Human cytomegalovirus long noncoding RNA4.9 regulates viral DNA replication
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ABSTRACT:
ORGANISM(S): Mus musculus Homo sapiens Human betaherpesvirus 5 Murid betaherpesvirus 1
PROVIDER: GSE134423 | GEO | 2020/03/03
REPOSITORIES: GEO
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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, compact genomes confer a selective advantage to viruses, yet human cytomegalovirus (HCMV) expresses the long non-coding RNAs (lncRNAs) RNA1.2, RNA2.7, RNA4.9, and RNA5.0. These lncRNAs account for majority of the viral transcriptome, but their functions remain largely unknown. Here, we showed that HCMV lncRNAs, except for RNA5.0, are required throughout the entire viral life cycle. Deletion of each lncRNA resulted in a decrease in viral progeny during lytic replication and failing to efficiently establish latent reservoirs and reactivate. Nanopore direct RNA sequencing of native lncRNA molecules revealed that each lncRNA exhibited a dynamic modification landscape, depending on the state of infection. Global analysis of the lncRNA interactome identified 32, 11, and 89 host factors that specifically bind to RNA1.2, RNA2.7, and RNA4.9, respectively. Moreover, 52 proteins commonly bound to the three lncRNAs were identified, including 11 antiviral immunity-related proteins. Our molecular analyses found that three lncRNAs are modified with N⁶-methyladenosine (m6A) and interact with m6A readers in all infection states. In-depth functional analysis revealed that m6A–mediated lncRNA stabilization as the key mechanism by which lncRNAs are maintained at high levels. Our study lays the groundwork for understanding viral lncRNA–mediated regulation of host-virus interaction throughout the HCMV life cycle.
2022-11-14 | PXD034587 | Pride
Project description:Human CMV (hCMV) establishes lifelong infections in most of us, causing developmental defects in human embryos and life-threatening disease in immunocompromised individuals. During productive infection, the viral >230,000-bp dsDNA genome is expressed widely and in a temporal cascade. The hCMV genome does not carry histones when encapsidated but has been proposed to form nucleosomes after release into the host cell nucleus. Here, we present hCMV genome-wide nucleosome occupancy and nascent transcript maps during infection of permissive human primary cells. We show that nucleosomes occupy nuclear viral DNA in a nonrandom and highly predictable fashion. At early times of infection, nucleosomes associate with the hCMV genome largely according to their intrinsic DNA sequence preferences, indicating that initial nucleosome formation is genetically encoded in the virus. However, as infection proceeds to the late phase, nucleosomes redistribute extensively to establish patterns mostly determined by nongenetic factors. We propose that these factors include key regulators of viral gene expression encoded at the hCMV major immediate-early (IE) locus. Indeed, mutant virus genomes defcient for IE1 expression exhibit globally increased nucleosome loads and reduced nucleosome dynamics compared with WT genomes. The temporal nucleosome occupancy differences between IE1-defcient and WT viruses correlate inversely with changes in the pattern of viral nascent and total transcript accumulation. These results provide a framework of spatial and temporal nucleosome organization across the genome of a major human pathogen and suggest that an hCMV major IE protein governs overall viral chromatin structure and function. [i] H3 ChIP-chip measurements of WT human cytomegalovirus (strain TB40E) following infection of MRC-5 cells. WT 8 hours postinfection, with corresponding mock IgG input control: 3 replicates; WT virus, 48 hours postinfection with corresponding mock IgG input contro: 3 replicates. [ii] MNase-chip measurements of WT and dlIE1 human cytomegalovirus nucleosomes following infection of MRC-5 cells. WT 8 hours postinfection, with corresponding sonicated DNA input control: 6 replicates; WT virus 48 hours postinfection, with corresponding sonicated DNA input control: 6 replicates; WT 96 hours postinfection, with corresponding sonicated DNA input control: 2 replicates; dlIE1 virus 8 hours postinfection, with corresponding sonicated DNA input control: 2 replicates; dlIE1 virus 96 hours postinfection, with corresponding sonicated DNA input control: 2 replicates. [iii] Total and nascent RNA measurements of WT and dlIE1 human cytomegalovirus transcripts following infection of MRC-5 cells. WT 8 hours postinfection: 2 replicates; WT virus 96 hours postinfection: 2 replicates; dlIE1 8 hours postinfection: 2 replicates; dlIE1 virus 96 hours postinfection: 2 replicates; sonicated DNA input control: 10 replicates.
