Secreted Oral Epithelial Cell Membrane Vesicles Induce Epstein-Barr Virus Reactivation in Latently Infected B Cells

ABSTRACTIn the oral epithelium, peripheral stores of Epstein-Barr virus (EBV) are transmitted from infiltrating B cells to epithelial cells. Once the virus is transmitted to epithelial cells, the highly permissive nature of this cell type for lytic replication allows virus amplification and exchange to other hosts. Since the initial transfer of EBV from B cells to epithelial cells requires transitioning of the B-cell to a state that induces virus reactivation, we hypothesized that there might be epithelium-specific signals that allow the infiltrating B cells to sense the appropriate environment to initiate reactivation and begin this exchange process. We previously found that the epithelium-specific miR-200 family of microRNAs promotes EBV lytic replication. Here we show that there are high levels of miR-200 family members in oral and tonsillar epithelia and in saliva. Analysis of cultured oral epithelial cells (OKF6) showed that they actively secrete membrane vesicles (exosomes) that are enriched with miR-200 family members. Coculturing of EBV-positive B cells with OKF6 cells induced viral reactivation. Further, treatment of EBV-positive B cells with OKF6 cell-derived membrane vesicles promoted reactivation. Using a cell system that does not naturally express miR-200 family members, we found that enforced expression of a miR-200 family member produced membrane vesicles that were able to induce the lytic cascade in EBV-positive B cells. We propose that membrane vesicles secreted by oral and tonsillar epithelial cells may serve as a tissue-specific environmental cue that initiates reactivation in B cells, promoting the transfer of virus from peripheral B-cell stores to the oral epithelium to facilitate virus amplification and exchange to other hosts.IMPORTANCEEpstein-Barr virus (EBV) is an important human pathogen that is causally associated with several lymphomas and carcinomas. The switch from latency to the lytic cycle is critical for successful host infection and for EBV pathogenesis. Although the EBV lytic cycle can be triggered by certain agentsin vitro, the mechanisms that signal reactivationin vivoare poorly understood. We previously reported that endogenously expressed miR-200 family members likely play a role in facilitating the lytic tendencies of EBV in epithelial cells. Here we show that membrane vesicles secreted from oral epithelial cells contain miR-200 family members and that they can be transmitted to proximal EBV-positive B cells, where they trigger reactivation. We propose that this intercellular communication pathway may serve as a sensor mechanism for infiltrating B cells to recognize an appropriate environment to initiate reactivation, thereby allowing the exchange of virus to the oral epithelium.

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Antibodies to gp350/220 Enhance the Ability of Epstein-Barr Virus To Infect Epithelial Cells

Journal of Virology ◽

10.1128/jvi.00622-06 ◽

2006 ◽

Vol 80 (19) ◽

pp. 9628-9633 ◽

Cited By ~ 41

Author(s):

Susan M. Turk ◽

Ru Jiang ◽

Liudmila S. Chesnokova ◽

Lindsey M. Hutt-Fletcher

Keyword(s):

B Cells ◽

Nasopharyngeal Carcinoma ◽

Epithelial Cells ◽

Epstein Barr Virus ◽

Lytic Cycle ◽

Virus Envelope ◽

Barr Virus ◽

High Risk Populations ◽

Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) is a persistent, orally transmitted herpesvirus that replicates in B cells and epithelial cells and is associated with lymphoid and epithelial malignancies. The virus binds to CD21 on B cells via glycoprotein gp350/220 and infects efficiently. Infection of cultured epithelial cells has not typically been efficient but can occur in the absence of gp350/220 and CD21 and in vivo is thought to be important to the development of nasopharyngeal carcinoma. We report here that antibodies to gp350/220, which inhibit EBV infection of B cells, enhance infection of epithelial cells. The effect is not mediated by Fc receptor binding but is further enhanced by antibody cross-linking, which may patch gp350/220 in the virus envelope. Saliva from EBV-seropositive individuals has similar effects that can be reversed by depletion of antibody. The results are consistent with a model in which gp350/220 interferes with the access of other important players to the epithelial cell surface. The results may have implications for the development of nasopharyngeal carcinoma in high-risk populations in which elevated titers of antibody to EBV lytic cycle proteins are prognostic.

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Features Distinguishing Epstein-Barr Virus Infections of Epithelial Cells and B Cells: Viral Genome Expression, Genome Maintenance, and Genome Amplification

Journal of Virology ◽

10.1128/jvi.00108-09 ◽

2009 ◽

Vol 83 (15) ◽

pp. 7749-7760 ◽

Cited By ~ 77

Author(s):

Claire Shannon-Lowe ◽

Emily Adland ◽

Andrew I. Bell ◽

Henri-Jacques Delecluse ◽

Alan B. Rickinson ◽

...

