Direct evidence of abortive lytic infection-mediated establishment of Epstein-Barr virus latency during B-cell infection

AbstractViral infection induces dynamic changes in transcriptional profiles. Virus-induced and anti-viral responses are intertwined during the infection. Epstein-Barr virus (EBV) is a human gammaherpesvirus that provides a model of herpesvirus latency. To measure the transcriptome changes during the establishment of EBV latency, EBV-negative Akata cells were infected with EBV-EGFP and observed by transcriptome sequencing (RNA-seq) at 0, 2, 4, 7, 10, and 14 days after infection. We found transient downregulation of mitotic division-related genes, reflecting reprograming of cell growth by EBV. Moreover, a burst of viral lytic gene expression was detected in the early phase of infection. Experimental and mathematical investigations demonstrated that infectious virions were not produced in the pre-latent phase, suggesting the presence of an abortive lytic infection. Finally, we conducted fate mapping using recombinant EBV, enabling the noninvasive, continuous observation of infected cells during EBV infection. Our tracking analysis provided direct evidence that the abortive lytic infection in the pre-latent phase converges to latent infection during EBV infection of B-cells, shedding light on novel roles of viral lytic gene(s) in establishing latency.Author summaryViral infection is a complex process that activates both virus-triggered and host anti-viral responses. This process has classically been studied by snapshot analysis such as microarray and RNA-seq at discrete time points as population averages. Snapshot data lead to invaluable findings in host-pathogen interactions. However, these “snapshot” omics, even from a single cell, lack temporal resolution. Because the behavior of infected cells is highly dynamic and heterogenous, continuous analysis is required for deciphering the fate of infected cells during viral infection. Here, we exploited fate mapping techniques with recombinant Epstein-Barr virus (EBV) to track the infected cells and recorded a log of lytic gene expression during EBV infection. Our continuous observation of infected cells revealed that EBV established latency in B-cells via an abortive lytic infection in the pre-latent phase.

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Direct Evidence of Abortive Lytic Infection-Mediated Establishment of Epstein-Barr Virus Latency During B-Cell Infection

Frontiers in Microbiology ◽

10.3389/fmicb.2020.575255 ◽

2021 ◽

Vol 11 ◽

Author(s):

Tomoki Inagaki ◽

Yoshitaka Sato ◽

Jumpei Ito ◽

Mitsuaki Takaki ◽

Yusuke Okuno ◽

...

Keyword(s):

Epstein Barr Virus ◽

Direct Evidence ◽

Lytic Infection ◽

Viral Gene ◽

Cell Infection ◽

Gene Expressions ◽

Lytic Gene ◽

Barr Virus ◽

Epstein Barr ◽

Latent Phase

Viral infection induces dynamic changes in transcriptional profiles. Virus-induced and antiviral responses are intertwined during the infection. Epstein-Barr virus (EBV) is a human gammaherpesvirus that provides a model of herpesvirus latency. To measure the transcriptome changes during the establishment of EBV latency, we infected EBV-negative Akata cells with EBV-EGFP and performed transcriptome sequencing (RNA-seq) at 0, 2, 4, 7, 10, and 14 days after infection. We found transient downregulation of mitotic division-related genes, reflecting reprogramming of cell growth by EBV, and a burst of viral lytic gene expression in the early phase of infection. Experimental and mathematical investigations demonstrate that infectious virions were not produced in the pre-latent phase, suggesting the presence of an abortive lytic infection. Fate mapping using recombinant EBV provided direct evidence that the abortive lytic infection in the pre-latent phase converges to latent infection during EBV infection of B-cells, shedding light on novel roles of viral lytic gene(s) in establishing latency. Furthermore, we find that the BZLF1 protein, which is a key regulator of reactivation, was dispensable for abortive lytic infection in the pre-latent phase, suggesting the divergent regulation of viral gene expressions from a productive lytic infection.

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Epstein-Barr virus tegument protein BGLF2 in exosomes released from virus-producer cells assists de novo infection by enhancing viral gene expression

10.1101/2020.06.22.166280 ◽

2020 ◽

Author(s):

Masahiro Yaguchi ◽

Yoshitaka Sato ◽

Yusuke Okuno ◽

Takayuki Murata ◽

Somi Ozaki ◽

...

