Epstein-Barr Virus Latent Membrane Protein 2B (LMP2B) Modulates LMP2A Activity

ABSTRACT Latent membrane protein 2A (LMP2A) and LMP2B are viral proteins 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 (BCR) signal transduction by associating with the cellular tyrosine kinases Lyn and Syk via specific phosphotyrosine motifs found within the LMP2A N-terminal tail domain. LMP2A has been shown to alter normal BCR signal transduction in B cells by reducing levels of Lyn and by blocking tyrosine phosphorylation and calcium mobilization following BCR cross-linking. Although little is currently known about the function of LMP2B in B cells, the similarity in structure between LMP2A and LMP2B suggests that they may localize to the same cellular compartments. To investigate the function of LMP2B, B-cell lines expressing LMP2A, LMP2B, LMP2A/LMP2B, and the relevant vector controls were analyzed. As was previously shown, cells expressing LMP2A had a dramatic block in normal BCR signal transduction as measured by calcium mobilization and tyrosine phosphorylation. There was no effect on BCR signal transduction in cells expressing LMP2B. Interestingly, when LMP2B was expressed in conjunction with LMP2A, there was a restoration of normal BCR signal transduction upon BCR cross-linking. The expression of LMP2B did not alter the cellular localization of LMP2A but did bind to and prevent the phosphorylation of LMP2A. A restoration of Lyn levels, but not a change in LMP2A levels, was also observed in cells coexpressing LMP2B with LMP2A. From these results, we conclude that LMP2B modulates LMP2A activity.

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Latent Membrane Protein 2B Regulates Susceptibility to Induction of Lytic Epstein-Barr Virus Infection

Journal of Virology ◽

10.1128/jvi.01723-07 ◽

2007 ◽

Vol 82 (4) ◽

pp. 1739-1747 ◽

Cited By ~ 26

Author(s):

Markus P. Rechsteiner ◽

Christoph Berger ◽

Ludwig Zauner ◽

Jürg A. Sigrist ◽

Matthias Weber ◽

...

Keyword(s):

B Cells ◽

Membrane Protein ◽

B Cell ◽

Epstein Barr Virus ◽

Latent Membrane Protein ◽

Cross Linking ◽

Epstein Barr Virus Infection ◽

Barr Virus ◽

Ebv Infection ◽

Epstein Barr

ABSTRACT The B-lymphotropic Epstein-Barr virus (EBV) encodes two isoforms of latent membrane protein 2 (LMP2), LMP2A and LMP2B, which are expressed during latency in B cells. The function of LMP2B is largely unknown, whereas LMP2A blocks B-cell receptor (BCR) signaling transduction and induction of lytic EBV infection, thereby promoting B-cell survival. Transfection experiments on LMP2B in EBV-negative B cells and the silencing of LMP2B in EBV-harboring Burkitt's lymphoma-derived Akata cells suggest that LMP2B interferes with the function of LMP2A, but the role of LMP2B in the presence of functional EBV has not been established. Here, LMP2B, LMP2A, or both were overexpressed in EBV-harboring Akata cells to study the function of LMP2B. The overexpression of LMP2B increased the magnitude of EBV switching from its latent to its lytic form upon BCR cross-linking, as indicated by a more-enhanced upregulation and expression of EBV lytic genes and significantly increased production of transforming EBV compared to Akata vector control cells or LMP2A-overexpressing cells. Moreover, LMP2B lowered the degree of BCR cross-linking required to induce lytic EBV infection. Finally, LMP2B colocalized with LMP2A as demonstrated by immunoprecipitation and immunofluorescence and restored calcium mobilization upon BCR cross-linking, a signaling process inhibited by LMP2A. Thus, our findings suggest that LMP2B negatively regulates the function of LMP2A in preventing the switch from latent to lytic EBV replication.

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c-Myc Represses Transcription of Epstein-Barr Virus Latent Membrane Protein 1 Early after Primary B Cell Infection

Journal of Virology ◽

10.1128/jvi.01178-17 ◽

2017 ◽

Vol 92 (2) ◽

Cited By ~ 9

Author(s):

Alexander M. Price ◽

Joshua E. Messinger ◽

Micah A. Luftig

Keyword(s):

