Infection of Epstein–Barr Virus in Type III Latency Modulates Biogenesis of Exosomes and the Expression Profile of Exosomal miRNAs in the Burkitt Lymphoma Mutu Cell Lines

Infection of Epstein–Barr virus (EBV), a ubiquitous human gamma herpesvirus, is associated with various malignancies in B lymphocytes and epithelial cells. EBV encodes 49 microRNAs in two separated regions, termed the BART and BHRF1 loci. Although accumulating evidence demonstrates that EBV infection regulates the profile of microRNAs in the cells, little is known about the microRNAs in exosomes released from infected cells. Here, we characterized the expression profile of intracellular and exosomal microRNAs in EBV-negative, and two related EBV-infected Burkitt lymphoma cell lines having type I and type III latency by next-generation sequencing. We found that the biogenesis of exosomes is upregulated in type III latently infected cells compared with EBV-negative and type I latently infected cells. We also observed that viral and several specific host microRNAs were predominantly incorporated in the exosomes released from the cells in type III latency. We confirmed that multiple viral microRNAs were transferred to the epithelial cells cocultured with EBV-infected B cells. Our findings indicate that EBV infection, in particular in type III latency, modulates the biogenesis of exosomes and the profile of exosomal microRNAs, potentially contributing to phenotypic changes in cells receiving these exosomes.

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MicroRNA-155 Is an Epstein-Barr Virus-Induced Gene That Modulates Epstein-Barr Virus-Regulated Gene Expression Pathways

Journal of Virology ◽

10.1128/jvi.02380-07 ◽

2008 ◽

Vol 82 (11) ◽

pp. 5295-5306 ◽

Cited By ~ 181

Author(s):

Qinyan Yin ◽

Jane McBride ◽

Claire Fewell ◽

Michelle Lacey ◽

Xia Wang ◽

...

Keyword(s):

Gene Expression ◽

Epstein Barr Virus ◽

Type I ◽

Type Iii ◽

Barr Virus ◽

Transcriptional Regulatory ◽

Infected Cells ◽

Epstein Barr ◽

Regulated Gene Expression ◽

In Cells

ABSTRACT The cellular microRNA miR-155 has been shown to be involved in lymphocyte activation and is expressed in Epstein-Barr virus (EBV)-infected cells displaying type III latency gene expression but not type I latency gene expression. We show here that the elevated levels of miR-155 in type III latency cells is due to EBV gene expression and not epigenetic differences in cell lines tested, and we show that expression in EBV-infected cells requires a conserved AP-1 element in the miR-155 promoter. Gene expression analysis was carried out in a type I latency cell line transduced with an miR-155-expressing retrovirus. This analysis identified both miR-155-suppressed and -induced cellular mRNAs and suggested that in addition to direct targeting of 3′ untranslated regions (UTRs), miR-155 alters gene expression in part through the alteration of signal transduction pathways. 3′ UTR reporter analysis of predicted miR-155 target genes identified the transcriptional regulatory genes encoding BACH1, ZIC3, HIVEP2, CEBPB, ZNF652, ARID2, and SMAD5 as miR-155 targets. Western blot analysis of the most highly suppressed of these, BACH1, showed lower expression in cells transduced with a miR-155 retrovirus. Inspection of the promoters from genes regulated in EBV-infected cells and in cells infected with an miR-155 retrovirus identified potential binding sequences for BACH1 and ZIC3. Together, these experiments suggest that the induction of miR-155 by EBV contributes to EBV-mediated signaling in part through the modulation of transcriptional regulatory factors.

