Induction of the Lytic Cycle Sensitizes Epstein-Barr Virus-Infected B Cells to NK Cell Killing That Is Counteracted by Virus-Mediated NK Cell Evasion Mechanisms in the Late Lytic Cycle

ABSTRACTEpstein-Barr Virus (EBV) persists for the lifetime of the infected host despite eliciting strong immune responses. This persistence requires a fine balance between the host immune system and EBV immune evasion. Accumulating evidence suggests an important role for natural killer (NK) cells in this balance. NK cells can kill EBV-infected cells undergoing lytic replicationin vitro, and studies in both humans and mice with reconstituted human immune systems have shown that NK cells can limit EBV replication and prevent infectious mononucleosis. We now show that NK cells, via NKG2D and DNAM-1 interactions, recognize and kill EBV-infected cells undergoing lytic replication and that expression of a single EBV lytic gene, BZLF1, is sufficient to trigger sensitization to NK cell killing. We also present evidence suggesting the possibility of the existence of an as-yet-unidentified DNAM-1 ligand which may be particularly important for killing lytically infected normal B cells. Furthermore, while cells entering the lytic cycle become sensitized to NK cell killing, we observed that cells in the late lytic cycle are highly resistant. We identified expression of the vBcl-2 protein, BHRF1, as one effective mechanism by which EBV mediates this protection. Thus, contrary to the view expressed in some reports, EBV has evolved the ability to evade NK cell responses.IMPORTANCEThis report extends our understanding of the interaction between EBV and host innate responses. It provides the first evidence that the susceptibility to NK cell lysis of EBV-infected B cells undergoing lytic replication is dependent upon the phase of the lytic cycle. Induction of the lytic cycle is associated with acquired sensitization to NK cell killing, while progress through the late lytic cycle is associated with acquired resistance to killing. We provide mechanistic explanations for this novel observation, indicating important roles for the BZLF1 immediate early transactivator, the BHRF1 vBcl-2 homologue, and a novel ligand for the DNAM-1 NK cell receptor.

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NK cells eliminate Epstein-Barr virus bound to B cells through a specific antibody-mediated uptake

PLoS Pathogens ◽

10.1371/journal.ppat.1009868 ◽

2021 ◽

Vol 17 (8) ◽

pp. e1009868

Author(s):

Elisenda Alari-Pahissa ◽

Michelle Ataya ◽

Ilias Moraitis ◽

Miriam Campos-Ruiz ◽

Mireia Altadill ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Nk Cells ◽

Epstein Barr Virus ◽

Actin Polymerization ◽

Nk Cell ◽

Lytic Cycle ◽

Barr Virus ◽

Early Endosomes ◽

Epstein Barr

Epstein Barr virus (EBV) causes a highly prevalent and lifelong infection contributing to the development of some malignancies. In addition to the key role played by T cells in controlling this pathogen, NK cells mediate cytotoxicity and IFNγ production in response to EBV-infected B cells in lytic cycle, both directly and through antibody (Ab)-dependent activation. We recently described that EBV-specific Ab-dependent NK cell interaction with viral particles (VP) bound to B cells triggered degranulation and TNFα secretion but not B cell lysis nor IFNγ production. In this report we show that NK cell activation under these conditions reduced B cell transformation by EBV. NK cells eliminated VP from the surface of B cells through a specific and active process which required tyrosine kinase activation, actin polymerization and Ca2+, being independent of proteolysis and perforin. VP were displayed at the NK cell surface before being internalized and partially shuttled to early endosomes and lysosomes. VP transfer was encompassed by a trogocytosis process including the EBV receptor CD21, together with CD19 and CD20. Our study reveals a novel facet of the antibody-dependent NK cell mediated response to this viral infection.

