scholarly journals Identification and Cloning of a New Western Epstein-Barr Virus Strain That Efficiently Replicates in Primary B Cells

2020 ◽  
Vol 94 (10) ◽  
Author(s):  
Susanne Delecluse ◽  
Remy Poirey ◽  
Martin Zeier ◽  
Paul Schnitzler ◽  
Uta Behrends ◽  
...  
Keyword(s):  
B Cells ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Sequence Information ◽  
World Population ◽  
Barr Virus ◽  
The World ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) causes human cancers, and epidemiological studies have shown that lytic replication is a risk factor for some of these tumors. This fits with the observation that EBV M81, which was isolated from a Chinese patient with nasopharyngeal carcinoma, induces potent virus production and increases the risk of genetic instability in infected B cells. To find out whether this property extends to viruses found in other parts of the world, we investigated 22 viruses isolated from Western patients. While one-third of the viruses hardly replicated, the remaining viruses showed variable levels of replication, with three isolates replicating at levels close to that of M81 in B cells. We cloned one strongly replicating virus into a bacterial artificial chromosome (BAC); the resulting recombinant virus (MSHJ) retained the properties of its nonrecombinant counterpart and showed similarities to M81, undergoing lytic replication in vitro and in vivo after 3 weeks of latency. In contrast, B cells infected with the nonreplicating Western B95-8 virus showed early but abortive replication accompanied by cytoplasmic BZLF1 expression. Sequencing confirmed that rMSHJ is a Western virus, being genetically much closer to B95-8 than to M81. Spontaneous replication in rM81- and rMSHJ-infected B cells was dependent on phosphorylated Btk and was inhibited by exposure to ibrutinib, opening the way to clinical intervention in patients with abnormal EBV replication. As rMSHJ contains the complete EBV genome and induces lytic replication in infected B cells, it is ideal to perform genetic analyses of all viral functions in Western strains and their associated diseases. IMPORTANCE The Epstein-Barr virus (EBV) infects the majority of the world population but causes different diseases in different countries. Evidence that lytic replication, the process that leads to new virus progeny, is linked to cancer development is accumulating. Indeed, viruses such as M81 that were isolated from Far Eastern nasopharyngeal carcinomas replicate strongly in B cells. We show here that some viruses isolated from Western patients, including the MSHJ strain, share this property. Moreover, replication of both M81 and of MSHJ was sensitive to ibrutinib, a commonly used drug, thereby opening an opportunity for therapeutic intervention. Sequencing of MSHJ showed that this virus is quite distant from M81 and is much closer to nonreplicating Western viruses. We conclude that Western EBV strains are heterogeneous, with some viruses being able to replicate more strongly and therefore being potentially more pathogenic than others, and that the virus sequence information alone cannot predict this property.

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 Nuclear Factor-κB Binds to the Epstein-Barr Virus LMP1 Promoter and Upregulates Its Expression

2008 ◽  
Vol 83 (3) ◽  
pp. 1393-1401 ◽  
Author(s):  
Pegah Johansson ◽  
Ann Jansson ◽  
Ulla Rüetschi ◽  
Lars Rymo
Keyword(s):  
B Cells ◽  
Epstein Barr Virus ◽  
Affinity Purification ◽  
Critical Role ◽  
Mobility Shift ◽  
Barr Virus ◽  
Exogenous Expression ◽  
Epstein Barr

ABSTRACT The latent membrane protein 1 (LMP1) oncogene carried by Epstein-Barr virus (EBV) is essential for transformation and maintenance of EBV-immortalized B cells in vitro, and it is expressed in most EBV-associated tumor types. The activation of the NF-κB pathway by LMP1 plays a critical role in the upregulation of antiapoptotic proteins. The EBV-encoded EBNA2 transactivator is required for LMP1 activation in latency III, while LMP1 itself appears to be critical for its activation in the latency II gene expression program. In both cases, additional viral and cellular transcription factors are required in mediating transcription activation of the LMP1 promoter. Using DNA affinity purification and chromatin immunoprecipitation assay, we showed here that members of the NF-κB transcription factor family bound to the LMP1 promoter in vitro and in vivo. Electrophoretic mobility shift assay analyses indicated the binding of the p50-p50 homodimer and the p65-p50 heterodimer to an NF-κB site in the LMP1 promoter. Transient transfections and reporter assays showed that the LMP1 promoter is activated by exogenous expression of NF-κB factors in both B cells and epithelial cells. Exogenous expression of NF-κB factors in the EBNA2-deficient P3HR1 cell line induced LMP1 protein expression. Overall, our data are consistent with the presence of a positive regulatory circuit between NF-κB activation and LMP1 expression.