Keyword(s):

Viral Infection ◽

Epstein Barr Virus ◽

Host Cells ◽

Tegument Protein ◽

Tegument Proteins ◽

Barr Virus ◽

Ebv Infection ◽

Cellular Environment ◽

Infected Cells ◽

Epstein Barr

AbstractViruses must adapt to the environment of their host cells to establish infection and persist. Diverse mammalian cells, including virus-infected cells, secrete extracellular vesicles such as exosomes containing proteins and miRNAs. These vesicles mediate intercellular communications, suggesting that they modulate viral infection by adapting cellular conditions. However, the roles of exosomes in viral infection remain unclear. Here we screened viral proteins to identify those responsible for the exosome-mediated upregulation of Epstein-Barr virus (EBV) infection. We found BGLF2 protein encapsulated in exosomes, which enhanced the EBV infection of Akata(-) B-cells. BGLF2 protein is a tegument protein that lines the space between the envelope and the nucleocapsid, and it is released shortly after infection into the cytoplasm. Therefore, tegument protein BGLF2 is encapsulated not only in viral particles, but also in exosomes secreted from infected cells, and plays crucial roles in establishing the EBV latent infection by modulating the cellular environment.ImportanceTegument proteins that line the space between the envelope and nucleocapsid are released shortly after infection into the cytoplasm to modulate the cellular environment. In this study, we identified that tegument protein BGLF2, which is conserved in all herpesviruses, is incorporated into exosomes and transferred to neighboring cells. Exosomes containing BGLF2 enhanced the infectivity of EBV. These findings suggest that this and perhaps other tegument proteins support viral infection not only between the envelope and nucleocapsid, but also in extra-virus particles such as exosomes.

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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

Cancers ◽

10.3390/cancers10070237 ◽

2018 ◽

Vol 10 (7) ◽

pp. 237 ◽

Cited By ~ 13

Author(s):

Asuka Nanbo ◽

Harutaka Katano ◽

Michiyo Kataoka ◽

Shiho Hoshina ◽

Tsuyoshi Sekizuka ◽

...

Keyword(s):

Epithelial Cells ◽

Burkitt Lymphoma ◽

Epstein Barr Virus ◽

Type I ◽

Type Iii ◽

Barr Virus ◽

Ebv Infection ◽

Latently Infected ◽

Infected Cells ◽

Epstein Barr

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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Heterogeneous Epstein-Barr virus infection patterns in peripheral T- cell lymphoma of angioimmunoblastic lymphadenopathy type

Blood ◽

10.1182/blood.v80.7.1804.1804 ◽

1992 ◽

Vol 80 (7) ◽

pp. 1804-1812 ◽

Cited By ~ 142

Author(s):

I Anagnostopoulos ◽

M Hummel ◽

T Finn ◽

M Tiemann ◽

P Korbjuhn ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cells ◽

Epstein Barr Virus ◽

Cell Lymphoma ◽

Angioimmunoblastic Lymphadenopathy ◽

Barr Virus ◽

Ebv Infection ◽

Infected Cells ◽

Epstein Barr

Abstract In this study, 32 cases of T-cell lymphoma of angioimmunoblastic lymphadenopathy type (AILD-TCL) were investigated for their association with Epstein-Barr virus (EBV). For this purpose, three different approaches were applied: polymerase chain reaction (PCR) for the presence of EBV-DNA, in situ hybridization (ISH) for EBV-encoded small nuclear RNAs (EBER), and immunohistology for EBV-encoded latent membrane protein (LMP). PCR and EBER-ISH produced almost identical results, showing that all but one case of AILD-TCL contained EBV genomes. Three distinctive patterns of EBV infection were observed after immunophenotypical characterization of EBER-positive cells: (1) in 26% of the cases, B and T cells were infected, the majority of which were B cells of immunoblastic morphology located in the remnants of lymphoid follicles; (2) in 42% of the cases, the vast majority of infected cells were neoplastic T cells diffusely distributed in the lymph nodes, but infected B cells were also present; and (3) in 32% of the cases, there were only a few infected small lymphoid cells. Detectable LMP was frequent in cases exhibiting patterns 1 and 2. These findings suggest that in AILD-TCL patients, B cells and especially T cells are highly susceptible to a persistent EBV infection, which often leads to a growth advantage of the infected cells. Thus EBV, in conjunction with genetic abnormalities and selective defects of the immune system, might be involved in the pathogenesis of AILD-TCL.

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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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Frequent expression of interleukin-10 by Epstein-Barr virus-harboring tumor cells of Hodgkin's disease

Blood ◽

10.1182/blood.v87.7.2918.bloodjournal8772918 ◽

1996 ◽

Vol 87 (7) ◽

pp. 2918-2929 ◽

Cited By ~ 142

Author(s):

H Herbst ◽

HD Foss ◽

J Samol ◽

I Araujo ◽

H Klotzbach ◽

...

