A Gammaherpesvirus MicroRNA Targets EWSR1 (Ewing Sarcoma Breakpoint Region 1) In Vivo To Promote Latent Infection of Germinal Center B Cells

ABSTRACT Gammaherpesviruses, including the human pathogens Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV), directly contribute to the genesis of multiple types of malignancies, including B cell lymphomas. In vivo, these viruses infect B cells and manipulate B cell biology to establish lifelong latent infection. To accomplish this, gammaherpesviruses employ an array of gene products, including microRNAs (miRNAs). Although numerous host mRNA targets of gammaherpesvirus miRNAs have been identified, the in vivo relevance of repression of these targets remains elusive due to species restriction. Murine gammaherpesvirus 68 (MHV68) provides a robust virus-host system to dissect the in vivo function of conserved gammaherpesvirus genetic elements. We identified here MHV68 mghv-miR-M1-7-5p as critical for in vivo infection and then validated host EWSR1 (Ewing sarcoma breakpoint region 1) as the predominant target for this miRNA. Using novel, target-specific shRNA-expressing viruses, we determined that EWSR1 repression in vivo was essential for germinal center B cell infection. These findings provide the first in vivo demonstration of the biological significance of repression of a specific host mRNA by a gammaherpesvirus miRNA. IMPORTANCE Gammaherpesviruses, including the human pathogens Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), directly contribute to the genesis of multiple types of malignancies. In vivo, these viruses infect B cells and manipulate B cell biology to establish lifelong infection. To accomplish this, gammaherpesviruses employ an array of gene products, including miRNAs, short noncoding RNAs that bind to and repress protein synthesis from specific target mRNAs. The in vivo relevance of repression of targets of gammaherpesvirus miRNAs remains highly elusive. Here, we identified a murine gammaherpesvirus miRNA as critical for in vivo infection and validated the host mRNA EWSR1 (Ewing sarcoma breakpoint region 1) as the predominant target for this miRNA. Using a novel technology, we demonstrated that repression of EWSR1 was essential for in vivo infection of the critical B cell reservoir. These findings provide the first in vivo demonstration of the significance of repression of a specific host mRNA by a gammaherpesvirus miRNA.

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

Journal of Virology ◽

10.1128/jvi.79.12.7355-7362.2005 ◽

2005 ◽

Vol 79 (12) ◽

pp. 7355-7362 ◽

Cited By ~ 39

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.

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Mouse model of Epstein–Barr virus LMP1- and LMP2A-driven germinal center B-cell lymphoproliferative disease

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1701836114 ◽

2017 ◽

Vol 114 (18) ◽

pp. 4751-4756 ◽

Cited By ~ 25

Author(s):

Takeharu Minamitani ◽

Yijie Ma ◽

Hufeng Zhou ◽

Hiroshi Kida ◽

Chao-Yuan Tsai ◽

...

Keyword(s):

B Cells ◽

Mouse Model ◽

B Cell ◽

Germinal Center ◽

Epstein Barr Virus ◽

Target Genes ◽

Nk Cell ◽

Barr Virus ◽

Epstein Barr

Epstein–Barr virus (EBV) is a major cause of immunosuppression-related B-cell lymphomas and Hodgkin lymphoma (HL). In these malignancies, EBV latent membrane protein 1 (LMP1) and LMP2A provide infected B cells with surrogate CD40 and B-cell receptor growth and survival signals. To gain insights into their synergistic in vivo roles in germinal center (GC) B cells, from which most EBV-driven lymphomas arise, we generated a mouse model with conditional GC B-cell LMP1 and LMP2A coexpression. LMP1 and LMP2A had limited effects in immunocompetent mice. However, upon T- and NK-cell depletion, LMP1/2A caused massive plasmablast outgrowth, organ damage, and death. RNA-sequencing analyses identified EBV oncoprotein effects on GC B-cell target genes, including up-regulation of multiple proinflammatory chemokines and master regulators of plasma cell differentiation. LMP1/2A coexpression also up-regulated key HL markers, including CD30 and mixed hematopoietic lineage markers. Collectively, our results highlight synergistic EBV membrane oncoprotein effects on GC B cells and provide a model for studies of their roles in immunosuppression-related lymphoproliferative diseases.

