Detection of RNA in purified cytomegalovirus virions

Sarcinella, Elizabeth; Brown, Martha; Tellier, Raymond; Petric, Martin; Mazzulli, Tony

doi:10.1016/j.virusres.2004.03.008

Cited by 12 publications

(8 citation statements)

References 19 publications

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“…However, it is difficult to produce DB preparations completely free of infectious virus and vice versa. In earlier studies, DB preparations were contaminated with virions [ 21, 27, 39 ] and were infectious [ 27 ]. The purity of our DB preparation was confirmed by the typical morphology of the particles by electron microscopy, the presence of pp65 protein and few copies of the HCMV genome, and the absence of the host cell genome (RNase P gene).…”

Section: Discussionmentioning

confidence: 99%

Human cytomegalovirus microRNAs are carried by virions and dense bodies and are delivered to target cells

Mohammad

Costa

Landázuri

et al. 2017

Journal of General Virology

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Human cytomegalovirus (HCMV) infection results in the production of virions, dense bodies (DBs) and non-infectious enveloped particles, all of which incorporate proteins and RNAs that can be transferred to host cells. Here, we investigated whether virions and DBs also carry microRNAs (miRNAs) and assessed their delivery and functionality in cells. Human lung fibroblasts (MRC-5) were infected with the HCMV strain AD169, and conditioned cell culture medium was collected and centrifuged. The pellets were treated with RNase-ONE, and the virions and DBs were purified with a potassium tartrate–glycerol gradient and dialysed. The virions and DBs were incubated with micrococcal nuclease, DNA and RNA were extracted and then analysed with TaqMan PCR assays, while the proteins were examined with Western blots. To assess the delivery of miRNAs to cells and their functionality, virions and DBs were irradiated with UV light. The purity of the virions and DBs was confirmed by typical morphology, the presence of the structural protein pp65 and the HCMV genome, the ability to infect MRC-5 cells and the absence of the host genome. RNA analysis revealed the presence of 14 HCMV-encoded miRNAs (UL22A-5p, US25-1-5p, UL22A-3p, US5-2-3p, UL112-3p, US25-2-3p, US25-2-5p, US33-3p, US5-1, UL36-5p, US4-5p, UL36-3p, UL70-5p and US25-1-3p), HCMV immediate-early mRNA and long non-coding RNA2.7, moreover, two host-encoded miRNAs (hsa-miR-218-5p and hsa-miR-21-5p) and beta-2-microglobulin RNA. UV-irradiated virions and DBs delivered viral miRNAs (US25-1-5p and UL112-3p) to the host cells, and miR-US25-1-5p was functional in a luciferase reporter assay. We conclude that virions and DBs carry miRNAs that are biologically functional and can be delivered to cells, which may affect cellular processes.

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Section: Discussionmentioning

confidence: 99%

Human cytomegalovirus microRNAs are carried by virions and dense bodies and are delivered to target cells

Mohammad

Costa

Landázuri

et al. 2017

Journal of General Virology

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“…Their downregulation was UV-sensitive, suggesting that it may be due to degradation by virion-associated nucleic acids [53], [54]. The GFI1 mRNA may be a direct target of virion-associated non-coding RNAs [55], [56]. The GFI1 protein may be degraded via the proteasome, which is known to play an important role in the transcription of the HCMV immediate early genes [57], [58].…”

Section: Discussionmentioning

confidence: 99%

The Downregulation of GFI1 by the EZH2-NDY1/KDM2B-JARID2 Axis and by Human Cytomegalovirus (HCMV) Associated Factors Allows the Activation of the HCMV Major IE Promoter and the Transition to Productive Infection