scholarly journals Epstein-Barr Virus WZhet DNA Can Induce Lytic Replication in Epithelial Cells in vitro, although WZhet Is Not Detectable in Many Human Tissues in vivo

Intervirology ◽  
2009 ◽  
Vol 52 (1) ◽  
pp. 8-16 ◽  
Author(s):  
Julie L. Ryan ◽  
Richard J. Jones ◽  
Sandra H. Elmore ◽  
Shannon C. Kenney ◽  
George Miller ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Human Tissues ◽  
Barr Virus ◽  
Epstein Barr

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

2006 ◽  
Vol 80 (14) ◽  
pp. 6764-6770 ◽  
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.

scholarly journals c-Myc and Rel/NF-κB Are the Two Master Transcriptional Systems Activated in the Latency III Program of Epstein-Barr Virus-Immortalized B Cells

2009 ◽  
Vol 83 (10) ◽  
pp. 5014-5027 ◽  
Author(s):  
Nathalie Faumont ◽  
Stéphanie Durand-Panteix ◽  
Martin Schlee ◽  
Sebastian Grömminger ◽  
Marino Schuhmacher ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Expression Patterns ◽  
Dominant Negative ◽  
Barr Virus ◽  
Conditional Expression ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) latency III program imposed by EBNA2 and LMP1 is directly responsible for immortalization of B cells in vitro and is thought to mediate most immunodeficiency-related posttransplant lymphoproliferative diseases in vivo. To answer the question whether and how this proliferation program is related to c-Myc, we have established the transcriptome of both c-Myc and EBV latency III proliferation programs using a Lymphochip specialized microarray. In addition to EBV-positive latency I Burkitt lymphoma lines and lymphoblastoid cell lines (LCLs), we used an LCL expressing an estrogen-regulatable EBNA2 fusion protein (EREB2-5) and derivative B-cell lines expressing a constitutively active or tetracycline-regulatable c-myc gene. A total of 897 genes were found to be fourfold or more up- or downregulated in either one or both proliferation programs compared to the expression profile of resting EREB2-5 cells. A total of 661 (74%) of these were regulated similarly in both programs. Numerous repressed genes were known targets of STAT1, and most induced genes were known to be upregulated by c-Myc and to be involved in cell proliferation. In keeping with the gene expression patterns, inactivation of c-Myc by a chemical inhibitor or by conditional expression of dominant-negative c-Myc and Max mutants led to proliferation arrest of LCLs. Most genes differently regulated in both proliferation programs corresponded to genes induced by NF-κB in LCLs, and many of them coded for immunoregulatory and/or antiapoptotic molecules. Thus, c-Myc and NF-κB are the two main transcription factors responsible for the phenotype, growth pattern, and biological properties of cells driven into proliferation by EBV.

scholarly journals Novel Therapeutics for Epstein–Barr Virus

Molecules ◽  
2019 ◽  
Vol 24 (5) ◽  
pp. 997 ◽  
Author(s):  
Graciela Andrei ◽  
Erika Trompet ◽  
Robert Snoeck
Keyword(s):  
B Cells ◽  
Epithelial Cells ◽  
Epstein Barr Virus ◽  
Adult Population ◽  
Antiviral Drug ◽  
Barr Virus ◽  
Epstein Barr ◽  
New Strategies

Epstein–Barr virus (EBV) is a human γ-herpesvirus that infects up to 95% of the adult population. Primary EBV infection usually occurs during childhood and is generally asymptomatic, though the virus can cause infectious mononucleosis in 35–50% of the cases when infection occurs later in life. EBV infects mainly B-cells and epithelial cells, establishing latency in resting memory B-cells and possibly also in epithelial cells. EBV is recognized as an oncogenic virus but in immunocompetent hosts, EBV reactivation is controlled by the immune response preventing transformation in vivo. Under immunosuppression, regardless of the cause, the immune system can lose control of EBV replication, which may result in the appearance of neoplasms. The primary malignancies related to EBV are B-cell lymphomas and nasopharyngeal carcinoma, which reflects the primary cell targets of viral infection in vivo. Although a number of antivirals were proven to inhibit EBV replication in vitro, they had limited success in the clinic and to date no antiviral drug has been approved for the treatment of EBV infections. We review here the antiviral drugs that have been evaluated in the clinic to treat EBV infections and discuss novel molecules with anti-EBV activity under investigation as well as new strategies to treat EBV-related diseases.

scholarly journals In vitro Stimulation with WT1 Peptide-Loaded Epstein-Barr Virus-Positive B Cells Elicits High Frequencies of WT1 Peptide-Specific T Cells with In vitro and In vivo Tumoricidal Activity

