scholarly journals Use of transmitochondrial cybrids to assign a complex I defect to the mitochondrial DNA-encoded NADH dehydrogenase subunit 6 gene mutation at nucleotide pair 14459 that causes Leber hereditary optic neuropathy and dystonia.

1996 ◽  
Vol 16 (3) ◽  
pp. 771-777 ◽  
Author(s):  
A S Jun ◽  
I A Trounce ◽  
M D Brown ◽  
J M Shoffner ◽  
D C Wallace
Keyword(s):  
Complex I ◽  
Epstein Barr Virus ◽  
Nadh Dehydrogenase ◽  
Specific Activity ◽  
Coenzyme Q ◽  
Wild Type ◽  
Leber Hereditary Optic Neuropathy ◽  
Nucleotide Pair ◽  
Barr Virus ◽  
Epstein Barr

A heteroplasmic G-to-A transition at nucleotide pair (np) 14459 within the mitochondrial DNA (mtDNA)-encoded NADH dehydrogenase subunit 6 (ND6) gene has been identified as the cause of Leber hereditary optic neuropathy (LHON) and/or pediatric-onset dystonia in three unrelated families. This ND6 np 14459 mutation changes a moderately conserved alanine to a valine at amino acid position 72 of the ND6 protein. Enzymologic analysis of mitochondrial NADH dehydrogenase (complex I) with submitochondrial particles isolated from Epstein-Barr virus-transformed lymphoblasts revealed a 60% reduction (P < 0.005) of complex I-specific activity in patient cell lines compared with controls, with no differences in enzymatic activity for complexes II plus III, III and IV. This biochemical defect was assigned to the ND6 np 14459 mutation by using transmitochondrial cybrids in which patient Epstein-Barr virus-transformed lymphoblast cell lines were enucleated and the cytoplasts were fused to a mtDNA-deficient (p 0) lymphoblastoid recipient cell line. Cybrids harboring the np 14459 mutation exhibited a 39% reduction (p < 0.02) in complex I-specific activity relative to wild-type cybrid lines but normal activity for the other complexes. Kinetic analysis of the np 14459 mutant complex I revealed that the Vmax of the enzyme was reduced while the Km remained the same as that of wild type. Furthermore, specific activity was inhibited by increasing concentrations of the reduced coenzyme Q analog decylubiquinol. These observations suggest that the np 14459 mutation may alter the coenzyme Q-binding site of complex I.

scholarly journals Genome-Wide Analysis of Wild-Type Epstein–Barr Virus Genomes Derived from Healthy Individuals of the 1000 Genomes Project

2014 ◽  
Vol 6 (4) ◽  
pp. 846-860 ◽  
Author(s):  
Gabriel Santpere ◽  
Fleur Darre ◽  
Soledad Blanco ◽  
Antonio Alcami ◽  
Pablo Villoslada ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Healthy Individuals ◽  
Wild Type ◽  
1000 Genomes Project ◽  
Genome Wide Analysis ◽  
Barr Virus ◽  
1000 Genomes ◽  
Genome Wide ◽  
Epstein Barr ◽  
Virus Genomes

scholarly journals The Epstein-Barr Virus BZLF1 Protein Inhibits Tumor Necrosis Factor Receptor 1 Expression through Effects on Cellular C/EBP Proteins

2010 ◽  
Vol 84 (23) ◽  
pp. 12362-12374 ◽  
Author(s):  
Jillian A. Bristol ◽  
Amanda R. Robinson ◽  
Elizabeth A. Barlow ◽  
Shannon C. Kenney
Keyword(s):  
Tumor Necrosis Factor ◽  
Tumor Necrosis ◽  
Epstein Barr Virus ◽  
Cytokine Signaling ◽  
Wild Type ◽  
Barr Virus ◽  
Early Protein ◽  
Epstein Barr ◽  
Necrosis Factor