Keyword(s):

B Cells ◽

Epithelial Cells ◽

Viral Genome ◽

Epstein Barr Virus ◽

Lytic Cycle ◽

Ags Cells ◽

Barr Virus ◽

Genome Expression ◽

Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) is associated with malignant diseases of lymphoid and epithelial cell origin. The tropism of EBV is due to B-cell-restricted expression of CD21, the major receptor molecule for the virus. However, efficient infection of CD21− epithelial cells can be achieved via transfer from EBV-coated B cells. We compare and contrast here the early events following in vitro infection of primary B cells and epithelial cells. Using sensitive, quantitative reverse transcription-PCR assays for several latent and lytic transcripts and two-color immunofluorescence staining to analyze expression at the single cell level, we confirmed and extended previous reports indicating that the two cell types support different patterns of transcription. Furthermore, whereas infection of B cells with one or two copies of EBV resulted in rapid amplification of the viral genome to >20 copies per cell, such amplification was not normally observed after infection of primary epithelial cells or undifferentiated epithelial lines. In epithelial cells, EBNA1 expression was detected in only ca. 40% of EBER+ cells, and the EBV genome was subsequently lost during prolonged culture. One exception was that infection of AGS, a gastric carcinoma line, resulted in maintenance of EBNA1 expression and amplification of the EBV episome. In contrast to B cells, where amplification of the EBV episome occurred even with a replication-defective BZLF1-knockout virus, amplification in AGS cells was dependent upon early lytic cycle gene expression. These data highlight the influence of the host cell on the outcome of EBV infection with regard to genome expression, amplification, and maintenance.

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Interaction sites of the Epstein–Barr virus Zta transcription factor with the host genome in epithelial cells

Access Microbiology ◽

10.1099/acmi.0.000282 ◽

2021 ◽

Vol 3 (11) ◽

Author(s):

Anja Godfrey ◽

Kay Osborn ◽

Alison J. Sinclair

Keyword(s):

Transcription Factor ◽

B Cells ◽

Epithelial Cells ◽

Binding Sites ◽

Epstein Barr Virus ◽

Lytic Replication ◽

Lytic Cycle ◽

Barr Virus ◽

Epstein Barr ◽

The Impact

Epstein–Barr virus (EBV) is present in a state of latency in infected memory B-cells and EBV-associated lymphoid and epithelial cancers. Cell stimulation or differentiation of infected B-cells and epithelial cells induces reactivation to the lytic replication cycle. In each cell type, the EBV transcription and replication factor Zta (BZLF1, EB1) plays a role in mediating the lytic cycle of EBV. Zta is a transcription factor that interacts directly with Zta response elements (ZREs) within viral and cellular genomes. Here we undertake chromatin-precipitation coupled to DNA-sequencing (ChIP-Seq) of Zta-associated DNA from cancer-derived epithelial cells. The analysis identified over 14 000 Zta-binding sites in the cellular genome. We assessed the impact of lytic cycle reactivation on changes in gene expression for a panel of Zta-associated cellular genes. Finally, we compared the Zta-binding sites identified in this study with those previously identified in B-cells and reveal substantial conservation in genes associated with Zta-binding sites.

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Differential Expression of the miR-200 Family MicroRNAs in Epithelial and B Cells and Regulation of Epstein-Barr Virus Reactivation by the miR-200 Family Member miR-429

Journal of Virology ◽

10.1128/jvi.00379-10 ◽

2010 ◽

Vol 84 (15) ◽

pp. 7892-7897 ◽

Cited By ~ 32

Author(s):

Zhen Lin ◽

Xia Wang ◽

Claire Fewell ◽

Jennifer Cameron ◽

Qinyan Yin ◽

...

Keyword(s):

B Cells ◽

Epithelial Cells ◽

Family Member ◽

Epstein Barr Virus ◽

Family Members ◽

Receptor Activation ◽

Virus Reactivation ◽

Barr Virus ◽

Ebv Reactivation ◽

Epstein Barr

ABSTRACT The miR-200 microRNA family is important for maintaining the epithelial phenotype, partially through suppressing ZEB1 and ZEB2. Since ZEB1 inhibits Epstein-Barr virus (EBV) reactivation, we hypothesized that expression of miR-200 family members in epithelial cells may partly account for higher levels of EBV reactivation in this tissue (relative to nonplasma B cells). Here we show that, whereas miR-200 family members are expressed in epithelial cells, their expression is low in latently infected B cells. Furthermore, the miR-200 family member miR-429 shows elevated expression in plasma cell lines and is induced by B-cell-receptor activation in Akata cells. Lastly, expression of miR-429 can break latency.