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LMP1 signaling can replace CD40 signaling in B cells in vivo and has unique features of inducing class-switch recombination to IgG1

Blood ◽

10.1182/blood-2007-10-117655 ◽

2008 ◽

Vol 111 (3) ◽

pp. 1448-1455 ◽

Cited By ~ 69

Author(s):

Julia Rastelli ◽

Cornelia Hömig-Hölzel ◽

Jane Seagal ◽

Werner Müller ◽

Andrea C. Hermann ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Function ◽

Somatic Hypermutation ◽

Class Switch Recombination ◽

Cd40 Ligand ◽

Barr Virus ◽

Class Switch ◽

Epstein Barr

AbstractThe Epstein-Barr virus (EBV) protein LMP1 is considered to be a functional homologue of the CD40 receptor. However, in contrast to the latter, LMP1 is a constitutively active signaling molecule. To compare B cell–specific LMP1 and CD40 signaling in an unambiguous manner, we generated transgenic mice conditionally expressing a CD40/LMP1 fusion protein, which retained the LMP1 cytoplasmic tail but has lost the constitutive activity of LMP1 and needs to be activated by the CD40 ligand. We show that LMP1 signaling can completely substitute CD40 signaling in B cells, leading to normal B-cell development, activation, and immune responses including class-switch recombination, germinal center formation, and somatic hypermutation. In addition, the LMP1-signaling domain has a unique property in that it can induce class-switch recombination to IgG1 independent of cytokines. Thus, our data indicate that LMP1 has evolved to imitate T-helper cell function allowing activation, proliferation, and differentiation of EBV-infected B cells independent of T cells.

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Use of a Virus-Encoded Enzymatic Marker Reveals that a Stable Fraction of Memory B Cells Expresses Latency-Associated Nuclear Antigen throughout Chronic Gammaherpesvirus Infection

Journal of Virology ◽

10.1128/jvi.02572-09 ◽

2010 ◽

Vol 84 (15) ◽

pp. 7523-7534 ◽

Cited By ~ 27

Author(s):

Michael S. Nealy ◽

Carrie B. Coleman ◽

Haiyan Li ◽

Scott A. Tibbetts

Keyword(s):

B Cells ◽

Germinal Center ◽

Maintenance Phase ◽

Memory B Cells ◽

Nuclear Antigen ◽

Murine Gammaherpesvirus ◽

Barr Virus ◽

Enzymatic Marker ◽

Epstein Barr

ABSTRACT An integral feature of gammaherpesvirus infections is the ability to establish lifelong latency in B cells. During latency, the viral genome is maintained as an extrachomosomal episome, with stable maintenance in dividing cells mediated by the viral proteins Epstein-Barr nuclear antigen 1 (EBNA-1) for Epstein-Barr virus and latency-associated nuclear antigen (LANA) for Kaposi's sarcoma-associated herpesvirus. It is believed that the expression of episome maintenance proteins is turned off in the predominant long-term latency reservoir of resting memory B cells, suggesting that chronic gammaherpesvirus infection is primarily dormant. However, the kinetics of LANA/EBNA-1 expression in individual B-cell subsets throughout a course of infection has not been examined. The infection of mice with murine gammaherpesvirus 68 (MHV68, γHV68) provides a model to determine the specific cellular and molecular events that occur in vivo during lifelong gammaherpesvirus latency. In work described here, we make use of a heterologously expressed enzymatic marker to define the types of B cells that express the LANA homolog (mLANA) during chronic MHV68 infection. Our data demonstrate that mLANA is expressed in a stable fraction of B cells throughout chronic infection, with a prominent peak at 28 days. The expression of mLANA was detected in naïve follicular B cells, germinal-center B cells, and memory B cells throughout infection, with germinal-center and memory B cells accounting for more than 80% of the mLANA-expressing cells during the maintenance phase of latency. These findings suggest that the maintenance phase of latency is an active process that involves the ongoing proliferation or reseeding of latently infected memory B cells.