ABSTRACT The Epstein-Barr virus immediate-early protein, BZLF1 (Z), initiates the switch between latent and lytic infection and plays an essential role in mediating viral replication. Z also inhibits expression of the major receptor for tumor necrosis factor (TNF), TNFR1, thus repressing TNF cytokine signaling, but the mechanism for this effect is unknown. Here, we demonstrate that Z prevents both C/EBPα- and C/EBPβ-mediated activation of the TNFR1 promoter (TNFR1p) by interacting directly with both C/EBP family members. We show that Z interacts directly with C/EBPα and C/EBPβ in vivo and that a Z mutant altered at alanine residue 204 in the bZIP domain is impaired for the ability to interact with both C/EBP proteins. Furthermore, we find that the Z(A204D) mutant is attenuated in the ability to inhibit the TNFR1p but mediates lytic viral reactivation and replication in vitro in 293 cells as well as wild-type Z. Although Z does not bind directly to the TNFR1p in EMSA studies, chromatin immunoprecipitation studies indicate that Z is complexed with this promoter in vivo. The Z(A204D) mutant has reduced interaction with the TNFR1p in vivo but is similar to wild-type Z in its ability to complex with the IL-8 promoter. Finally, we show that the effect of Z on C/EBPα- and C/EBPβ-mediated activation is promoter dependent. These results indicate that Z modulates the effects of C/EBPα and C/EBPβ in a promoter-specific manner and that in some cases (including that of the TNFR1p), Z inhibits C/EBPα- and C/EBPβ-mediated activation.

scholarly journals Epstein-Barr Virus Small RNAs Potentiate Tumorigenicity of Burkitt Lymphoma Cells Independently of an Effect on Apoptosis

2000 ◽  
Vol 74 (21) ◽  
pp. 10223-10228 ◽  
Author(s):  
Ingrid K. Ruf ◽  
Paul W. Rhyne ◽  
Chunying Yang ◽  
John L. Cleveland ◽  
Jeffery T. Sample
Keyword(s):  
Cell Survival ◽  
Cell Line ◽  
Burkitt Lymphoma ◽  
Small Rnas ◽  
Epstein Barr Virus ◽  
Wild Type ◽  
Barr Virus ◽  
Tumorigenic Potential ◽  
Epstein Barr ◽  
Dependent Mechanism

ABSTRACT The tumorigenic potential of the Burkitt lymphoma (BL) cell line Akata is dependent on the restricted latency program of Epstein-Barr virus (EBV) that is characteristically maintained in BL tumors. Within these cells, EBV-mediated inhibition of apoptosis correlates with an up-regulation of BCL-2 levels in concert with a down-regulation in c-MYC expression that occurs under growth-limiting conditions. Here we addressed whether EBV's effects on apoptosis and tumorigenicity are mediated by the EBV small RNAs EBER-1 and EBER-2. Stable expression of the EBERs in EBV-negative Akata BL cells, at levels comparable to those in EBV-positive cells, significantly enhanced the tumorigenic potential of EBV-negative BL cells in SCID mice, but did not fully restore tumorigenicity relative to EBV-positive Akata cells. Furthermore, wild-type or greater levels of EBER expression in EBV-negative Akata cells did not promote BL cell survival. These data therefore suggest that EBV can contribute to BL through at least two avenues: an EBER-dependent mechanism that enhances tumorigenic potential independent of a direct effect on apoptosis, and a second mechanism, mediated by an as-yet-unidentified EBV gene(s), that offsets the proapoptotic consequences of deregulated c-MYC in BL.

scholarly journals Notch1IC Partially Replaces EBNA2 Function in B Cells Immortalized by Epstein-Barr Virus

2001 ◽  
Vol 75 (13) ◽  
pp. 5899-5912 ◽  
Author(s):  
Alexey V. Gordadze ◽  
RongSheng Peng ◽  
Jie Tan ◽  
GuoZhen Liu ◽  
Richard Sutton ◽  
...  
Keyword(s):  
B Cells ◽  
Signaling Pathways ◽  
Epstein Barr Virus ◽  
Cellular Proliferation ◽  
Wild Type ◽  
Type Iii ◽  
Barr Virus ◽  
Viral Genes ◽  
Estrogen Withdrawal ◽  
Epstein Barr