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The Epstein-Barr virus (EBV) nuclear antigen 1 BamHI F promoter is activated on entry of EBV-transformed B cells into the lytic cycle.

Journal of Virology ◽

10.1128/jvi.66.12.7461-7468.1992 ◽

1992 ◽

Vol 66 (12) ◽

pp. 7461-7468 ◽

Cited By ~ 41

Author(s):

A L Lear ◽

M Rowe ◽

M G Kurilla ◽

S Lee ◽

S Henderson ◽

...

Keyword(s):

B Cells ◽

Epstein Barr Virus ◽

Lytic Cycle ◽

Nuclear Antigen ◽

Barr Virus ◽

Epstein Barr

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Histone Loaders CAF1 and HIRA Restrict Epstein-Barr Virus B-Cell Lytic Reactivation

mBio ◽

10.1128/mbio.01063-20 ◽

2020 ◽

Vol 11 (5) ◽

Author(s):

Yuchen Zhang ◽

Chang Jiang ◽

Stephen J. Trudeau ◽

Yohei Narita ◽

Bo Zhao ◽

...

Keyword(s):

B Cells ◽

Burkitt Lymphoma ◽

Epstein Barr Virus ◽

Chromatin Assembly ◽

Lytic Cycle ◽

Histone Chaperones ◽

Viral Genomes ◽

Barr Virus ◽

Lytic Reactivation ◽

Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) infects 95% of adults worldwide and causes infectious mononucleosis. EBV is associated with endemic Burkitt lymphoma, Hodgkin lymphoma, posttransplant lymphomas, nasopharyngeal and gastric carcinomas. In these cancers and in most infected B-cells, EBV maintains a state of latency, where nearly 80 lytic cycle antigens are epigenetically suppressed. To gain insights into host epigenetic factors necessary for EBV latency, we recently performed a human genome-wide CRISPR screen that identified the chromatin assembly factor CAF1 as a putative Burkitt latency maintenance factor. CAF1 loads histones H3 and H4 onto newly synthesized host DNA, though its roles in EBV genome chromatin assembly are uncharacterized. Here, we found that CAF1 depletion triggered lytic reactivation and virion secretion from Burkitt cells, despite also strongly inducing interferon-stimulated genes. CAF1 perturbation diminished occupancy of histones 3.1 and 3.3 and of repressive histone 3 lysine 9 and 27 trimethyl (H3K9me3 and H3K27me3) marks at multiple viral genome lytic cycle regulatory elements. Suggestive of an early role in establishment of latency, EBV strongly upregulated CAF1 expression in newly infected primary human B-cells prior to the first mitosis, and histone 3.1 and 3.3 were loaded on the EBV genome by this time point. Knockout of CAF1 subunit CHAF1B impaired establishment of latency in newly EBV-infected Burkitt cells. A nonredundant latency maintenance role was also identified for the DNA synthesis-independent histone 3.3 loader histone regulatory homologue A (HIRA). Since EBV latency also requires histone chaperones alpha thalassemia/mental retardation syndrome X-linked chromatin remodeler (ATRX) and death domain-associated protein (DAXX), EBV coopts multiple host histone pathways to maintain latency, and these are potential targets for lytic induction therapeutic approaches. IMPORTANCE Epstein-Barr virus (EBV) was discovered as the first human tumor virus in endemic Burkitt lymphoma, the most common childhood cancer in sub-Saharan Africa. In Burkitt lymphoma and in 200,000 EBV-associated cancers per year, epigenetic mechanisms maintain viral latency, during which lytic cycle factors are silenced. This property complicated EBV’s discovery and facilitates tumor immunoevasion. DNA methylation and chromatin-based mechanisms contribute to lytic gene silencing. Here, we identified histone chaperones CAF1 and HIRA, which have key roles in host DNA replication-dependent and replication-independent pathways, respectively, as important for EBV latency. EBV strongly upregulates CAF1 in newly infected B-cells, where viral genomes acquire histone 3.1 and 3.3 variants prior to the first mitosis. Since histone chaperones ATRX and DAXX also function in maintenance of EBV latency, our results suggest that EBV coopts multiple histone pathways to reprogram viral genomes and highlight targets for lytic induction therapeutic strategies.