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CD8+ immunodominance among Epstein-Barr virus lytic cycle antigens directly reflects the efficiency of antigen presentation in lytically infected cells

Journal of Experimental Medicine ◽

10.1084/jem.20041542 ◽

2005 ◽

Vol 201 (3) ◽

pp. 349-360 ◽

Cited By ~ 90

Author(s):

Victoria A. Pudney ◽

Alison M. Leese ◽

Alan B. Rickinson ◽

Andrew D. Hislop

Keyword(s):

T Cell ◽

Epstein Barr Virus ◽

Cd8 T Cell ◽

Lytic Replication ◽

Lytic Cycle ◽

E Proteins ◽

Barr Virus ◽

Infected Cells ◽

Epstein Barr ◽

L Proteins

Antigen immunodominance is an unexplained feature of CD8+ T cell responses to herpesviruses, which are agents whose lytic replication involves the sequential expression of immediate early (IE), early (E), and late (L) proteins. Here, we analyze the primary CD8 response to Epstein-Barr virus (EBV) infection for reactivity to 2 IE proteins, 11 representative E proteins, and 10 representative L proteins, across a range of HLA backgrounds. Responses were consistently skewed toward epitopes in IE and a subset of E proteins, with only occasional responses to novel epitopes in L proteins. CD8+ T cell clones to representative IE, E, and L epitopes were assayed against EBV-transformed lymphoblastoid cell lines (LCLs) containing lytically infected cells. This showed direct recognition of lytically infected cells by all three sets of effectors but at markedly different levels, in the order IE > E ≫ L, indicating that the efficiency of epitope presentation falls dramatically with progress of the lytic cycle. Thus, EBV lytic cycle antigens display a hierarchy of immunodominance that directly reflects the efficiency of their presentation in lytically infected cells; the CD8+ T cell response thereby focuses on targets whose recognition leads to maximal biologic effect.

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B Cell Receptor-Responsive miR-141 Enhances Epstein-Barr Virus Lytic Cycle via FOXO3 Inhibition

mSphere ◽

10.1128/msphere.00093-21 ◽

2021 ◽

Vol 6 (2) ◽

Author(s):

Yan Chen ◽

Devin N. Fachko ◽

Nikita S. Ivanov ◽

Rebecca L. Skalsky

Keyword(s):

B Cells ◽

B Cell ◽

Epstein Barr Virus ◽

Cell Receptor ◽

Lytic Replication ◽

B Cell Receptor ◽

Lytic Cycle ◽

Cross Linking ◽

Barr Virus ◽

Epstein Barr

ABSTRACT Antigen recognition by the B cell receptor (BCR) is a physiological trigger for reactivation of Epstein-Barr virus (EBV) and can be recapitulated in vitro by cross-linking of surface immunoglobulins. Previously, we identified a subset of EBV microRNAs (miRNAs) that attenuate BCR signal transduction and subsequently dampen lytic reactivation in B cells. The roles of host miRNAs in the EBV lytic cycle are not completely understood. Here, we profiled the small RNAs in reactivated Burkitt lymphoma cells and identified several miRNAs, such as miR-141, that are induced upon BCR cross-linking. Notably, EBV encodes a viral miRNA, miR-BART9, with sequence homology to miR-141. To better understand the functions of these two miRNAs, we examined their molecular targets and experimentally validated multiple candidates commonly regulated by both miRNAs. Targets included B cell transcription factors and known regulators of EBV immediate-early genes, leading us to hypothesize that these miRNAs modulate kinetics of the lytic cascade in B cells. Through functional assays, we identified roles for miR-141 and EBV miR-BART9 and one specific target, FOXO3, in progression of the lytic cycle. Our data support a model whereby EBV exploits BCR-responsive miR-141 and further mimics activity of this miRNA family via a viral miRNA to promote productive lytic replication. IMPORTANCE EBV is a human pathogen associated with several malignancies. A key aspect of lifelong virus persistence is the ability to switch between latent and lytic replication modes. The mechanisms governing latency, reactivation, and progression of the lytic cycle are only partly understood. This study reveals that specific miRNAs can act to support the EBV lytic phase following BCR-mediated reactivation triggers. Furthermore, this study identifies a role for FOXO3, commonly suppressed by both host and viral miRNAs, in modulating progression of the EBV lytic cycle.