ABSTRACT Immortalization of B cells by Epstein-Barr virus (EBV) depends on the virally encoded EBNA2 protein. Although not related by sequence, the cellular Notch protein and EBNA2 share several biochemical and functional properties, such as interaction with CBF1 and the ability to activate transcription of a number of cellular and viral genes. Whether these similarities are coincidental or exemplify EBNA2 mimicry of evolutionarily conserved cellular signaling pathways is unclear. We therefore investigated whether activated forms of Notch could substitute for EBNA2 in maintaining the immortalized phenotype of EBV-infected B cells. To address this question, we devised a transcomplementation system using EREB2.5 cells. EREB2.5 cells are immortalized by EBV expressing a conditional estrogen receptor EBNA2 fusion protein (EREBNA2), and cellular proliferation is dependent on the availability of estrogen. Withdrawal of estrogen results in inactivation of EREBNA2, leading to growth arrest and eventually to cell death. Transduction of EREB2.5 cells with a lentiviral vector expressing wild-type EBNA2 rescued EREB2.5 cells from the growth-inhibitory effects of estrogen deprivation, in contrast to transduction with the lentivirus vector alone. EREB2.5 cells were also rescued by enforced expression of human Notch1IC after estrogen starvation, but this effect was restricted to cells expressing high levels of the transcription factor. Compared to wild-type EBNA2-expressing EREB2.5 cells, the Notch-expressing cells expanded more slowly after estrogen starvation, and once established, they continued to display a lower proliferation rate. Analysis of viral and cellular gene expression from transduced EREB2.5 cells after estrogen withdrawal indicated that both wild-type EBNA2- and Notch1IC-positive cells expressed c-Myc at levels similar to those found in parental EREB2.5 cells. However, the latter cells expressed LMP-1 far less efficiently than cells transduced with the wild-type EBNA2 gene. Cells rescued by either wild-type EBNA2 or Notch1IC expressed surface CD21 and CD23 proteins, but not CD10, indicating that induction of relevant type III latency markers was maintained. The data imply that both Notch and EBNA2 activate an important subset of cellular genes associated with type III latency and B-cell growth, while EBNA2 more efficiently induces important viral genes, such as LMP-1. Thus, exploitation of conserved Notch-related signaling pathways may represent a key mechanism by which EBNA2 contributes to EBV-induced cell immortalization.

scholarly journals MDM2-Dependent Inhibition of p53 Is Required for Epstein-Barr Virus B-Cell Growth Transformation and Infected-Cell Survival

2009 ◽  
Vol 83 (6) ◽  
pp. 2491-2499 ◽  
Author(s):  
Eleonora Forte ◽  
Micah A. Luftig
Keyword(s):  
B Cells ◽  
Epstein Barr Virus ◽  
Target Genes ◽  
Wild Type ◽  
P53 Pathway ◽  
Protein Levels ◽  
Barr Virus ◽  
Growth Transformation ◽  
Wild Type P53 ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) growth transformation of primary B lymphocytes into indefinitely proliferating lymphoblastoid cell lines (LCLs) depends on the concerted activities of a subset of viral proteins expressed during latency. EBV drives quiescent B cells into S phase, and consequently, a host response is activated that includes expression of p53 and its target genes. Since LCLs retain wild-type p53, it was of interest to determine what contribution the p53 pathway may have in controlling established LCL growth and EBV-mediated transformation of primary B cells. We found that liberation of p53 through chemical antagonism of one of its major ubiquitin ligases, MDM2, using the small-molecule Nutlin-3 led to apoptosis of established LCLs and suppressed EBV-mediated transformation of primary B cells. The activation of latent p53 induced target genes associated with apoptosis. Furthermore, MDM2 antagonism synergized with NF-κB inhibition in killing LCLs. NF-κB was important to increase steady-state MDM2 protein levels rather than in affecting p53-dependent transcription, suggesting a unique mechanism by which LCLs survive in the presence of a primed p53 pathway. Nutlin sensitivity of EBV-infected cells provides a novel system for studying the pathways that dictate LCL survival and regulate EBV transformation. Finally, MDM2 antagonists may be considered for therapeutic intervention in EBV-associated malignancies expressing wild-type p53.

scholarly journals Epstein–Barr virus lacking latent membrane protein 2 immortalizes B cells with efficiency indistinguishable from that of wild-type virus