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Knockout of Epstein-Barr Virus BPLF1 Retards B-Cell Transformation and Lymphoma Formation in Humanized Mice

mBio ◽

10.1128/mbio.01574-15 ◽

2015 ◽

Vol 6 (5) ◽

Cited By ~ 29

Author(s):

Christopher B. Whitehurst ◽

Guangming Li ◽

Stephanie A. Montgomery ◽

Nathan D. Montgomery ◽

Lishan Su ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Epstein Barr Virus ◽

Cell Transformation ◽

Lytic Cycle ◽

Humanized Mice ◽

Barr Virus ◽

Epstein Barr ◽

Knockout Virus ◽

Large B Cell

ABSTRACTBPLF1 of Epstein-Barr virus (EBV) is classified as a late lytic cycle protein but is also found in the viral tegument, suggesting its potential involvement at both initial and late stages of viral infection. BPLF1 possesses both deubiquitinating and deneddylating activity located in its N-terminal domain and is involved in processes that affect viral infectivity, viral DNA replication, DNA repair, and immune evasion. A recently constructed EBV BPLF1-knockout (KO) virus was used in conjunction with a humanized mouse model that can be infected with EBV, enabling the first characterization of BPLF1 functionin vivo. Results demonstrate that the BPLF1-knockout virus is approximately 90% less infectious than wild-type (WT) virus. Transformation of human B cells, a hallmark of EBV infection, was delayed and reduced with BPLF1-knockout virus. Humanized mice infected with EBV BPLF1-knockout virus showed less weight loss and survived longer than mice infected with equivalent infectious units of WT virus. Additionally, splenic tumors formed in 100% of mice infected with WT EBV but in only 25% of mice infected with BPLF1-KO virus. Morphological features of spleens containing tumors were similar to those in EBV-induced posttransplant lymphoproliferative disease (PTLD) and were almost identical to cases seen in human diffuse large B-cell lymphoma. The presence of EBV genomes was detected in all mice that developed tumors. The results implicate BPLF1 in human B-cell transformation and tumor formation in humanized mice.IMPORTANCEEpstein-Barr virus infects approximately 90% of the world's population and is the causative agent of infectious mononucleosis. EBV also causes aggressive lymphomas in individuals with acquired and innate immune disorders and is strongly associated with diffuse large B-cell lymphomas, classical Hodgkin lymphoma, Burkitt lymphoma, and nasopharyngeal carcinoma (NPC). Typically, EBV initially infects epithelial cells in the oropharynx, followed by a lifelong persistent latent infection in B-cells, which may develop into lymphomas in immunocompromised individuals. This work is the first of its kind in evaluating the effects of EBV's BPLF1 in terms of pathogenesis and lymphomagenesis in humanized mice and implicates BPLF1 in B-cell transformation and tumor development. Currently, there is no efficacious treatment for EBV, and therapeutic targeting of BPLF1 may lead to a new path to treatment for immunocompromised individuals or transplant recipients infected with EBV.

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Compatibility of the gH homologues of Epstein–Barr virus and related lymphocryptoviruses

Journal of General Virology ◽

10.1099/vir.0.82949-0 ◽

2007 ◽

Vol 88 (8) ◽

pp. 2129-2136 ◽

Cited By ~ 7

Author(s):

Liguo Wu ◽

Lindsey M. Hutt-Fletcher

Keyword(s):

B Cells ◽

Epithelial Cells ◽

B Cell ◽

Cell Fusion ◽

Epstein Barr Virus ◽

Common Marmoset ◽

Barr Virus ◽

Human B Cells ◽

The Common ◽

Epstein Barr

Glycoprotein gH, together with its chaperone gL and a third glycoprotein gB, is essential for cell–cell fusion and virus–cell fusion mediated by herpesviruses. Epstein–Barr virus (EBV), the prototype human lymphocryptovirus, requires a fourth glycoprotein gp42 to support fusion with B cells in addition to epithelial cells. Two other lymphocryptoviruses, the rhesus lymphocryptovirus (Rh-LCV) and the common marmoset lymphocryptovirus (CalHV3), have been sequenced in their entirety and each has a gp42 homologue. Combinations of proteins from EBV, Rh-LCV and CalHV3 were able to mediate fusion of epithelial cells, but, even when complexed with EBV gp42, only Rh-LCV and not CalHV3 proteins were able to mediate fusion with human B cells. CalHV3 gL was also unable to function effectively as a chaperone for EBV or Rh-LCV gH. The Rh-LCV gH homologue supported more fusion than EBV gH with an epithelial cell and supported the highest levels of fusion with a B cell. Chimeric constructs made from Rh-LCV gH and EBV gH that have 85.4 % sequence identity should prove useful for mapping the regions of gH that are of importance to fusion as a whole and to B-cell fusion in particular.

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