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Morphology, immunophenotype, and distribution of latently and/or productively Epstein-Barr virus-infected cells in acute infectious mononucleosis: implications for the interindividual infection route of Epstein-Barr virus

Blood ◽

10.1182/blood.v85.3.744.bloodjournal853744 ◽

1995 ◽

Vol 85 (3) ◽

pp. 744-750 ◽

Cited By ~ 155

Author(s):

I Anagnostopoulos ◽

M Hummel ◽

C Kreschel ◽

H Stein

Keyword(s):

Epithelial Cells ◽

Infectious Mononucleosis ◽

Epstein Barr Virus ◽

Lymphoid Cells ◽

Productive Infection ◽

Barr Virus ◽

Ebv Infection ◽

Infected Cells ◽

Epstein Barr ◽

Acute Infectious Mononucleosis

The present study was undertaken to unequivocally demonstrate the morphology, immunophenotype, and localization of Epstein Barr virus (EBV)-infected cells as well as the type of infection (latent versus productive) in tonsils of acute infectious mononucleosis. Paraffin sections from nine cases with clinical, serologic, and morphologic evidence of EBV infection were analyzed for the detection of small transcripts, designated EBER1 & 2, and BHLF1 by in situ hybridization (ISH) using nonisotopically labeled probes. ISH was combined with immunohistology, employing a broad panel of antibodies against B-, T-, epithelial-, macrophage-, and follicular dendritic cell (FDC)-antigens. All EBER-positive cells could be identified as lymphocytes, as they did not exhibit any morphologic or immunologic characteristics of epithelial cells, macrophages, or FDCs. A preferential accumulation of EBER-positive cells was noted around crypts, within surface squamous epithelium, and in the surroundings of necrosis. The majority of these lymphocytes could be shown to be B cells, which morphologically included Reed-Sternberg (RS)-like cells, immunoblasts, medium-sized lymphoid cells, as well as cells with plasmacytoid differentiation. In all cases, a varying number of EBER-positive T cells could be identified. ISH for BHLF1-RNA detection showed that almost all cases contained single positive small lymphoid cells, indicating a transition from latent to productive infection cycle. Such cells could also be detected within the crypt epithelium reaching up to its surface. Additional screening of 123 oropharyngeal mucosa samples from patients without evidence of acute EBV-infection, using the polymerase chain reaction for EBV-DNA detection combined with EBER- and BHLF1-ISH showed single latently infected lymphocytes in only one case. Our data imply that infected lymphocytes and not epithelial cells are, in fact, the reservoir for EBV infection, and that these are the cells that participate in the interindividual virus transfer.

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Epstein-Barr virus (EBV)–encoded small RNA is released from EBV-infected cells and activates signaling from toll-like receptor 3

Journal of Experimental Medicine ◽

10.1084/jem.20081761 ◽

2009 ◽

Vol 206 (10) ◽

pp. 2091-2099 ◽

Cited By ~ 182

Author(s):

Dai Iwakiri ◽

Li Zhou ◽

Mrinal Samanta ◽

Misako Matsumoto ◽

Takashi Ebihara ◽

...

Keyword(s):

Proinflammatory Cytokines ◽

Small Rna ◽

Epstein Barr Virus ◽

Type I ◽

Toll Like Receptor ◽

Double Stranded Rna ◽

Barr Virus ◽

Ebv Infection ◽

Infected Cells ◽

Epstein Barr

Epstein-Barr virus–encoded small RNA (EBER) is nonpolyadenylated, noncoding RNA that forms stem-loop structure by intermolecular base-pairing, giving rise to double-stranded RNA (dsRNA)–like molecules, and exists abundantly in EBV-infected cells. Here, we report that EBER induces signaling from the Toll-like receptor 3 (TLR3), which is a sensor of viral double-stranded RNA (dsRNA) and induces type I IFN and proinflammatory cytokines. A substantial amount of EBER, which was sufficient to induce signaling from TLR3, was released from EBV-infected cells, and the majority of the released EBER existed as a complex with a cellular EBER-binding protein La, suggesting that EBER was released from the cells by active secretion of La. Sera from patients with infectious mononucleosis (IM), chronic active EBV infection (CAEBV), and EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), whose general symptoms are caused by proinflammatory cytokines contained EBER, and addition of RNA purified from the sera into culture medium induced signaling from TLR3 in EBV-transformed lymphocytes and peripheral mononuclear cells. Furthermore, DCs treated with EBER showed mature phenotype and antigen presentation capacity. These findings suggest that EBER, which is released from EBV-infected cells, is responsible for immune activation by EBV, inducing type I IFN and proinflammatory cytokines. EBER-induced activation of innate immunity would account for immunopathologic diseases caused by active EBV infection.