1999 ◽  
Vol 80 (8) ◽  
pp. 2193-2203 ◽  
Author(s):  
Peter Speck ◽  
Kimberly A. Kline ◽  
Paul Cheresh ◽  
Richard Longnecker
Keyword(s):  
B Cells ◽  
Membrane Protein ◽  
Epstein Barr Virus ◽  
Culture Medium ◽  
Cell Transformation ◽  
Wild Type Virus ◽  
Wild Type ◽  
Barr Virus ◽  
Epstein Barr ◽  
Latent Membrane Protein 2

Epstein–Barr virus (EBV) is a human herpesvirus that efficiently transforms and immortalizes human primary B lymphocytes. In this study, the role of latent membrane protein 2 (LMP2) in EBV growth transformation was investigated. LMP2 is a virally encoded membrane protein expressed in EBV-immortalized B cells previously shown to be nonessential for EBV transformation. However, a recent study reported that LMP2 may be an important determinant for efficient B cell transformation (Brielmeier et al., Journal of General Virology 77, 2807–2818, 1996). In this study a deletion mutation was introduced into the LMP2 gene using an E. coli mini-EBV construct containing sufficient EBV DNA to result in growth transformation of primary B cells. In an alternative approach, the introduction of the gene encoding the enhanced green fluorescent protein (EGFP) by homologous recombination into the LMP2 gene of EBV strain B95-8, generating the same LMP2 deletion mutation is reported. Careful quantification of B cell transformation using the EGFP+LMP2− recombinant virus determined that in liquid culture medium or in culture medium containing soft agarose there was no difference in the ability of LMP2− virus to immortalize primary human B cells when compared to that of wild-type virus.

scholarly journals Molecular Basis of the Interaction between Complement Receptor Type 2 (CR2/CD21) and Epstein-Barr Virus Glycoprotein gp350

2008 ◽  
Vol 82 (22) ◽  
pp. 11217-11227 ◽  
Author(s):  
Kendra A. Young ◽  
Andrew P. Herbert ◽  
Paul N. Barlow ◽  
V. Michael Holers ◽  
Jonathan P. Hannan
Keyword(s):  
Epstein Barr Virus ◽  
Receptor Type ◽  
Complement Receptor ◽  
Wild Type ◽  
Virus Glycoprotein ◽  
Complement Receptor Type ◽  
Barr Virus ◽  
Epstein Barr ◽  
Mutant Forms

ABSTRACT The binding of the Epstein-Barr virus glycoprotein gp350 by complement receptor type 2 (CR2) is critical for viral attachment to B lymphocytes. We set out to test hypotheses regarding the molecular nature of this interaction by developing an enzyme-linked immunosorbent assay (ELISA) for the efficient analysis of the gp350-CR2 interaction by utilizing wild-type and mutant forms of recombinant gp350 and also of the CR2 N-terminal domains SCR1 and SCR2 (designated CR2 SCR1-2). To delineate the CR2-binding site on gp350, we generated 17 gp350 single-site substitutions targeting an area of gp350 that has been broadly implicated in the binding of both CR2 and the major inhibitory anti-gp350 monoclonal antibody (MAb) 72A1. These site-directed mutations identified a novel negatively charged CR2-binding surface described by residues Glu-21, Asp-22, Glu-155, Asp-208, Glu-210, and Asp-296. We also identified gp350 amino acid residues involved in non-charge-dependent interactions with CR2, including Tyr-151, Ile-160, and Trp-162. These data were supported by experiments in which phycoerythrin-conjugated wild-type and mutant forms of gp350 were incubated with CR2-expressing K562 cells and binding was assessed by flow cytometry. The ELISA was further utilized to identify several positively charged residues (Arg-13, Arg-28, Arg-36, Lys-41, Lys-57, Lys-67, Arg-83, and Arg-89) within SCR1-2 of CR2 that are involved in the binding interaction with gp350. These experiments allowed a comparison of those CR2 residues that are important for binding gp350 to those that define the epitope for an effective inhibitory anti-CR2 MAb, 171 (Asn-11, Arg-13, Ser-32, Thr-34, Arg-36, and Tyr-64). The mutagenesis data were used to calculate a model of the CR2-gp350 complex using the soft-docking program HADDOCK.

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