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Copies of Donor Killer Immunoglobulin-like Receptor Genes and Motifs Titrate Natural Killer (NK) Cells' Functional Response to Epstein - Barr Virus Infections and Influence the Risk of Developing Post-Transplant Lymphoproliferative Disease (PTLD) after Allogeneic Hematopoietic Cell Transplantation

Blood ◽

10.1182/blood.v126.23.741.741 ◽

2015 ◽

Vol 126 (23) ◽

pp. 741-741

Author(s):

Rehan Mujeeb Faridi ◽

Taylor J Kemp ◽

Poonam Dharmani ◽

Victor A. Lewis ◽

Noureddine Berka ◽

...

Keyword(s):

Nk Cells ◽

Functional Response ◽

Epstein Barr Virus ◽

Viral Infections ◽

Nk Cell ◽

Barr Virus ◽

Infected Cells ◽

Ifn Γ ◽

Epstein Barr ◽

Kir Gene

Abstract BACKGROUND: Recipientsof allogeneic HCT remain vulnerable to a heightened risk of reactivation of otherwise latent viral infections owing to a compromised immune system early after transplantation. Uncontrolled reactivation of Epstein-Barr virus (EBV) leading to post-transplant lymphoproliferative disorder (PTLD) is one of such major complications after T-cell depleted HCT. Recovering within weeks after transplantation and being first in line of defense against viral infections, natural killer (NK) cells are deemed important in the immunopathogenesis of EBV complications. Their role however remains elusive. NK cell responses are regulated by a series of activating and inhibitory cell surface receptors, central to which are the Killer Immunoglobulin-like Receptors (KIR). Through these receptors NK cells discriminate healthy cells from 'altered' self-cells by scaling the perturbations in HLA expression after viral transformation of the target cell. Here, we set out to determine whether and how KIR gene and motifs' content of HCT donors and/or recipients influences the development of PTLD after allo-HCT. STUDY DESIGN: Hypothesizing that diverse NK cell receptor repertoires can titrate NK cell functional responses to EBV infections/reactivation and can potentially modify the risk of developing PTLD, we determined the KIR gene repertoires of 356 HLA-matched donor-recipient pairs of first allo-HCT and 50 healthy donors through Next Generation Sequencing of the KIR locus on the Illumina MiSeq platform. Based on the presence/absence and number of copies of individual genes, the KIR genotypes were determined and classified into four common centromeric (cA01, cB01, cB02 and cB03) and two telomeric (tA01 and tB01) motifs along with their variants. PBMNCs from KIR typed healthy volunteers were stimulated with EBV-transformed target cells to enumerate NK cell response to EBV (degranulation and/or IFNγ production) as a function of KIR gene content and motifs' distribution using a multicolor flow cytometry-based assay. Effect of KIR gene profile on development of PTLD was analyzed using binomial competing risks regression statistics. Distribution of NK cell functional response across various KIR characterized groups was analyzed using Mann-Whitney U statistics. RESULTS: Donor telomeric A motifs (tA01, KIR3DL1+ve KIR2DS4+ve; KIR3DS1/2DS1+/-ve), strongly protected against PTLD (p=0.0001, SHR=0.17; Figure 1). An increased protection against PTLD with increasing number of tA01 was noted with at least one copy required for a significant protective effect (Figure 1B). Copy number analysis of tA01 gene contents yielded similar associations. Further, the number of EBV induced functional NK cell subsets were significantly higher in individuals with than without KIR genotypes containing tA01 motifs (Figure 2 A-C) and was found to be increasing with an increasing number of tA01 copies (Figure 2 A'-C'). There was no influence of recipients' KIR repertoire on the risk of developing PTLD CONCLUSIONS: NK cell responsiveness, a function of KIR gene repertoire has a profound effect on the development of PTLD. Appropriately characterized KIR gene profile based identification of HCT recipients at high risk of developing PTLD will enable closer monitoring of EBV DNAemia and facilitate prompt therapy. Figure 1. Donor KIR telomeric A motif (tA01) protects against the risk of developing PTLD (A). Presence of at least one copy of donor KIR tA01 motif confers significant protection from PTLD (B) Figure 1. Donor KIR telomeric A motif (tA01) protects against the risk of developing PTLD (A). Presence of at least one copy of donor KIR tA01 motif confers significant protection from PTLD (B) Figure 2. KIR telomeric A motifs (tA01) titrate NK cells' functional response to Epstein-Barr virus infected cells (A-C), with and increasing %functional NK cells and subsets (measures as expressing CD107a, IFN-γ, or both) are observed with increasing tA01 motifs' copies (A'-C') Figure 2. KIR telomeric A motifs (tA01) titrate NK cells' functional response to Epstein-Barr virus infected cells (A-C), with and increasing %functional NK cells and subsets (measures as expressing CD107a, IFN-γ, or both) are observed with increasing tA01 motifs' copies (A'-C') Disclosures No relevant conflicts of interest to declare.

