scholarly journals Rewiring of B cell receptor signaling by Epstein–Barr virus LMP2A

2020 ◽  
Vol 117 (42) ◽  
pp. 26318-26327
Author(s):  
Kamonwan Fish ◽  
Federico Comoglio ◽  
Arthur L. Shaffer ◽  
Yanlong Ji ◽  
Kuan-Ting Pan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Barr Virus ◽  
Human B Cells ◽  
Bcr Signaling ◽  
Epstein Barr

Epstein–Barr virus (EBV) infects human B cells and reprograms them to allow virus replication and persistence. One key viral factor in this process is latent membrane protein 2A (LMP2A), which has been described as a B cell receptor (BCR) mimic promoting malignant transformation. However, how LMP2A signaling contributes to tumorigenesis remains elusive. By comparing LMP2A and BCR signaling in primary human B cells using phosphoproteomics and transcriptome profiling, we identified molecular mechanisms through which LMP2A affects B cell biology. Consistent with the literature, we found that LMP2A mimics a subset of BCR signaling events, including tyrosine phosphorylation of the kinase SYK, the calcium initiation complex consisting of BLNK, BTK, and PLCγ2, and its downstream transcription factor NFAT. However, the majority of LMP2A-induced signaling events markedly differed from those induced by BCR stimulation. These included differential phosphorylation of kinases, phosphatases, adaptor proteins, transcription factors such as nuclear factor κB (NF-κB) and TCF3, as well as widespread changes in the transcriptional output of LMP2A-expressing B cells. LMP2A affected apoptosis and cell-cycle checkpoints by dysregulating the expression of apoptosis regulators such as BCl-xL and the tumor suppressor retinoblastoma-associated protein 1 (RB1). LMP2A cooperated with MYC and mutant cyclin D3, two oncogenic drivers of Burkitt lymphoma, to promote proliferation and survival of primary human B cells by counteracting MYC-induced apoptosis and by inhibiting RB1 function, thereby promoting cell-cycle progression. Our results indicate that LMP2A is not a pure BCR mimic but rather rewires intracellular signaling in EBV-infected B cells that optimizes cell survival and proliferation, setting the stage for oncogenic transformation.

scholarly journals Drug Modulators of B Cell Signaling Pathways and Epstein-Barr Virus Lytic Activation

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
John G. Kosowicz ◽  
Jaeyeun Lee ◽  
Brandon Peiffer ◽  
Zufeng Guo ◽  
Jianmeng Chen ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Barr Virus ◽  
Viral Expression ◽  
Bcr Signaling ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) is a ubiquitous human gammaherpesvirus that establishes a latency reservoir in B cells. In this work, we show that ibrutinib, idelalisib, and dasatinib, drugs that block B cell receptor (BCR) signaling and are used in the treatment of hematologic malignancies, block BCR-mediated lytic induction at clinically relevant doses. We confirm that the immunosuppressive drugs cyclosporine and tacrolimus also inhibit BCR-mediated lytic induction but find that rapamycin does not inhibit BCR-mediated lytic induction. Further investigation shows that mammalian target of rapamycin complex 2 (mTORC2) contributes to BCR-mediated lytic induction and that FK506-binding protein 12 (FKBP12) binding alone is not adequate to block activation. Finally, we show that BCR signaling can activate EBV lytic induction in freshly isolated B cells from peripheral blood mononuclear cells (PBMCs) and that activation can be inhibited by ibrutinib or idelalisib. IMPORTANCE EBV establishes viral latency in B cells. Activation of the B cell receptor pathway activates lytic viral expression in cell lines. Here we show that drugs that inhibit important kinases in the BCR signaling pathway inhibit activation of lytic viral expression but do not inhibit several other lytic activation pathways. Immunosuppressant drugs such as cyclosporine and tacrolimus but not rapamycin also inhibit BCR-mediated EBV activation. Finally, we show that BCR activation of lytic infection occurs not only in tumor cell lines but also in freshly isolated B cells from patients and that this activation can be blocked by BCR inhibitors.

scholarly journals Highly efficient CRISPR-Cas9-mediated gene knockout in primary human B cells for functional genetic studies of Epstein-Barr virus infection