et al. 2014

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Earlier studies had suggested that epigenetic mechanisms play an important role in the control of human cytomegalovirus (HCMV) infection. Here we show that productive HCMV infection is indeed under the control of histone H3K27 trimethylation. The histone H3K27 methyltransferase EZH2, and its regulators JARID2 and NDY1/KDM2B repress GFI1, a transcriptional repressor of the major immediate-early promoter (MIEP) of HCMV. Knocking down EZH2, NDY1/KDM2B or JARID2 relieves the repression and results in the upregulation of GFI1. During infection, the incoming HCMV rapidly downregulates the GFI1 mRNA and protein in both wild-type cells and in cells in which EZH2, NDY1/KDM2B or JARID2 were knocked down. However, since the pre-infection levels of GFI1 in the latter cells are significantly higher, the virus fails to downregulate it to levels permissive for MIEP activation and viral infection. Following the EZH2-NDY1/KDM2B-JARID2-independent downregulation of GFI1 in the early stages of infection, the virus also initiates an EZH2-NDY1/ΚDM2Β-JARID2-dependent program that represses GFI1 throughout the infection cycle. The EZH2 knockdown also delays histone H3K27 trimethylation in the immediate early region of HCMV, which is accompanied by a drop in H3K4 trimethylation that may contribute to the shEZH2-mediated repression of the major immediate early HCMV promoter. These data show that HCMV uses multiple mechanisms to allow the activation of the HCMV MIEP and to prevent cellular mechanisms from blocking the HCMV replication program.

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“…While the presence and role of “virion-associated RNAs” remain controversial (Marcinowski et al, 2012;Sarcinella et al, 2004), these RNAs appear to be packaged selectively over host RNAs (Cliffe, Nash and Dutia, 2009;Greijer, Dekkers and Middeldorp, 2000;Sciortino et al, 2001) and virion-associated RNAs are incorporated in the proportions they are found in the host cell (Terhune, Schroer and Shenk, 2004). Virion-associated RNAs have been identified in the related herpesviruses: herpes simplex virus type 1 (HSV1), cytomegalovirus (CMV), murine gamma-herpesvirus (MHV68), and Kaposi’s sarcoma-associated herpesvirus (KSHV) and most recently in EBV (Jochum et al, 2012b).…”

Section: Virion-associated Rnas and Very Early Events In Infectionmentioning

confidence: 99%

Dynamic Epstein–Barr Virus Gene Expression on the Path to B-Cell Transformation

Price

Luftig

2014

Advances in Virus Research

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Epstein-Barr Virus is an oncogenic human herpesvirus in the γ-herpesvirinae sub-family that contains a 170–180 kb double stranded DNA genome. In vivo, EBV commonly infects B and epithelial cells and persists for the life of the host in a latent state in the memory B cell compartment of the peripheral blood. EBV can be reactivated from its latent state leading to increased expression of lytic genes that primarily encode for enzymes necessary to replicate the viral genome as well as structural components of the virion. Lytic cycle proteins also aid in immune evasion, inhibition of apoptosis, and the modulation of other host responses to infection. In vitro, EBV has the potential to infect primary human B cells and induce cellular proliferation to yield effectively immortalized lymphoblastoid cell lines, or LCLs. EBV immortalization of B cells in vitro serves as a model system for studying EBV-mediated lymphomagenesis. While much is known about the steady state viral gene expression within EBV immortalized LCLs and other EBV-positive cell lines, relatively little is known about the early events after primary B-cell infection. It was previously thought that upon latent infection EBV only expressed the well-characterized latency associated transcripts found in LCLs. However, recent work has characterized the early, but transient, expression of lytic genes necessary for efficient transformation as well as delayed responses in the known latency genes. This review summarizes these recent findings that show how dynamic and controlled expression of multiple EBV genes can control the activation of B cells, entry into the cell cycle, inhibition of apoptosis, and control of innate and adaptive immune responses.

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Detection of RNA in purified cytomegalovirus virions

Cited by 12 publications

References 19 publications

Human cytomegalovirus microRNAs are carried by virions and dense bodies and are delivered to target cells

Human cytomegalovirus microRNAs are carried by virions and dense bodies and are delivered to target cells

The Downregulation of GFI1 by the EZH2-NDY1/KDM2B-JARID2 Axis and by Human Cytomegalovirus (HCMV) Associated Factors Allows the Activation of the HCMV Major IE Promoter and the Transition to Productive Infection

Dynamic Epstein–Barr Virus Gene Expression on the Path to B-Cell Transformation

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