Transcription Factor ◽

B Cells ◽

Membrane Protein ◽

B Cell ◽

Epstein Barr Virus ◽

Latent Membrane Protein ◽

Cell Infection ◽

Barr Virus ◽

Epstein Barr ◽

Factor C

ABSTRACTRecent evidence has shown that the Epstein-Barr virus (EBV) oncogene LMP1 is not expressed at high levels early after EBV infection of primary B cells, despite its being essential for the long-term outgrowth of immortalized lymphoblastoid cell lines (LCLs). In this study, we found that expression of LMP1 increased 50-fold between 7 days postinfection and the LCL state. Metabolic labeling of nascent transcribed mRNA indicated that this was primarily a transcription-mediated event. EBNA2, the key viral transcription factor regulating LMP1, and CTCF, an important chromatin insulator, were recruited to the LMP1 locus similarly early and late after infection. However, the activating histone H3K9Ac mark was enriched at the LMP1 promoter in LCLs relative to that in infected B cells early after infection. We found that high c-Myc activity in EBV-infected lymphoma cells as well as overexpression of c-Myc in an LCL model system repressed LMP1 transcription. Finally, we found that chemical inhibition of c-Myc both in LCLs and early after primary B cell infection increased LMP1 expression. These data support a model in which high levels of endogenous c-Myc activity induced early after primary B cell infection directly repress LMP1 transcription.IMPORTANCEEBV is a highly successful pathogen that latently infects more than 90% of adults worldwide and is also causally associated with a number of B cell malignancies. During the latent life cycle, EBV expresses a set of viral oncoproteins and noncoding RNAs with the potential to promote cancer. Critical among these is the viral latent membrane protein LMP1. Prior work suggests that LMP1 is essential for EBV to immortalize B cells, but our recent work indicates that LMP1 is not produced at high levels during the first few weeks after infection. Here we show that transcription of the LMP1 gene can be negatively regulated by a host transcription factor, c-Myc. Ultimately, understanding the regulation of EBV oncogenes will allow us to better treat cancers that rely on these viral products for survival.

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Latent Membrane Protein 1 Regulates STAT1 through NF-κB-Dependent Interferon Secretion in Epstein-Barr Virus-Immortalized B Cells

Journal of Virology ◽

10.1128/jvi.79.8.4936-4943.2005 ◽

2005 ◽

Vol 79 (8) ◽

pp. 4936-4943 ◽

Cited By ~ 38

Author(s):

Imen Najjar ◽

Fanny Baran-Marszak ◽

Christophe Le Clorennec ◽

Christelle Laguillier ◽

Olivier Schischmanoff ◽

...

Keyword(s):

B Cells ◽

Membrane Protein ◽

Tyrosine Phosphorylation ◽

Epstein Barr Virus ◽

Latent Membrane Protein ◽

Cell Culture Supernatant ◽

Latent Membrane Protein 1 ◽

Barr Virus ◽

Ifn Γ ◽

Epstein Barr

ABSTRACT Constitutive activation of signal transducer and activator of transcription 1 (STAT1) is a distinctive feature of Epstein-Barr virus (EBV)-immortalized B cells (lymphoblastoid cell lines [LCLs]). The expression of STAT1 in these cells is modulated by the latent membrane protein 1 (LMP1), but the mechanism of STAT1 activation has remained unclear. We demonstrate that the tyrosine phosphorylation of STAT1 in LCLs results from an indirect pathway encompassing an NF-κB-dependent secretion of interferons (IFNs). The cell culture supernatant of LCLs induced tyrosine phosphorylation of STAT1 in cells with no constitutively activated STAT1. Moreover, removal of supernatant from LCLs was sufficient to decrease the phosphorylation of STAT1. Inhibition of NF-κB activity by different pharmacological inhibitors (i.e., parthenolide, MG132 and BAY 11-7082) and by overexpressed mutated IκBα prevented the activation of STAT1. To identify the factors involved, we performed macroarray cDNA profiling with or without inhibition of NF-κB. The expression of several cytokines was NF-κB dependent among those alpha and gamma IFNs (IFN-α and IFN-γ), known activators of STAT1. By real-time PCR and enzyme-linked immunosorbent assay we show that IFN-α and IFN-γ are expressed and released by LCLs in an NF-κB-dependent manner. Finally, the blocking of the IFN-α and IFN-γ by neutralizing antibodies led to the complete inhibition of tyrosine phosphorylation of STAT1. Taken together, our results clearly show that LMP1-induced tyrosine phosphorylation of STAT1 is almost exclusively due to the NF-κB-dependent secretion of IFNs. Whether this response, which is usually considered to be antiviral, is in fact required for the persistence of the virus remains to be elucidated.