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Epstein-Barr Virus LMP2A Enhances B-Cell Responses In Vivo and In Vitro

Journal of Virology ◽

10.1128/jvi.00433-06 ◽

2006 ◽

Vol 80 (14) ◽

pp. 6764-6770 ◽

Cited By ~ 27

Author(s):

Michelle Swanson-Mungerson ◽

Rebecca Bultema ◽

Richard Longnecker

Keyword(s):

B Cells ◽

B Cell ◽

Epstein Barr Virus ◽

Plasma Cells ◽

Barr Virus ◽

Epstein Barr ◽

Hen Egg ◽

Hen Egg Lysozyme

ABSTRACT Epstein-Barr virus (EBV) establishes latent infections in a significant percentage of the population. Latent membrane protein 2A (LMP2A) is an EBV protein expressed during latency that inhibits B-cell receptor signaling in lymphoblastoid cell lines. In the present study, we have utilized a transgenic mouse system in which LMP2A is expressed in B cells that are specific for hen egg lysozyme (E/HEL-Tg). To determine if LMP2A allows B cells to respond to antigen, E/HEL-Tg mice were immunized with hen egg lysozyme. E/HEL-Tg mice produced antibody in response to antigen, indicating that LMP2A allows B cells to respond to antigen. In addition, E/HEL-Tg mice produced more antibody and an increased percentage of plasma cells after immunization compared to HEL-Tg littermates, suggesting that LMP2A increased the antibody response in vivo. Finally, in vitro studies determined that LMP2A acts directly on the B cell to increase antibody production by augmenting the expansion and survival of the activated B cells, as well as increasing the percentage of plasma cells generated. Taken together, these data suggest that LMP2A enhances, not diminishes, B-cell-specific antibody responses in vivo and in vitro in the E/HEL-Tg system.

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Identification of an alternative CD20 transcript variant in B-cell malignancies coding for a novel protein associated to rituximab resistance

Blood ◽

10.1182/blood-2009-06-229112 ◽

2010 ◽

Vol 115 (12) ◽

pp. 2420-2429 ◽

Cited By ~ 44

Author(s):

Carole Henry ◽

Marina Deschamps ◽

Pierre-Simon Rohrlich ◽

Jean-René Pallandre ◽

Jean-Paul Rémy-Martin ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Cell Lineage ◽

Specific Marker ◽

Reading Frame ◽

Barr Virus ◽

Before And After ◽

Epstein Barr

AbstractHuman CD20 is a B-cell lineage–specific marker expressed by normal and leukemic B cells from the pre-B to the plasma-cell stages and is a target for rituximab (RTX) immunotherapy. A CD20 reverse transcriptase–polymerase chain reaction (PCR) on B-cell lines cDNA yielded a short PCR product (ΔCD20) corresponding to a spliced mRNA transcript linking the exon 3 and exon 7 ends. We established here that this novel, alternatively spliced CD20 transcript is expressed and detectable at various levels in leukemic B cells, lymphoma B cells, in vivo tonsil- or in vitro CD40L-activated B cells, and Epstein-Barr virus (EBV)–transformed B cells, but not in resting CD19+- or CD20+-sorted B cells from peripheral blood or bone marrow of healthy donors. The truncated CD20 sequence is within the reading frame, codes a protein of 130 amino acids (∼ 15-17 kDa) lacking large parts of the 4 transmembrane segments, suggesting that ΔCD20 is a nonanchored membrane protein. We demonstrated the translation into a ΔCD20 protein which is associated with the membrane CD20 protein and showed its involvement in RTX resistance. Study of patient samples before and after RTX resistance or escape confirms our in vitro findings.

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Epstein–Barr virus EBER1 and murine gammaherpesvirus TMER4 share conserved in vivo function to promote B cell egress and dissemination

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1915752116 ◽

2019 ◽

Vol 116 (51) ◽

pp. 25392-25394 ◽

Cited By ~ 2

Author(s):

Brett A. Hoffman ◽

Yiping Wang ◽

Emily R. Feldman ◽

Scott A. Tibbetts

Keyword(s):

B Cells ◽

Noncoding Rna ◽

Epstein Barr Virus ◽

Peripheral Circulation ◽

Murine Gammaherpesvirus ◽

Barr Virus ◽

Long Latency ◽

Epstein Barr ◽

Gammaherpesvirus 68

The oncogenic gammaherpesviruses, including human Epstein–Barr virus (EBV), human Kaposi’s sarcoma-associated herpesvirus (KSHV), and murine gammaherpesvirus 68 (MHV68, γHV68, MuHV-4) establish life-long latency in circulating B cells. The precise determinants that mediate in vivo gammaherpesvirus latency and tumorigenesis remain unclear. The EBV-encoded RNAs (EBERs) are among the first noncoding RNAs ever identified and have been the subject of decades of studies; however, their biological roles during in vivo infection remain unknown. Herein, we use a series of refined virus mutants to define the active isoform of MHV68 noncoding RNA TMER4 and demonstrate that EBV EBER1 functionally conserves this activity in vivo to promote egress of infected B cells from lymph nodes into peripheral circulation.