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Negative Autoregulation of Epstein-Barr Virus (EBV) Replicative Gene Expression by EBV SM Protein

Journal of Virology ◽

10.1128/jvi.00382-09 ◽

2009 ◽

Vol 83 (16) ◽

pp. 8041-8050 ◽

Cited By ~ 11

Author(s):

Dinesh Verma ◽

Chen Ling ◽

Eric Johannsen ◽

Tirumuru Nagaraja ◽

Sankar Swaminathan

Keyword(s):

Epstein Barr Virus ◽

Feedback Regulation ◽

Lytic Replication ◽

Lytic Cycle ◽

Immediate Early ◽

Mrna Levels ◽

Barr Virus ◽

Infected Cells ◽

Epstein Barr ◽

Sm Protein

ABSTRACT The Epstein-Barr virus (EBV) SM protein is essential for lytic EBV DNA replication and virion production. When EBV replication is induced in cells infected with an SM-deleted recombinant EBV, approximately 50% of EBV genes are expressed inefficiently. When EBV replication is rescued by transfection of SM, SM enhances expression of these genes by direct and indirect mechanisms. While expression of most EBV genes is either unaffected or enhanced by SM, expression of several genes is decreased in the presence of SM. Expression of BHRF1, a homolog of cellular bcl-2, is particularly decreased in the presence of SM. Investigation of the mechanism of BHRF1 downregulation revealed that SM downregulates expression of the immediate-early EBV transactivator R. In EBV-infected cells, R-responsive promoters, including the BHRF1 and SM promoters, were less active in the presence of SM, consistent with SM inhibition of R expression. SM decreased spliced R mRNA levels, supporting a posttranscriptional mechanism of R inhibition. R and BHRF1 expression were also found to decrease during later stages of EBV lytic replication in EBV-infected lymphoma cells. These data indicate that feedback regulation of immediate-early and early genes occurs during the lytic cycle of EBV regulation.

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The Switch from Latent to Productive Infection in Epstein-Barr Virus-Infected B Cells Is Associated with Sensitization to NK Cell Killing

Journal of Virology ◽

10.1128/jvi.01777-06 ◽

2006 ◽

Vol 81 (2) ◽

pp. 474-482 ◽

Cited By ~ 93

Author(s):

Isabel Y. Pappworth ◽

Eddie C. Wang ◽

Martin Rowe

Keyword(s):

B Cells ◽

Nk Cells ◽

Mhc Class I ◽

Epstein Barr Virus ◽

Nk Cell ◽

Class I ◽

Inhibitory Receptors ◽

Barr Virus ◽

Epstein Barr ◽

Mhc Class I Molecules

ABSTRACT Following activation of Epstein-Barr virus (EBV)-infected B cells from latent to productive (lytic) infection, there is a concomitant reduction in the level of cell surface major histocompatibility complex (MHC) class I molecules and an impaired antigen-presenting function that may facilitate evasion from EBV-specific CD8+ cytotoxic T cells. In some other herpesviruses studied, most notably human cytomegalovirus (HCMV), evasion of virus-specific CD8+ effector responses via downregulation of surface MHC class I molecules is supplemented with specific mechanisms for evading NK cells. We now report that EBV differs from HCMV in this respect. While latently infected EBV-positive B cells were resistant to lysis by two NK lines and by primary polyclonal NK cells from peripheral blood, these effectors efficiently killed cells activated into the lytic cycle. Susceptibility to NK lysis coincided not only with downregulation of HLA-A, -B, and -C molecules that bind to the KIR family of inhibitory receptors on NK cells but also with downregulation of HLA-E molecules binding the CD94/NKG2A inhibitory receptors. Conversely, ULBP-1 and CD112, ligands for the NK cell-activating receptors NKG2D and DNAM-1, respectively, were elevated. Susceptibility of the virus-producing target cells to NK cell lysis was partially reversed by blocking ULBP-1 or CD112 with specific antibodies. These results highlight a fundamental difference between EBV and HCMV with regards to evasion of innate immunity.