2004 ◽  
Vol 10 (21) ◽  
pp. 7207-7219 ◽  
Author(s):  
Ekaterina S. Doubrovina ◽  
Mikhail M. Doubrovin ◽  
Sangyull Lee ◽  
Jae-Hung Shieh ◽  
Glen Heller ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Epstein Barr Virus ◽  
Tumoricidal Activity ◽  
High Frequencies ◽  
Barr Virus ◽  
Epstein Barr

scholarly journals Methylation of Transcription Factor Binding Sites in the Epstein-Barr Virus Latent Cycle Promoter Wp Coincides with Promoter Down-Regulation during Virus-Induced B-Cell Transformation

2000 ◽  
Vol 74 (22) ◽  
pp. 10468-10479 ◽  
Author(s):  
R. J. Tierney ◽  
H. E. Kirby ◽  
J. K. Nagra ◽  
J. Desmond ◽  
A. I. Bell ◽  
...  
Keyword(s):  
B Cells ◽  
Binding Sites ◽  
Epstein Barr Virus ◽  
Regulatory Region ◽  
Nuclear Antigen ◽  
Regulatory Sequences ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Two Epstein-Barr virus latent cycle promoters for nuclear antigen expression, Wp and Cp, are activated sequentially during virus-induced transformation of B cells to B lymphoblastoid cell lines (LCLs) in vitro. Previously published restriction enzyme studies have indicated hypomethylation of CpG dinucleotides in the Wp and Cp regions of the viral genome in established LCLs, whereas these same regions appeared to be hypermethylated in Burkitt's lymphoma cells, where Wp and Cp are inactive. Here, using the more sensitive technique of bisulfite genomic sequencing, we reexamined the situation in established LCLs with the typical pattern of dominant Cp usage; surprisingly, this showed substantial methylation in the 400-bp regulatory region upstream of the Wp start site. This was not an artifact of long-term in vitro passage, since, in cultures of recently infected B cells, we found progressive methylation of Wp (but not Cp) regulatory sequences occurring between 7 and 21 days postinfection, coincident with the period in which dominant nuclear antigen promoter usage switches from Wp to Cp. Furthermore, in the equivalent in vivo situation, i.e., in the circulating B cells of acute infectious mononucleosis patients undergoing primary EBV infection, we again frequently observed selective methylation of Wp but not Cp sequences. An effector role for methylation in Wp silencing was supported by methylation cassette assays of Wp reporter constructs and by bandshift assays, where the binding of two sets of transcription factors important for Wp activation in B cells, BSAP/Pax5 and CREB/ATF proteins, was shown to be blocked by methylation of their binding sites.

scholarly journals Epstein-Barr Virus SM Protein Interacts with mRNA In Vivo and Mediates a Gene-Specific Increase in Cytoplasmic mRNA

2001 ◽  
Vol 75 (13) ◽  
pp. 6033-6041 ◽  
Author(s):  
Vivian Ruvolo ◽  
Ashish K. Gupta ◽  
Sankar Swaminathan
Keyword(s):  
Epstein Barr Virus ◽  
Rna Binding ◽  
Lytic Replication ◽  
Nuclear Accumulation ◽  
Rich Region ◽  
Rnase Protection ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT SM is an Epstein-Barr virus (EBV) gene expressed during early lytic replication of EBV. SM encodes a nuclear phosphoprotein that functions as a posttranscriptional regulator of gene expression. SM has been implicated in several aspects of gene regulation, including nuclear mRNA stabilization, posttranscriptional processing, and nuclear mRNA export. Activation by SM is promoter independent but gene specific. The mechanism by which SM selectively activates some EBV target genes or heterologous reporter genes remains to be determined. SM binds RNA in vitro, suggesting that sequence- or structure-specific mRNA interactions might mediate SM specificity. We have further analyzed RNA binding by SM and demonstrated that proteolytic cleavage of SM and consequent exposure of an arginine-rich region are necessary to allow RNA binding in vitro. However, SM mutants with deletions of this arginine-rich region localized normally in the nucleus and were fully functional in gene activation. We therefore developed an assay to study in vivo interactions of SM with target mRNAs based on immunoprecipitation of SM from cell lysates followed by RNase protection analysis. Using this assay, we demonstrated that SM forms complexes with specific mRNAs in vivo. SM binds mRNAs from both SM-responsive as well as nonresponsive intronless genes and increases the nuclear accumulation of both types of mRNAs. In addition, SM preferentially associates with newly transcribed mRNAs. These data indicate that SM forms complexes with mRNAs in the nucleus and enhances their nuclear accumulation. However, SM does not enhance cytoplasmic accumulation of all transcripts that it binds to the same degree, suggesting that additional mRNA-specific characteristics, such as nuclear retention motifs or binding sites for cellular proteins, also determine responsiveness to SM.

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