2013-07-17 | E-GEOD-48875 | biostudies-arrayexpress
Project description:Herpesvirus late promoters activate gene expression after viral DNA synthesis has begun. Alphaherpesviruses utilize a viral immediate-early protein to do this, whereas beta- and gammaherpesviruses primarily use a 6-member set of viral late-acting transcription factors (LTF) that are drawn to a TATT sequence in the late promoter. The betaherpesvirus, human cytomegalovirus (HCMV), produces three immediate-early 2 protein isoforms, IE2-86, IE2-60, IE2-40, in late infection, but whether they activate late viral promoters is unknown. Here, we quickly degrade the IE2 proteins in late infection and analyze effects on transcription using customized PRO-Seq and computational methods combined with multiple validation methods. We discover that the IE2 proteins selectively drive RNA Pol II transcription initiation at a subset of viral early-late and late promoters common to different HCMV strains, but do not substantially affect Pol II transcription of the 9,942 expressed host genes. Most of the IE2-activated viral late infection promoters lack the TATT sequence bound by the HCMV UL87-encoded LTF. The HCMV TATT-binding protein is not mechanistically involved in late RNA expression from the IE2-activated TATT-less UL83 (pp65) promoter, as it is for the TATT-containing UL82 (pp71) promoter. While antecedent viral DNA synthesis is necessary for transcription from the late infection viral promoters, continued viral DNA synthesis is unnecessary. We conclude that the IE2 proteins target a distinct subset of late infection HCMV promoters for transcription initiation by RNA Pol II. Commencement of viral DNA replication renders the HCMV genome late promoters susceptible to late-acting viral transcription factors, which do not appreciably affect host transcription during this late time.
2020-03-05 | GSE139114 | GEO
Project description:Interferon-stimulated gene products (ISGs) play a crucial role in early infection control. The ISG zinc finger CCCH-type antiviral protein 1 (ZAP/ZC3HAV1) antagonises several RNA viruses by binding to CG-rich RNA sequences, whereas its effect on DNA viruses is largely unknown. Here, we decipher the role of ZAP in the context of human cytomegalovirus (HCMV) infection, a β-herpesvirus that is associated with high morbidity in immunosuppressed individuals and newborns. We show that expression of the two major isoforms of ZAP, the long (ZAP-L) and short (ZAP-S), is induced during HCMV infection and that both negatively affect HCMV replication. Transcriptome and proteome analyses demonstrated that the expression of ZAP decelerates the progression of HCMV infection. SLAM-sequencing revealed that ZAP restricts HCMV at early stages of infection by destabilising a distinct subset of viral transcripts with low CG content. In summary, this report provides evidence of an important antiviral role for ZAP in host defense against HCMV infection and highlights its differentiated function during DNA virus infection.
2021-04-22 | PXD023559 | Pride
Project description:Purpose: To identify interactions of the viral DNA-binding protein, UL34, with the viral genome during lytic-phase replication. Methods: Human fibroblasts were infected with human cytomegalovirus expressing Myc-tagged UL34 in biological duplicate. At 48 hours post infection, ChIP was performed with anti-Myc tag antibody (Cell Signaling) and the resulting DNA fragments were sequenced on Illumina MiSeq. Reads were trimmed and duplicates removed. Reads passing quality filter were aligned to a custom reference genome containing both the human genome with the human cytomegalovirus genome as an additional chromosome to prevent read assignment bias with CLC Genomics Workbench verison 9.0. Enrichment of UL34 in the viral origin of lytic replication (oriLyt) was calculated by dividing read counts from the ChIP'd samples (Myc-UL34-1, Myc-UL34-2) by those from non-ChIP whole cell lysate (Input) with a 125bp sliding window (25bp overhang) after adjusting for total read count variances. An additional sample containing DNA that was subjected to the ChIP protocol in the absence of antibody (No Antibody) was compared to the Input to determine background enrichment. Results: Several peaks were identified when comparing the Myc-UL34-ChIP samples to Input. UL34 is bound to the viral oriLyt at 48 hours post infection near the three previously defined UL34-binding sites to varying degrees, as well as additional locations within the region. No peaks were identified when comparing the No Antibody and Input samples.