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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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Rapid identification and characterization of infected cells in blood during chronic active Epstein-Barr virus infection

Journal of Experimental Medicine ◽

10.1084/jem.20192262 ◽

2020 ◽

Vol 217 (11) ◽

Author(s):

Benjamin Fournier ◽

David Boutboul ◽

Julie Bruneau ◽

Charline Miot ◽

Cécile Boulanger ◽

...

Keyword(s):

Native American ◽

Nk Cells ◽

Epstein Barr Virus ◽

Nk Cell ◽

Rapid Identification ◽

Effector Memory ◽

Epstein Barr Virus Infection ◽

Barr Virus ◽

Infected Cells ◽

Epstein Barr

Epstein-Barr virus (EBV) preferentially infects epithelial cells and B lymphocytes and sometimes T and NK lymphocytes. Persistence of EBV-infected cells results in severe lymphoproliferative disorders (LPDs). Diagnosis of EBV-driven T or NK cell LPD and chronic active EBV diseases (CAEBV) is difficult, often requiring biopsies. Herein, we report a flow-FISH cytometry assay that detects cells expressing EBV-encoded small RNAs (EBERs), allowing rapid identification of EBV-infected cells among PBMCs. EBV-infected B, T, and/or NK cells were detectable in various LPD conditions. Diagnosis of CAEBV in 22 patients of Caucasian and African origins was established. All exhibited circulating EBV-infected T and/or NK cells, highlighting that CAEBV is not restricted to native American and Asian populations. Proportions of EBV-infected cells correlated with blood EBV loads. We showed that EBV-infected T cells had an effector memory activated phenotype, whereas EBV-infected B cells expressed plasma cell differentiation markers. Thus, this method achieves accurate and unambiguous diagnoses of different forms of EBV-driven LPD and represents a powerful tool to study their pathophysiological mechanisms.

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Identification of Epstein–Barr virus-infected CD27+ memory B-cells in liver or stem cell transplant patients

Journal of General Virology ◽

10.1099/vir.0.033712-0 ◽

2011 ◽

Vol 92 (11) ◽

pp. 2590-2595 ◽

Cited By ~ 3

Author(s):

Yoshinori Ito ◽

Shinji Kawabe ◽

Seiji Kojima ◽

Fumihiko Nakamura ◽

Yukihiro Nishiyama ◽

...

Keyword(s):

Liver Transplantation ◽

Stem Cell ◽

B Cells ◽

Epstein Barr Virus ◽

Peripheral Blood Lymphocytes ◽

Haematopoietic Stem Cell ◽

Cell Transplant ◽

Barr Virus ◽

Infected Cells ◽

Epstein Barr

To analyse the phenotype of Epstein–Barr virus (EBV)-infected lymphocytes in EBV-associated infections, cells from eight haematopoietic stem cell/liver transplantation recipients with elevated EBV viral loads were examined by a novel quantitative assay designed to identify EBV-infected cells by using a flow cytometric detection of fluorescent in situ hybridization (FISH) assay. By this assay, 0.05–0.78 % of peripheral blood lymphocytes tested positive for EBV, and the EBV-infected cells were CD20+ B-cells in all eight patients. Of the CD20+ EBV-infected lymphocytes, 48–83 % of cells tested IgD positive and 49–100 % of cells tested CD27 positive. Additionally, the number of EBV-infected cells assayed by using FISH was significantly correlated with the EBV-DNA load, as determined by real-time PCR (r 2 = 0.88, P<0.0001). The FISH assay enabled us to characterize EBV-infected cells and perform a quantitative analysis in patients with EBV infection after stem cell/liver transplantation.

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