A Single Amino Acid in EBNA-2 Determines Superior B Lymphoblastoid Cell Line Growth Maintenance by Epstein-Barr Virus Type 1 EBNA-2

Tzellos, Stelios; Correia, Paulo B.; Karstegl, Claudio Elgueta; Cancian, Laila; Cano-Flanagan, Julian; McClellan, Michael J.; West, Michelle J.; Farrell, Patrick J.

doi:10.1128/jvi.01000-14

Cited by 47 publications

(64 citation statements)

References 33 publications

(42 reference statements)

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“…Thus, gene repression due to binding at EBNA3-only sites may still be attributable to impaired (abrogated) EBNA2 binding. This phenomenon may explain the recent discovery that the increased transforming effects of EBNA2 from type I EBV compared to that from type II EBV are due to increased binding of EBNA2 at ETS/IRF4 sites (12). The "novel" binding sites observed with type I EBNA2 may actually be due to an enhanced ability to compete with EBNA3C for binding at ETS/IRF4 sites.…”

Section: Discussionmentioning

confidence: 78%

“…EBNA2 upregulates the other EBV latent gene products, as well as cell oncogenes, such as c-myc, required for LCL growth (5)(6)(7)(8). EBNA2 effects are substantially similar, but not identical, to those of ICN (9)(10)(11)(12). Unlike that of ICN, EBNA2 activation is constitutive and ligand independent.…”

Section: Epstein-barr Virus (Ebv) Latent Gene Products Cause Human Camentioning

confidence: 73%

“…Notably, we did not observe enrichment for the RBPJ binding motif at EBNA3B-bound sites. The RBPJ motif is also not enriched at EBNA3A-or EBNA3C-bound sites, even though it can be readily detected at EBNA2-bound sites (12,42).…”

Section: Genome-wide Binding Analysis Of Ebna3 Proteins In Lclsmentioning

confidence: 99%

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Epstein-Barr Virus Nuclear Antigen 3 (EBNA3) Proteins Regulate EBNA2 Binding to Distinct RBPJ Genomic Sites

Wang

Welch

et al. 2016

J Virol

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Latent infection of B lymphocytes by Epstein-Barr virus (EBV) in vitro results in their immortalization into lymphoblastoid cell lines (LCLs); this latency program is controlled by the EBNA2 viral transcriptional activator, which targets promoters via RBPJ, a DNA binding protein in the Notch signaling pathway. Three other EBNA3 proteins (EBNA3A, EBNA3B, and EBNA3C) interact with RBPJ to regulate cell gene expression. The mechanism by which EBNAs regulate different genes via RBPJ remains unclear. Our chromatin immunoprecipitation with deep sequencing (ChIP-seq) analysis of the EBNA3 proteins analyzed in concert with prior EBNA2 and RBPJ data demonstrated that EBNA3A, EBNA3B, and EBNA3C bind to distinct, partially overlapping genomic locations. Although RBPJ interaction is critical for EBNA3A and EBNA3C growth effects, only 30 to 40% of EBNA3-bound sites colocalize with RBPJ. Using LCLs conditional for EBNA3A or EBNA3C activity, we demonstrate that EBNA2 binding at sites near EBNA3A-or EBNA3C-regulated genes is specifically regulated by the respective EBNA3. To investigate EBNA3 binding specificity, we identified sequences and transcription factors enriched at EBNA3A-, EBNA3B-, and EBNA3C-bound sites. This confirmed the prior observation that IRF4 is enriched at EBNA3A-and EBNA3C-bound sites and revealed IRF4 enrichment at EBNA3B-bound sites. Using IRF4-negative BJAB cells, we demonstrate that IRF4 is essential for EBNA3C, but not EBNA3A or EBNA3B, binding to specific sites. These results support a model in which EBNA2 and EBNA3s compete for distinct subsets of RBPJ sites to regulate cell genes and where EBNA3 subset specificity is determined by interactions with other cell transcription factors. IMPORTANCE Epstein-Barr virus (EBV) latent gene products cause human cancers and transform B lymphocytes into immortalized lymphoblastoid cell lines in vitro.EBV nuclear antigens (EBNAs) and membrane proteins constitutively activate pathways important for lymphocyte growth and survival. An important unresolved question is how four different EBNAs (EBNA2, -3A, -3B, and -3C) exert unique effects via a single transcription factor, RBPJ. Here, we report that each EBNA binds to distinct but partially overlapping sets of genomic sites. EBNA3A and EBNA3C specifically regulate EBNA2's access to different RBPJ sites, providing a mechanism by which each EBNA can regulate distinct cell genes. We show that IRF4, an essential regulator of B cell differentiation, is critical for EBNA3C binding specificity; EBNA3A and EBNA3B specificities are likely due to interactions with other cell transcription factors. EBNA3 titration of EBNA2 transcriptional function at distinct sites likely limits cell defenses that would be triggered by unchecked EBNA2 prooncogenic activity. E pstein-Barr virus (EBV) is a herpesvirus that infects over 90% of the population by adulthood. Primary EBV infection usually presents as a nonspecific illness in early childhood but often manifests as infectious mononucleosis in adolescents (1). Thereafter, EBV es...