2018-05-17 | GSE106211 | GEO
Project description:Reactivation of latent HCMV is a significant infectious complication of organ transplantation, and current therapies target viral replication once reactivation of transcriptionally silent, latent virus has already occurred. The specific molecular pathways that activate viral gene expression are not well understood. Our studies aim to identify these factors, with the goal of developing novel therapies that prevent transcriptional reactivation in transplant recipients. MCMV is a valuable model for studying latency and reactivation of CMV induced by organ transplantation. We previously demonstrated that transplantation of MCMV-latently infected kidneys into allogeneic recipients induces transcriptional reactivation of immediate early (IE) gene expression within 48 hr. We used microarrays to profile global mouse kidney gene expression associated with transcriptional reactivation of MCMV immediate early gene expression at 48 hr after transplant of MCMV latently infected kidneys into allogeneic recipients. We identified differentially expressed genes and pathways associated with both early acute rejection and reactivation of MCMV. control vs. allograft
2015-08-27 | E-GEOD-72392 | biostudies-arrayexpress
Project description:Human cytomegalovirus (HCMV) is an important pathogen and a paradigm of viral immune evasion. Here, we describe a multiplexed approach to discover proteins with novel innate immune functions on the basis of their active proteasomal or lysosomal degradation during the early phase of HCMV infection. Using three orthogonal proteomic/transcriptomic screens to quantify protein degradation, we identified 35 proteins with high confidence that were enriched in known antiviral restriction factors. A final screen facilitated global analysis of the mechanism of viral immune-modulation by predicting which viral genes target >250 human proteins. Helicase-like Transcription Factor (HLTF), a DNA helicase important in error-free post-replication DNA repair, was rapidly degraded during infection and potently inhibited early viral gene expression. HCMV UL145, a protein of previously unknown function, recruits the Cullin 4 E3 ligase complex to degrade HLTF. Our approach and data will enable further identifications of innate pathways targeted by HCMV and other viruses.
2018-08-16 | PXD009945 | Pride
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:Primary infection with Human cytomegalovirus (HCMV) results in a persistent lifelong infection due to its ability to establish latent infection. During productive HCMV infection, viral genes are expressed in a coordinated cascade that is characteristic of all herpesviruses and traditionally relies on the dependencies of viral genes on protein synthesis and viral DNA replication. In contrast, the transcriptional landscape associated with HCMV latency is still disputed and poorly understood. Here, we examine viral transcriptomic dynamics during the establishment of both productive and latent HCMV infections. These temporal measurements reveal that viral gene expression dynamics along productive infection and their dependencies on protein synthesis and viral DNA replication, do not fully align. This illustrates that the regulation of herpesvirus genes does not represent a simple sequential transcriptional cascade and surprisingly many viral genes are regulated by multiple independent modules. Using our improved classification of viral gene expression kinetics in conjunction with transcriptome-wide measurements of the effects of a wide array of chromatin modifiers, we unbiasedly show that a defining characteristic of latent cells is the unique repression of immediate early (IE) genes. In particular, we demonstrate that IE1 (a central IE protein) expression is the principal barrier for achieving a full productive cycle. Altogether, our findings provide an unbiased and elaborate definition of HCMV gene expression in lytic and latent infection states.
2022-03-29 | GSE193467 | GEO