Keyword(s):

Tumor Cells ◽

Hodgkin's Disease ◽

Epstein Barr Virus ◽

Hodgkin’S Disease ◽

Interleukin 10 ◽

Cell Mediated Immunity ◽

Barr Virus ◽

Ebv Infection ◽

Infected Cells ◽

Epstein Barr

Tumor cells of Epstein-Barr virus (EBV)-associated Hodgkin's disease (HD) express the viral protein, latent infection membrane protein-1 (LMP1), but evade cytotoxic responses normally directed at this antigen. We tested whether local production of the immunoregulatory interleukins (IL)-4 and -10 may have a role in this process. IL-4 RNA was not detectable in any of the HD cases. By contrast, isotopic in situ hybridization and correlation with the presence of EBV gene products showed significantly higher proportions of cases with IL-10 expressing tumor cells in LMP1-positive (17 of 26, 66%) as compared with LMP1-negative HD cases (six of 37, 16%). Absence of EBV BCRF1 RNA indicated that the transcripts originated from the cellular IL-10 gene. Similarly, an association between IL-10 expression and EBV-infection of tumor cells was found in AIDS-related malignant non-Hodgkin lymphomas (ARL). Very small proportions of EBV-infected cells, mainly blasts, expressed IL-10 in infectious mononucleosis tonsils. Thus, although not entirely exclusive to EBV-positive cases, IL-10 expression is frequently associated with EBV-infection in HD and ARL and appears to be upregulated by EBV, most likely through LMP1. In view of the established inhibitory effects of IL-10 on cell mediated immunity, it is suggested that IL-10 expression may contribute to evasion of LMP1- positive cells from cytotoxicity directed at viral antigens.

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Epstein-Barr Virus Induces Expression of the LPAM-1 Integrin in B CellsIn VitroandIn Vivo

Journal of Virology ◽

10.1128/jvi.01618-18 ◽

2018 ◽

Vol 93 (5) ◽

Cited By ~ 1

Author(s):

Susanne Delecluse ◽

Ming-Han Tsai ◽

Anatoliy Shumilov ◽

Maja Bencun ◽

Sebastian Arrow ◽

...

Keyword(s):

B Cells ◽

Lymph Nodes ◽

B Cell ◽

Epstein Barr Virus ◽

Digestive System ◽

The Body ◽

Barr Virus ◽

Ebv Infection ◽

Infected Cells ◽

Epstein Barr

ABSTRACTEpstein-Barr virus (EBV) infects the oropharynx but, surprisingly, frequently induces B cell proliferation in the gut of immunosuppressed individuals. We found that EBV infectionin vitroinduces the expression of the LPAM-1 integrin on tonsillar B cells and increases it on peripheral blood cells. Similarly, LPAM-1 was induced in the tonsils of patients undergoing primary infectious mononucleosis. EBV-induced LPAM-1 bound to the MAdCAM-1 addressin, which allows B cell homing to the gastrointestinal mucosa-associated lymphoid tissue (GALT). Thus, we hypothesized that EBV-induced LPAM-1 could induce relocation of infected B cells from the tonsil to the GALT.In situhybridization with an EBER-specific probe revealed the frequent presence of EBV-infected cells in the pericolic lymph nodes of healthy individuals. Relocation of infected B cells into the GALT would expand the EBV reservoir, possibly protecting it from T cells primed in the oropharynx, and explain why EBV induces lymphoid tumors in the gut.IMPORTANCEEBV causes tumors in multiple organs, particularly in the oro- and nasopharyngeal area but also in the digestive system. This virus enters the body in the oropharynx and establishes a chronic infection in this area. The observation that the virus causes tumors in the digestive system implies that the infected cells can move to this organ. We found that EBV infection induces the expression of integrin beta 7 (ITGB7), an integrin that associates with integrin alpha 4 to form the LPAM-1 dimer. LPAM-1 is key for homing of B cells to the gastrointestinal tract, suggesting that induction of this molecule is the mechanism through which EBV-infected cells enter this organ. In favor of this hypothesis, we could also detect EBV-infected cells in the lymph nodes adjacent to the colon and in the appendix.

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Epstein-Barr Virus BZLF1 Gene, a Switch from Latency to Lytic Infection, Is Expressed as an Immediate-Early Gene after Primary Infection of B Lymphocytes

Journal of Virology ◽

10.1128/jvi.01416-06 ◽

2006 ◽

Vol 81 (2) ◽

pp. 1037-1042 ◽

Cited By ~ 73

Author(s):

Wangrong Wen ◽

Dai Iwakiri ◽

Koji Yamamoto ◽

Seiji Maruo ◽

Teru Kanda ◽

...