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The Intersection of Epstein-Barr Virus with the Germinal Center

Journal of Virology ◽

10.1128/jvi.02609-08 ◽

2009 ◽

Vol 83 (8) ◽

pp. 3968-3976 ◽

Cited By ~ 86

Author(s):

Jill E. Roughan ◽

David A. Thorley-Lawson

Keyword(s):

B Cells ◽

Germinal Center ◽

Epstein Barr Virus ◽

Barr Virus ◽

Ebv Infection ◽

Latently Infected ◽

Infected Cells ◽

Epstein Barr ◽

Transcription Program

ABSTRACT The current model of Epstein-Barr virus (EBV) infection and persistence in vivo proposes that EBV uses the germinal center (the GC model) to establish a quiescent latent infection in otherwise-normal memory B cells. However, the evidence linking EBV-infected cells and the GC is only indirect and limited. Therefore, a key portion of the model, that EBV-infected cells physically reside and participate in GCs, has yet to be verified. Furthermore, recent experiments suggested that upon infection of GC cells the viral growth latency transcription program is dominant and GC functionality and phenotype are ablated, i.e., EBV infection is not consistent with GC function. In this study we show that in vivo, EBV-infected B cells in the tonsils retain expression of functional and phenotypic markers of GC cells, including bcl-6 and AID. Furthermore, these cells are physically located in the GC and express a restricted form of latency, the default latency program. Thus, the EBV default latency transcription program, unlike the growth latency program, is consistent with the retention of GC functionality in vivo. This work verifies key components of the GC model of EBV persistence and suggests that EBV and the GC can interact to produce the latently infected memory cells found in the periphery. Furthermore, it identifies latently infected GC B cells as a potential pathogenic nexus for the development of the EBV-positive, GC-associated lymphomas Hodgkin's disease and Burkitt's lymphoma.

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Epstein-Barr Virus miR-BART6-3p Inhibits the RIG-I Pathway

Journal of Innate Immunity ◽

10.1159/000479749 ◽

2017 ◽

Vol 9 (6) ◽

pp. 574-586 ◽

Cited By ~ 33

Author(s):

Yuanjun Lu ◽

Zailong Qin ◽

Jia Wang ◽

Xiang Zheng ◽

Jianhong Lu ◽

...

Keyword(s):

Gene Expression ◽

Immune Response ◽

Epstein Barr Virus ◽

Type I ◽

Receptor Signaling ◽

Viral Pathogen ◽

Infection Period ◽

Barr Virus ◽

Ebv Infection ◽

Epstein Barr

Recognition of viral pathogen-associated molecular patterns by pattern recognition receptors (PRRs) is the first step in the initiation of a host innate immune response. As a PRR, RIG-I detects either viral RNA or replication transcripts. Avoiding RIG-I recognition is a strategy employed by viruses for immune evasion. Epstein-Barr virus (EBV) infects the majority of the human population worldwide. During the latent infection period there are only a few EBV proteins expressed, whereas EBV-encoded microRNAs, such as BART microRNAs, are highly expressed. BART microRNAs regulate both EBV and the host's gene expression, modulating virus proliferation and the immune response. Here, through gene expression profiling, we found that EBV miR-BART6-3ps inhibited genes of RIG-I-like receptor signaling and the type I interferon (IFN) response. We demonstrated that miR-BART6-3p rather than other BARTs specifically suppressed RIG-I-like receptor signaling-mediated IFN-β production. RNA-seq was used to analyze the global transcriptome change upon EBV infection and miR-BART6-3p mimics transfection, which revealed that EBV infection-triggered immune response signaling can be repressed by miR-BART6-3p overexpression. Furthermore, miR-BART6-3p inhibited the EBV-triggered IFN-β response and facilitated EBV infection through targeting the 3′UTR of RIG-I mRNA. These findings provide new insights into the mechanism underlying the strategies employed by EBV to evade immune surveillance.

