytokines induced during viral infection play major roles in the regulation of the cellular interactions involving cells of the immune and inflammatory systems and consequently determine the pathogenic outcome of infection. The chemokine RANTES (Regulated on activation, normal T-cell expressed and secreted) attracts immune cells during inflammation and the immune response, indicating a role for RANTES in viral pathogenesis. Here, we show that RANTES was downregulated in human foreskin fibroblast (HFF) cells at a later stage after infection with the Toledo strain but not after infection with the AD169 strain. miR-UL148D, the only miRNA predicted from the UL/b' sequences of the Toledo genome, targeted the 3'-untranslated region of RANTES and induced degradation of RANTES mRNA during infection. While wild-type Toledo inhibited expression of RANTES in HFF cells, Toledo mutant virus in which miR-UL148D is specifically abrogated did not repress RANTES expression. Furthermore, miR-UL148D-mediated downregulation of RANTES was inhibited by treatment with a miR-UL148D-specific inhibitor designed to bind to the miR-UL148D sequence via an antisense mechanism, supporting the potential value of antisense agents as therapeutic tools directed against HCMV. Our findings identify a viral microRNA as a novel negative regulator of the chemokine RANTES and provide clues for understanding the pathogenesis of the clinical strains of HCMV.
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Project description:Responses to human cytomegalovirus (HCMV) infection are largely individual and cell type specific. We investigated molecular profiles in 2 primary cell cultures of human fibroblasts, which are highly or marginally sensitive to HCMV infection, respectively. We screened expression of genes and microRNAs (miRs) at the early (3 hours) stage of infection. To assess molecular pathway activation profiles, we applied bioinformatic algorithms OncoFinder and MiRImpact. In both cell types, pathway regulation properties at mRNA and miR levels were markedly different. Surprisingly, in the infected highly sensitive cells, we observed a "freeze" of miR expression profiles compared to uninfected controls. Our results evidence that in the sensitive cells, HCMV blocks intracellular regulation of microRNA expression already at the earliest stage of infection. These data suggest somewhat new functions for HCMV products and demonstrate dependence of miR expression arrest on the host-encoded factors.
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Project description:As the first line of antiviral defense, type I interferon (IFN) binds IFN receptor 1 (IFNAR1) and IFNAR2 to activate the Jak-STAT signal transduction pathway, producing IFN-stimulated genes (ISGs) to control viral infection. The mechanisms by which human cytomegalovirus (HCMV) counteracts the IFN pathway are only partially defined. We show that miR-US33as-5p encoded by HCMV is expressed in both lytic and latent infection. By analysis with RNA hybrid and screening with luciferase reporter assays, we identified IFNAR1 as a target of hcmv-miR-US33as-5p, which was further verified by examining the expression of two IFNAR1 mutants and the binding of IFNAR1 to miR-US33as-5p/miR-US33as-5p-M1/miR-US33as-5p-M2. We found that after the transfection of miR-US33as-5p mimics into different cell lines, the phosphorylation of downstream proteins and ISG expression were downregulated. Immunofluorescence showed that the miR-US33as-5p mimics also inhibited STAT1 translocation into the nucleus. Furthermore, we constructed HCMV with mutant miR-US33as-5p and determined that the mutation did not affect HCMV replication. We found that MRC-5/human foreskin fibroblast (HFF) cells infected with ΔmiRNA HCMV exhibited higher IFNAR1 and ISG expression and a reduced viral load in the presence of exogenous IFN than cells infected with WT HCMV did, confirming that the knockout of miR-US33as-5p impaired viral resistance to IFN. Finally, we tested the effect of ΔmiRNA HCMV on THP-1 and d-THP-1 cells, common in vitro models of latent infection and reactivation, respectively. Again, we found that cells infected with ΔmiRNA HCMV showed a reduced viral load in the presence of IFN than the control cells did, confirming that miR-US33as-5p also affects IFN resistance during both latency and reactivation. These results indicate a new microRNA (miRNA)-based immune evasion mechanism employed by HCMV to achieve lifelong infection.
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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: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