2021 ◽  
Vol 17 (4) ◽  
pp. e1009117
Author(s):  
Ezgi Akidil ◽  
Manuel Albanese ◽  
Alexander Buschle ◽  
Adrian Ruhle ◽  
Dagmar Pich ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Gene Editing ◽  
Barr Virus ◽  
Ebv Infection ◽  
Highly Efficient ◽  
Human B Cells ◽  
Epstein Barr

Gene editing is now routine in all prokaryotic and metazoan cells but has not received much attention in immune cells when the CRISPR-Cas9 technology was introduced in the field of mammalian cell biology less than ten years ago. This versatile technology has been successfully adapted for gene modifications in human myeloid cells and T cells, among others, but applications to human primary B cells have been scarce and limited to activated B cells. This limitation has precluded conclusive studies into cell activation, differentiation or cell cycle control in this cell type. We report on highly efficient, simple and rapid genome engineering in primary resting human B cells using nucleofection of Cas9 ribonucleoprotein complexes, followed by EBV infection or culture on CD40 ligand feeder cells to drive in vitro B cell survival. We provide proof-of-principle of gene editing in quiescent human B cells using two model genes: CD46 and CDKN2A. The latter encodes the cell cycle regulator p16INK4a which is an important target of Epstein-Barr virus (EBV). Infection of B cells carrying a knockout of CDKN2A with wildtype and EBNA3 oncoprotein mutant strains of EBV allowed us to conclude that EBNA3C controls CDKN2A, the only barrier to B cell proliferation in EBV infected cells. Together, this approach enables efficient targeting of specific gene loci in quiescent human B cells supporting basic research as well as immunotherapeutic strategies.

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

mSphere ◽  
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.

scholarly journals Epstein-Barr virus latent membrane protein 2A is a B-cell receptor mimic and essential for B-cell survival

Blood ◽  
2007 ◽  
Vol 110 (10) ◽  
pp. 3715-3721 ◽  
Author(s):  
Christoph Mancao ◽  
Wolfgang Hammerschmidt
Keyword(s):  
B Cells ◽  
Membrane Protein ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Cell Receptor ◽  
Latent Membrane Protein ◽  
B Cell Receptor ◽  
Latent Membrane Protein 2A ◽  
Barr Virus ◽  
Epstein Barr

AbstractMany cells latently infected with Epstein-Barr virus (EBV), including certain virus-associated tumors, express latent membrane protein 2A (LMP2A), suggesting an important role for this protein in viral latency and oncogenesis. LMP2A mimics B-cell receptor signaling but can also act as a decoy receptor blocking B-cell receptor (BCR) activation. Studies of peripheral B cells have not resolved this apparent contradiction because LMP2A seems to be dispensable for EBV-induced transformation of these B cells in vitro. We show here that LMP2A is essential for growth transformation of germinal center B cells, which do not express the genuine BCR because of deleterious somatic hypermutations in their immunoglobulin genes. BCR-positive (BCR+) and BCR-negative (BCR−) B cells are readily transformed with a recombinant EBV encoding a conditional, floxed LMP2A allele, but the survival and continued proliferation of both BCR+ and BCR− B cells is strictly dependent on LMP2A. These findings indicate that LMP2A has potent, distinct antiapoptotic and/or transforming characteristics and point to its role as an indispensable BCR mimic in certain B cells from which human B-cell tumors such as Hodgkin lymphoma originate.

scholarly journals Signatures of B-cell receptor diversity in B lymphocytes following Epstein-Barr virus transformation

2019 ◽  
Vol 51 (6) ◽  
pp. 197-207
Author(s):  
Meimei Lai ◽  
Qiongdan Wang ◽  
Yutian Lu ◽  
Xi Xu ◽  
Ying Xia ◽  
...  
Keyword(s):  
B Cell ◽  
Cell Line ◽  
Epstein Barr Virus ◽  
High Throughput Sequencing ◽  
Cell Receptor ◽  
B Cell Receptor ◽  
Human Virus ◽  
Barr Virus ◽  
Ebv Infection ◽  
Epstein Barr