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Section: Discussionmentioning

confidence: 78%

Section: Epstein-barr Virus (Ebv) Latent Gene Products Cause Human Camentioning

confidence: 73%

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Epstein-Barr Virus Nuclear Antigen 3 (EBNA3) Proteins Regulate EBNA2 Binding to Distinct RBPJ Genomic Sites

Wang

Welch

et al. 2016

J Virol

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“…The polymorphisms we found in the EBNA2 gene may have functional consequences as supported by recent data showing that a single amino acid in EBNA2 determines superior B lymphoblastoid cell line growth maintenance. 29 The allelic variants we described may affect the host-virus interplay at different levels: (1) the same region is involved in interactions with cellular proteins such as Nur77 30 and SMARCB1 31 that have been associated with MS susceptibility 32 and antiviral responses 33 ; (2) the gene region we investigated is important for successful strategies of EBV immune evasion 34,35 ; (3) last, but not least, the EBNA2 messenger RNA, where we found MS-related variants, belongs to the targetome of human micro-RNAs that may serve to keep in check EBV infection. 36 MS may resemble other immune-mediated diseases in which infectious agents are thought to have a key role.…”

mentioning

confidence: 99%

Epstein-Barr virus genetic variants are associated with multiple sclerosis

et al. 2015

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Objective: We analyzed the Epstein-Barr nuclear antigen 2 (EBNA2) gene, which contains the most variable region of the viral genome, in persons with multiple sclerosis (MS) and control subjects to verify whether virus genetic variants are involved in disease development. 84-14.32; p 5 0.016) and underrepresentation of 1.3B allele (OR 5 0.23; 95% CI 0.08-0.51; p 5 0.0006). We identified new genetic variants, mostly 1.2 allele-and MS-associated (especially amino acid variation at position 245; OR 5 9.4; 95% CI 1.19-78.72; p 5 0.0123). In all cases, the consensus sequence from deep sequencing confirmed Sanger sequencing (including the cosegregation of newly identified variants with known EBNA2 alleles) and showed that the extent of genotype intraindividual variability was higher than expected: rare EBNA2 variants were detected in all HDs and patients with MS (range 1-17 and 3-19, respectively). EBNA2 variants did not seem to correlate with human leucocyte antigen typing or clinical/MRI features. MethodsConclusions: Our study unveils a strong association between Epstein-Barr virus genomic variants and MS, reinforcing the idea that Epstein-Barr virus contributes to disease development. Despite converging evidence supporting an etiologic role for Epstein-Barr virus (EBV) in multiple sclerosis (MS), we still do not know through which mechanisms the virus may contribute to disease development. 1 The potential pathogenic role of EBV genetic variants in MS may be in keeping with epidemiologic observations, in particular the geographic gradient of MS and the change in MS risk in migrants, 2 and with the reported association between virus genetic variants and EBV-related malignancies with different geographic distribution.3-5 Also, the discrepancy between the global diffusion of EBV infection and the low prevalence of MS could be at least in part explained by EBV genomic diversity that differently affects MS development.Major methodologic difficulties hinder the study of EBV variants through sequencing of the whole viral genome. To date, only 8 complete genome sequences have been described: 5 from patients with EBV-related diseases and 3 from healthy individuals.6 This is mainly attributable

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“…More detailed analysis showed that converting a single amino acid of type 2 EBNA2 from serine to the aspartate found in type 1 EBNA2 (S442D) was sufficient to give the type 1 growth maintenance of LCLs (Tzellos et al 2014). That amino acid is in the transactivation domain (TAD) of EBNA2, and the TAD of type 1 EBNA2 was about 2-fold more active in a standard TAD reporter assay.…”

Section: Functional Difference Between Type 1 and Type 2 Ebna2mentioning

confidence: 98%

Epstein–Barr Virus Strain Variation

Farrell

2015

Current Topics in Microbiology and Immunology

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What is wild-type Epstein-Barr virus and are there genetic differences in EBV strains that contribute to some of the EBV-associated diseases? Recent progress in DNA sequencing has resulted in many new Epstein-Barr virus (EBV) genome sequences becoming available. EBV isolates worldwide can be grouped into type 1 and type 2, a classification based on the EBNA2 gene sequence. Type 1 transforms human B cells into lymphoblastoid cell lines much more efficiently than type 2 EBV and molecular mechanisms that may account for this difference in cell transformation are now becoming understood. Study of geographic variation of EBV strains independent of the type 1/type 2 classification and systematic investigation of the relationship between viral strains, infection and disease are now becoming possible. So we should consider more directly whether viral sequence variation might play a role in the incidence of some EBV-associated diseases.

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A Single Amino Acid in EBNA-2 Determines Superior B Lymphoblastoid Cell Line Growth Maintenance by Epstein-Barr Virus Type 1 EBNA-2

Cited by 47 publications

References 33 publications

Epstein-Barr Virus Nuclear Antigen 3 (EBNA3) Proteins Regulate EBNA2 Binding to Distinct RBPJ Genomic Sites

Epstein-Barr Virus Nuclear Antigen 3 (EBNA3) Proteins Regulate EBNA2 Binding to Distinct RBPJ Genomic Sites

Epstein-Barr virus genetic variants are associated with multiple sclerosis

Epstein–Barr Virus Strain Variation

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