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

Blood ◽

10.1182/blood-2007-05-090142 ◽

2007 ◽

Vol 110 (10) ◽

pp. 3715-3721 ◽

Cited By ~ 134

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.

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Cd40 doesn't Complement The Function Of Epstein-barr Virus (Ebv) Latent Membrane Protein 1 In B Cells Transformation

Bangladesh Journal of Medical Science ◽

10.3329/bjms.v11i2.8175 ◽

2012 ◽

Vol 11 (2) ◽

pp. 112-116

Author(s):

Mohammed Nazmul Ahsan ◽

Anwarul A Akhand

Keyword(s):

B Cells ◽

Membrane Protein ◽

B Cell ◽

Epstein Barr Virus ◽

Medical Science ◽

Latent Membrane Protein ◽

Latent Membrane Protein 1 ◽

Barr Virus ◽

Epstein Barr

Objective: EpsteinBarr virus (EBV) latent membrane protein 1 (LMP1) is known to plays important role in B cells growth and transformation. LMP1 is considered to be a functional homologue of the CD40 receptor and they can activate many overlapping signaling pathways. In this study, we compared the function of CD40 with that of LMP1 in B cells transformation. Materials and methods: Expression of CD40L was observed in infected B cells with LMP1 mutated EBV. To observe the expression reverse transcription-PCR were performed. Results: This expression of CD40L did not support proliferation and transformation of B cell. Even in vitro proliferation and transformation of B cell infected with LMP1 deleted EBV supplemented with CD40L were also not observed. Conclusion: Despite many similarities shared between CD40 and LMP1, CD40-CD40L interaction didnt complement on LMP1 mediated B cells transformation in vitro. DOI: http://dx.doi.org/10.3329/bjms.v11i2.8175 Bangladesh Journal of Medical Science Vol. 11 No. 02 April 2012: 112-116

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Tyrosine 112 of Latent Membrane Protein 2A Is Essential for Protein Tyrosine Kinase Loading and Regulation of Epstein-Barr Virus Latency

Journal of Virology ◽

10.1128/jvi.72.10.7796-7806.1998 ◽

1998 ◽

Vol 72 (10) ◽

pp. 7796-7806 ◽

Cited By ~ 95

Author(s):

Sara Fruehling ◽

Rachel Swart ◽

KristIne M. Dolwick ◽

Elisabeth Kremmer ◽

Richard Longnecker

Keyword(s):

Signal Transduction ◽

Membrane Protein ◽

Tyrosine Kinase ◽

B Cell ◽

Epstein Barr Virus ◽

Protein Tyrosine Kinase ◽

Latent Membrane Protein ◽

Latent Membrane Protein 2A ◽

Barr Virus ◽

Epstein Barr

ABSTRACT Latent membrane protein 2A (LMP2A) of Epstein-Barr virus (EBV) is expressed on the plasma membrane of B lymphocytes latently infected with EBV and blocks B-cell receptor (BCR) signal transduction in EBV-immortalized B cells in vitro. The LMP2A amino-terminal domain that is essential for the LMP2A-mediated block on BCR signal transduction contains eight tyrosine residues. Association of Syk protein tyrosine kinase (PTK) with LMP2A occurs at the two tyrosines of the LMP2A immunoreceptor tyrosine-based activation motif, and it is hypothesized that Lyn PTK associates with the YEEA amino acid motif at LMP2A tyrosine 112 (Y112). To examine the specific association of Lyn PTK to LMP2A, a panel of LMP2A cDNA expression vectors containing LMP2A mutations were transfected into an EBV-negative B-cell line and analyzed for Lyn and LMP2A coimmunoprecipitation. Lyn associates with wild-type LMP2A and other LMP2A mutant constructs, but Lyn association is lost in the LMP2A construct containing a tyrosine (Y)-to-phenylalanine (F) mutation at LMP2A residue Y112 (LMP2AY112F). Next, the LMP2AY112F mutation was recombined into the EBV genome to generate stable lymphoblastoid cell lines (LCLs) transformed with the LMP2AY112F mutant virus. Analysis of BCR-mediated signal transduction in the LMP2AY112F LCLs revealed loss of the LMP2A-mediated block in BCR signal transduction. In addition, LMP2A was not tyrosine phosphorylated in LMP2AY112F LCLs. Together these data indicate the importance of the LMP2A Y112 residue in the ability of LMP2A to block BCR-mediated signal transduction and place the role of this residue and its interaction with Lyn PTK as essential to LMP2A phosphorylation, PTK loading, and down-modulation of PTKs involved in BCR-mediated signal transduction.

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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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