Epstein-Barr virus (EBV) is a widespread human virus that establishes latent infection, potentially leading to tumors, hematological disorders, and other severe diseases. EBV infections are associated with diverse symptoms and affect various organs; therefore, early diagnosis and treatment are crucial. B cell receptor (BCR) repertoires of B cell surface immunoglobulins have been widely studied for their association with various infectious diseases. However, the specific genetic changes that modulate the BCR repertoires after an EBV infection are still poorly understood. In this study, we employed high-throughput sequencing (HTS) to investigate the diversity of BCR repertoires in an EBV-transformed lymphoblastic cell line (LCL). Compared with the noninfected control B cell line, the LCL exhibited a decrease in overall BCR diversity but displayed an increase in the expansion of some dominant rearrangements such as IGHV4-31/IGHJ4, IGHV4-59/IGHJ4, IGHV5-51/IGHJ3, and IGHV3-74/IGHJ3. A higher frequency of occurrence of these rearrangement types was confirmed in patients with EBV infection. Interestingly, the IGHV3-74 rearrangement was only detected in EBV-infected children, suggesting that our experimental observations were not coincidental. In addition, we identified a highly dominant consensus motif, CAR(xRx)YGSG(xYx)FD, in complementarity-determining region 3 (CDR3) sequences of the heavy chain in the LCL. Our findings demonstrated the utility of HTS technology for studying the variations in signature motifs of the BCR repertoires after EBV infection. We propose that the analysis of BCR repertoire sequences represents a promising method for diagnosing early EBV infections and developing novel antibody- and vaccine-based therapies against such infections.

scholarly journals Epstein-Barr Virus LMP2A Alters In Vivo and In Vitro Models of B-Cell Anergy, but Not Deletion, in Response to Autoantigen

2005 ◽  
Vol 79 (12) ◽  
pp. 7355-7362 ◽  
Author(s):  
Michelle A. Swanson-Mungerson ◽  
Robert G. Caldwell ◽  
Rebecca Bultema ◽  
Richard Longnecker
Keyword(s):  
B Cells ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Nuclear Translocation ◽  
Cell Receptor ◽  
Barr Virus ◽  
Low Levels ◽  
Epstein Barr

ABSTRACT A significant percentage of the population latently harbors Epstein-Barr virus (EBV) in B cells. One EBV-encoded protein, latent membrane protein 2A (LMP2A), is expressed in tissue culture models of EBV latent infection, in human infections, and in many of the EBV-associated proliferative disorders. LMP2A constitutively activates proteins involved in the B-cell receptor (BCR) signal transduction cascade and inhibits the antigen-induced activation of these proteins. In the present study, we investigated whether LMP2A alters B-cell receptor signaling in primary B cells in vivo and in vitro. LMP2A does not inhibit antigen-induced tolerance in response to strong stimuli in an in vivo tolerance model in which B cells are reactive to self-antigen. In contrast, LMP2A bypasses anergy induction in response to low levels of soluble hen egg lysozyme (HEL) both in vivo and in vitro as determined by the ability of LMP2A-expressing HEL-specific B cells to proliferate and induce NF-κB nuclear translocation after exposure to low levels of antigen. Furthermore, LMP2A induces NF-κB nuclear translocation independent of BCR cross-linking. Since NF-κB is required to bypass tolerance induction, this LMP2A-dependent NF-κB activation may complete the tolerogenic signal induced by low levels of soluble HEL. Overall, the findings suggest that LMP2A may not inhibit BCR-induced signals under all conditions as previously suggested by studies with EBV immortalized B cells.

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

2007 ◽  
Vol 88 (8) ◽  
pp. 2129-2136 ◽  
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.

scholarly journals Calcium-dependent signalling in B-cell lymphomas

Oncogene ◽  
2021 ◽  
Author(s):  
Fedor Berditchevski ◽  
Eanna Fennell ◽  
Paul G. Murray
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Receptor ◽  
Receptor Activation ◽  
B Cell Receptor ◽  
Signalling Pathways ◽  
B Cell Lymphomas ◽  
Calcium Dependent ◽  
Barr Virus ◽  
Epstein Barr