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Hippo signaling effectors YAP and TAZ induce Epstein-Barr Virus (EBV) lytic reactivation through TEADs in epithelial cells

PLoS Pathogens ◽

10.1371/journal.ppat.1009783 ◽

2021 ◽

Vol 17 (8) ◽

pp. e1009783

Author(s):

Nicholas Van Sciver ◽

Makoto Ohashi ◽

Nicholas P. Pauly ◽

Jillian A. Bristol ◽

Scott E. Nelson ◽

...

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

B Cell ◽

Epstein Barr Virus ◽

Hippo Signaling ◽

Oral Keratinocytes ◽

Barr Virus ◽

Ebv Infection ◽

Lytic Reactivation ◽

Epstein Barr

The Epstein-Barr virus (EBV) human herpesvirus is associated with B-cell and epithelial-cell malignancies, and both the latent and lytic forms of viral infection contribute to the development of EBV-associated tumors. Here we show that the Hippo signaling effectors, YAP and TAZ, promote lytic EBV reactivation in epithelial cells. The transcriptional co-activators YAP/TAZ (which are inhibited by Hippo signaling) interact with DNA-binding proteins, particularly TEADs, to induce transcription. We demonstrate that depletion of either YAP or TAZ inhibits the ability of phorbol ester (TPA) treatment, cellular differentiation or the EBV BRLF1 immediate-early (IE) protein to induce lytic EBV reactivation in oral keratinocytes, and show that over-expression of constitutively active forms of YAP and TAZ reactivate lytic EBV infection in conjunction with TEAD family members. Mechanistically, we find that YAP and TAZ interact with, and activate, the EBV BZLF1 immediate-early promoter. Furthermore, we demonstrate that YAP, TAZ, and TEAD family members are expressed at much higher levels in epithelial cell lines in comparison to B-cell lines, and find that EBV infection of oral keratinocytes increases the level of activated (dephosphorylated) YAP and TAZ. Finally, we have discovered that lysophosphatidic acid (LPA), a known YAP/TAZ activator that plays an important role in inflammation, induces EBV lytic reactivation in epithelial cells through a YAP/TAZ dependent mechanism. Together these results establish that YAP/TAZ are powerful inducers of the lytic form of EBV infection and suggest that the ability of EBV to enter latency in B cells at least partially reflects the extremely low levels of YAP/TAZ and TEADs in this cell type.

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Epstein–Barr Virus Permissively Infects Human Syncytiotrophoblastsin Vitroand Induces Replication of Human T Cell Leukemia-Lymphoma Virus Type I in Dually Infected Cells

Virology ◽

10.1006/viro.1997.8449 ◽

1997 ◽

Vol 229 (2) ◽

pp. 400-414 ◽

Cited By ~ 14

Author(s):

Ferenc D. Tóth ◽

George Aboagye-Mathiesen ◽

József Nemes ◽

Xiangdong Liu ◽

István Andirkó ◽

...

Keyword(s):

T Cell ◽

Virus Type ◽

Epstein Barr Virus ◽

Type I ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Barr Virus ◽

Human T Cell ◽

Infected Cells ◽

Epstein Barr

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Nascent Transcriptomics Reveal Cellular Prolytic Factors Upregulated Upstream of the Latent-to-Lytic Switch Protein of Epstein-Barr Virus

Journal of Virology ◽

10.1128/jvi.01966-19 ◽

2020 ◽

Vol 94 (7) ◽

Author(s):

Tiffany R. Frey ◽

Jozan Brathwaite ◽

Xiaofan Li ◽

Sandeepta Burgula ◽

Ibukun A. Akinyemi ◽

...