Keyword(s):

Protein Synthesis ◽

B Lymphocytes ◽

Epstein Barr Virus ◽

Lytic Infection ◽

Early Gene ◽

Immediate Early ◽

Barr Virus ◽

Ebv Infection ◽

Bzlf1 Gene ◽

Epstein Barr

ABSTRACT We demonstrate here that the Epstein-Barr virus (EBV) BZLF1 gene, a switch from latent infection to lytic infection, is expressed as early as 1.5 h after EBV infection in Burkitt's lymphoma-derived, EBV-negative Akata and Daudi cells and primary B lymphocytes. Since BZLF1 mRNA is expressed even when the cells are infected with EBV in the presence of anisomycin, an inhibitor of protein synthesis, its expression does not require prerequisite protein synthesis, indicating that BZLF1 is expressed as an immediate-early gene following primary EBV infection of B lymphocytes.

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Epstein-Barr Virus-Specific CD8 T Cells Selectively Infiltrate the Brain in Multiple Sclerosis and Interact Locally with Virus-Infected Cells: Clue for a Virus-Driven Immunopathological Mechanism

Journal of Virology ◽

10.1128/jvi.00980-19 ◽

2019 ◽

Vol 93 (24) ◽

Cited By ~ 8

Author(s):

Barbara Serafini ◽

Barbara Rosicarelli ◽

Caterina Veroni ◽

Gina Adriana Mazzola ◽

Francesca Aloisi

Keyword(s):

T Cells ◽

Cd8 T Cells ◽

Epstein Barr Virus ◽

Cd8 T Cell ◽

Cns Injury ◽

Barr Virus ◽

Ebv Infection ◽

Infected Cells ◽

Epstein Barr ◽

The Brain

ABSTRACT Epstein-Barr virus (EBV) is a ubiquitous herpesvirus strongly associated with multiple sclerosis (MS), a chronic inflammatory disease of the central nervous system (CNS). However, the mechanisms linking EBV infection to MS pathology are uncertain. Neuropathological and immunological studies suggest that a persistent EBV infection in the CNS can stimulate a CD8 T-cell response aimed at clearing the virus but inadvertently causing CNS injury. Inasmuch as in situ demonstration of EBV-specific CD8 T cells and their effector function is missing, we searched for EBV-specific CD8 T cells in MS brain tissue using the pentamer technique. Postmortem brain samples from 12 donors with progressive MS and known HLA class I genotype were analyzed. Brain sections were stained with HLA-matched pentamers coupled with immunogenic peptides from EBV-encoded proteins, control virus (cytomegalovirus and influenza A virus) proteins, and myelin basic protein. CD8 T cells recognizing proteins expressed in the latent and lytic phases of the EBV life cycle were visualized in white matter lesions and/or meninges of 11/12 MS donors. The fraction (median value) of CD8 T cells recognizing individual EBV epitopes ranged from 0.5 to 2.5% of CNS-infiltrating CD8 T cells. Cytomegalovirus-specific CD8 T cells were detected at a lower frequency (≤0.3%) in brain sections from 4/12 MS donors. CNS-infiltrating EBV-specific CD8 T cells were CD107a positive, suggesting a cytotoxic phenotype, and stuck to EBV-infected cells. Together with local EBV dysregulation, selective enrichment of EBV-specific CD8 T cells in the MS brain supports the notion that skewed immune responses toward EBV contribute to inflammation causing CNS injury. IMPORTANCE EBV establishes a lifelong and asymptomatic infection in most individuals and more rarely causes infectious mononucleosis and malignancies, like lymphomas. The virus is also strongly associated with MS, a chronic neuroinflammatory disease with unknown etiology. Infectious mononucleosis increases the risk of developing MS, and immune reactivity toward EBV is higher in persons with MS, indicating inadequate control of the virus. Previous studies have suggested that persistent EBV infection in the CNS stimulates an immunopathological response, causing bystander neural cell damage. To verify this, we need to identify the immune culprits responsible for the detrimental antiviral response in the CNS. In this study, we analyzed postmortem brains donated by persons with MS and show that CD8 cytotoxic T cells recognizing EBV enter the brain and interact locally with the virus-infected cells. This antiviral CD8 T cell-mediated immune response likely contributes to MS pathology.

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