AbstractInduced waves of calcium fluxes initiate multiple signalling pathways that play an important role in the differentiation and maturation of B-cells. Finely tuned transient Ca+2 fluxes from the endoplasmic reticulum in response to B-cell receptor (BCR) or chemokine receptor activation are followed by more sustained calcium influxes from the extracellular environment and contribute to the mechanisms responsible for the proliferation of B-cells, their migration within lymphoid organs and their differentiation. Dysregulation of these well-balanced mechanisms in B-cell lymphomas results in uncontrolled cell proliferation and resistance to apoptosis. Consequently, several cytotoxic drugs (and anti-proliferative compounds) used in standard chemotherapy regimens for the treatment of people with lymphoma target calcium-dependent pathways. Furthermore, ~10% of lymphoma associated mutations are found in genes with functions in calcium-dependent signalling, including those affecting B-cell receptor signalling pathways. In this review, we provide an overview of the Ca2+-dependent signalling network and outline the contribution of its key components to B cell lymphomagenesis. We also consider how the oncogenic Epstein-Barr virus, which is causally linked to the pathogenesis of a number of B-cell lymphomas, can modify Ca2+-dependent signalling.

scholarly journals Frequencies of the separate human B cell subsets activatable to Ig secretion by Epstein-Barr virus and pokeweed mitogen.

1983 ◽  
Vol 157 (6) ◽  
pp. 1808-1814 ◽  
Author(s):  
O Martínez-Maza ◽  
S Britton
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Human Peripheral Blood ◽  
Pokeweed Mitogen ◽  
Barr Virus ◽  
Human B Cells ◽  
Epstein Barr ◽  
B Cell Subsets

We have developed a microculture system suitable for limiting dilution analysis of Epstein-Barr virus (EBV)- and pokeweed mitogen (PWM)-induced activation of immunoglobulin secretion by human B cells. It was found that exogenous filler cells were not required to obtain optimal EBV-induced B cell precursor frequency (PF) estimates, although filler T cells were required for optimal PWM activation. In fact, when autologous T cells were used as filler cells, a marked decrease in the EBV-induced IgM PF was noted. Treatment of the T cells with cyclosporin A partially eliminated, and irradiation of the T cells completely eliminated, this decrease. The calculated PF of B cells activated by EBV was from 1/290 to 1/3,700 for IgM, and from 1/920 to 1/3,250 for IgG secretion. PWM activated from 1/140 to 1/3,200 B cells to IgM secretion. The results of experiments in which EBV and PWM were mixed, indicated that these two polyclonal activators operated on different B cell subpopulations. Therefore, both these agents seem to activate small, discrete subpopulations of human peripheral blood B cells to Ig secretion.

scholarly journals Limiting dilution analysis of Epstein-Barr virus-induced immunoglobulin production by human B cells.

1983 ◽  
Vol 157 (1) ◽  
pp. 1-14 ◽  
Author(s):  
R Yarchoan ◽  
G Tosato ◽  
R M Blaese ◽  
R M Simon ◽  
D L Nelson
Keyword(s):  
B Cells ◽  
B Cell ◽  
Peripheral Blood ◽  
Epstein Barr Virus ◽  
Cell Activation ◽  
B Cell Activation ◽  
Barr Virus ◽  
Human B Cells ◽  
Epstein Barr ◽  
Limiting Dilution

The Epstein-Barr virus (EBV) is a herpes virus that has the capacity to infect human B cells and to induce them to secrete immunoglobulin (Ig). In the current experiments, Poisson analysis of limiting dilution cultures has been used to study the activation of human peripheral B cells by the B95-8 strain of EBV. Under the culture conditions used, 0.2-1% of peripheral blood B cells were activated by EBV to secrete IgM or IgG. In addition, when multiple replicate cultures containing limited numbers of B cells were tested for IgM and for IgG production, the precursors for IgM and IgG segregated independently; thus, individual B cell precursors matured into cells secreting IgM or IgG but not both classes of Ig. Additional experiments using limiting dilutions of EBV were undertaken to study the viral requirements for B cell activation. These studies indicated that B cell activation by EBV to produce Ig was consistent with a "one-hit" model and inconsistent with a "two-hit" model. Taken together, these results indicate that infection by one EBV virion is sufficient to induce a precursor peripheral blood B cell to secrete Ig and that only one isotype of Ig is then secreted.

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