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Human B Cells Immortalized with Epstein-Barr Virus Upregulate CCR6 and CCR10 and Downregulate CXCR4 and CXCR5

Journal of Virology ◽

10.1128/jvi.76.6.3072-3077.2002 ◽

2002 ◽

Vol 76 (6) ◽

pp. 3072-3077 ◽

Cited By ~ 54

Author(s):

Takashi Nakayama ◽

Ryuichi Fujisawa ◽

Dai Izawa ◽

Kunio Hieshima ◽

Kenzo Takada ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Epstein Barr Virus ◽

Receptor Expression ◽

Stable Expression ◽

B Cell Differentiation ◽

Barr Virus ◽

Human B Cells ◽

Epstein Barr

ABSTRACT Compared to peripheral blood resting B cells, Epstein-Barr virus (EBV)-immortalized B cells consistently express CCR6 and CCR10 at high levels and CXCR4 and CXCR5 at low levels. Accordingly, these cells vigorously responded to the ligands of CCR6 and CCR10 but not to those of CXCR4 and CXCR5. In a human EBV-negative B-cell line, BJAB, stable expression of EBNA2 upregulated CCR6, while stable expression of EBNA2 as well as LMP1 downregulated CXCR4. On the other hand, upregulation of CCR10 or downregulation of CXCR5 was not induced in BJAB by stable expression of EBNA2 or LMP1. Thus, these changes may be due to a plasmablast-like stage of B-cell differentiation fixed by EBV immortalization. EBV-infected B cells in infectious mononucleosis are known to avoid germinal centers and accumulate under the mucosal surfaces. EBV-associated opportunistic lymphomas also tend to occur in extranodal sites. These preferred sites of in vivo localization are consistent with the unique profile of chemokine receptor expression exhibited by EBV-immortalized B cells.

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Epstein-Barr Virus Latent Membrane Protein 2A Preferentially Signals through the Src Family Kinase Lyn

Journal of Virology ◽

10.1128/jvi.00843-08 ◽

2008 ◽

Vol 82 (17) ◽

pp. 8520-8528 ◽

Cited By ~ 24

Author(s):

Mark Rovedo ◽

Richard Longnecker

Keyword(s):

B Cells ◽

Membrane Protein ◽

B Cell ◽

Epstein Barr Virus ◽

Sh2 Domain ◽

Latent Membrane Protein ◽

Latent Membrane Protein 2A ◽

Barr Virus ◽

Epstein Barr

ABSTRACT Latent membrane protein 2A (LMP2A) is a viral protein expressed during Epstein-Barr virus (EBV) latency in EBV-infected B cells both in cell culture and in vivo. LMP2A has important roles in modulating B-cell receptor signal transduction and provides survival and developmental signals to B cells in vivo. Although Lyn has been shown to be important in mediating LMP2A signaling, it is still unclear if Lyn is used preferentially or if LMP2A associates promiscuously with other Src family kinase (SFK) members. To investigate the role of various SFKs in LMP2A signaling, we crossed LMP2A transgenic mice (TgE) with Lyn−/−, Fyn−/−, or Blk−/− mice. TgE Lyn−/− mice had a larger immunoglobulin M (IgM)-positive B-cell population than TgE mice, suggesting that the absence of Lyn prevents LMP2A from delivering survival and developmental signals to the B cells. Both TgE Fyn−/− and TgE Blk−/− mice have an IgM-negative population of splenic B cells, similar to the TgE mice. LMP2A was also transiently transfected into the human EBV-negative B-cell line BJAB to determine which SFK members associate with LMP2A. Lyn was detected in LMP2A immunoprecipitates, whereas Fyn was not. Both Lyn and Fyn were able to bind to an LMP2A mutant which contained a sequence shown previously to bind tightly to the SH2 domain of multiple SFK members. From these results, we conclude that LMP2A preferentially associates with and signals through Lyn compared to its association with other SFKs. This preferential association is due in part to the SH2 domain of Lyn associating with LMP2A.

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