Keyword(s):

Epstein Barr Virus ◽

Human Herpesvirus ◽

Lytic Cycle ◽

Barr Virus ◽

Viral Spread ◽

Cellular Genes ◽

Latently Infected ◽

Infected Cells ◽

Host Shutoff ◽

Epstein Barr

ABSTRACT Lytic activation from latency is a key transition point in the life cycle of herpesviruses. Epstein-Barr virus (EBV) is a human herpesvirus that can cause lymphomas, epithelial cancers, and other diseases, most of which require the lytic cycle. While the lytic cycle of EBV can be triggered by chemicals and immunologic ligands, the lytic cascade is activated only when expression of the EBV latent-to-lytic switch protein ZEBRA is turned on. ZEBRA then transcriptionally activates other EBV genes and, together with some of those gene products, ensures completion of the lytic cycle. However, not every latently infected cell exposed to a lytic trigger turns on the expression of ZEBRA, resulting in responsive and refractory subpopulations. What governs this dichotomy? By examining the nascent transcriptome following exposure to a lytic trigger, we find that several cellular genes are transcriptionally upregulated temporally upstream of ZEBRA. These genes regulate lytic susceptibility to various degrees in latently infected cells that respond to mechanistically distinct lytic triggers. While increased expression of these cellular genes defines a prolytic state, such upregulation also runs counter to the well-known mechanism of viral-nuclease-mediated host shutoff that is activated downstream of ZEBRA. Furthermore, a subset of upregulated cellular genes is transcriptionally repressed temporally downstream of ZEBRA, indicating an additional mode of virus-mediated host shutoff through transcriptional repression. Thus, increased transcription of a set of host genes contributes to a prolytic state that allows a subpopulation of cells to support the EBV lytic cycle. IMPORTANCE Transition from latency to the lytic phase is necessary for herpesvirus-mediated pathology as well as viral spread and persistence in the population at large. Yet, viral genomes in only some cells in a population of latently infected cells respond to lytic triggers, resulting in subpopulations of responsive/lytic and refractory cells. Our investigations into this partially permissive phenotype of the herpesvirus Epstein-Barr virus (EBV) indicate that upon exposure to lytic triggers, certain cellular genes are transcriptionally upregulated, while viral latency genes are downregulated ahead of expression of the viral latent-to-lytic switch protein. These cellular genes contribute to lytic susceptibility to various degrees. Apart from indicating that there may be a cellular “prolytic” state, our findings indicate that (i) early transcriptional upregulation of cellular genes counters the well-known viral-nuclease-mediated host shutoff and (ii) subsequent transcriptional downregulation of a subset of early upregulated cellular genes is a previously undescribed mode of host shutoff.

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Epstein–Barr Virus Infection of Pseudostratified Nasopharyngeal Epithelium Disrupts Epithelial Integrity

Cancers ◽

10.3390/cancers12092722 ◽

2020 ◽

Vol 12 (9) ◽

pp. 2722

Author(s):

Fenggang Yu ◽

Yanan Lu ◽

Yingying Li ◽

Yuji Uchio ◽

Utomo Andi Pangnguriseng ◽

...

Keyword(s):

Epithelial Cells ◽

Epstein Barr Virus ◽

Basal Layer ◽

Infection Model ◽

Hybridization Technique ◽

Epstein Barr Virus Infection ◽

Barr Virus ◽

Ebv Infection ◽

Epstein Barr ◽

Nasopharyngeal Epithelial

Epstein–Barr virus (EBV) is a human oncogenic virus that causes several types of tumor, such as Burkitt’s lymphoma and nasopharyngeal carcinoma (NPC). NPC tumor cells are clonal expansions of latently EBV-infected epithelial cells. However, the mechanisms by which EBV transforms the nasopharyngeal epithelium is hampered, because of the lack of good in vitro model to pursue oncogenic process. Our primary nasopharyngeal epithelial cell cultures developed pseudostratified epithelium at the air-liquid interface, which was susceptible to EBV infection. Using the highly sensitive RNA in situ hybridization technique, we detected viral infection in diverse cell types, including ciliated cells, goblet cells, and basal cells. EBV-encoded small RNA-positive cells were more frequently detected in the suprabasal layer than in the basal layer. We established the most physiologically relevant EBV infection model of nasopharyngeal epithelial cells. This model will advance our understanding of EBV pathogenesis in the development of NPC.

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