scholarly journals Low NADPH oxidase activity in Epstein-Barr-virus-immortalized B-lymphocytes is due to a post-transcriptional block in expression of cytochrome b558

1995 ◽  
Vol 306 (1) ◽  
pp. 141-145 ◽  
Author(s):  
M Chetty ◽  
A J Thrasher ◽  
A Abo ◽  
C M Casimir
Keyword(s):  
Nadph Oxidase ◽  
B Cell ◽  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
Hl60 Cells ◽  
Cytochrome B558 ◽  
Barr Virus ◽  
Generating Capacity ◽  
Cell Cytosol ◽  
Epstein Barr

The NADPH oxidase of phagocytes is known to be expressed in Epstein-Barr-virus-transformed B-lymphocytes, albeit at levels only approx. 5% of those found in neutrophils. We have investigated the basis of this low level of expression and find that all four specific components of the NADPH oxidase are expressed in B-lymphocytes, but only p47-phox protein attains levels equivalent with those found in neutrophils. This component was shown to phosphorylate and translocate to the membrane normally on activation. The other cytosolic component, p67-phox, did show a deficit, and by supplementing a B-cell cytosol extract with recombinant p67-phox, this was shown to account for the somewhat reduced activity of B-cell cytosol in a cell-free oxidase system. The cell-free analysis also clearly located the major deficiency in superoxide-generating capacity of B-lymphocytes to the membrane. Western blotting of membrane proteins revealed major reductions in the amount of cytochrome b558. Analysis of the levels of mRNA for both subunits of cytochrome b558, however, showed levels greater than expected. Significantly more mRNA for gp91-phox was present in B-cells than in undifferentiated HL60 cells, although it was not quite as abundant as in differentiated HL60 cells, which are capable of producing large amounts of superoxide. We conclude that the failure of B-lymphocytes to generate amounts of superoxide equivalent to those generated by neutrophils is primarily due to a post-transcriptionally determined block to the accumulation of cytochrome b558.

scholarly journals Epstein-Barr virus transformed B lymphocytes produce interleukin-5

Blood ◽  
1990 ◽  
Vol 75 (7) ◽  
pp. 1400-1403 ◽  
Author(s):  
CC Paul ◽  
JR Keller ◽  
JM Armpriester ◽  
MA Baumann
Keyword(s):  
B Cell ◽  
B Lymphocytes ◽  
Messenger Rna ◽  
Epstein Barr Virus ◽  
Northern Analysis ◽  
Interleukin 5 ◽  
Barr Virus ◽  
Epstein Barr ◽  
Semi Solid ◽  
Culture Supernatants

Abstract Interleukin-5 (IL-5) has previously been isolated only as a product of T lymphocytes. We have found that Epstein-Barr virus transformed B lymphocytes produce large amounts of IL-5 activity in culture supernatants, inducing proliferation of murine BCL1 cells, and supporting the selective growth of eosinophil colonies in semi-solid culture. Production of IL-5 messenger RNA by transformed B-cell lines was verified by Northern analysis using a 3.2-kilobase cloned DNA fragment containing the full-length human IL-5 gene, and immunoreactive IL-5 was detected in B-cell culture supernatants. These findings suggest a possible expanded role for the B cell in the induction of eosinophilia, and should serve as a focus for additional investigation into possible roles for IL-5 in human B-cell proliferation and differentiation.

scholarly journals NADPH oxidase of Epstein-Barr-virus immortalized B lymphocytes.

2001 ◽  
Vol 268 (19) ◽  
pp. 5197-5208 ◽  
Author(s):  
Marie-Hélène Paclet ◽  
Anthony W. Coleman ◽  
James Burritt ◽  
Françoise Morel
Keyword(s):  
Nadph Oxidase ◽  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
Barr Virus ◽  
Epstein Barr

scholarly journals Increased association between Epstein-Barr virus EBNA2 from type 2 strains and the transcriptional repressor BS69 restricts B cell growth

2018 ◽  
Author(s):  
Rajesh Ponnusamy ◽  
Ritika Khatri ◽  
Paulo B. Correia ◽  
Erika Mancini ◽  
Paul J. Farrell ◽  
...  
Keyword(s):  
B Cell ◽  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
Natural Variation ◽  
B Lymphocyte ◽  
Barr Virus ◽  
Growth Promoting ◽  
Epstein Barr

AbstractNatural variation separates Epstein-Barr virus (EBV) into type 1 and type 2 strains. Type 2 EBV is less transformingin vitrodue to sequence differences in the EBV transcription factor EBNA2. This correlates with reduced activation of the EBV oncogene LMP1 and some cell genes. Transcriptional activation by type 1 EBNA2 can be suppressed through the binding of two PXLXP motifs in its transactivation domain (TAD) to the dimeric coiled-coil MYND domain (CC-MYND) of the BS69 repressor protein (ZMYND11). We identified a third conserved PXLXP motif in type 2 EBNA2. We found that type 2 EBNA2 peptides containing this motif bound BS69CC-MYNDefficiently and that the type 2 EBNA2TADbound an additional BS69CC-MYNDmolecule. Full-length type 2 EBNA2 also bound BS69 more efficiently in pull-down assays. Molecular weight analysis and low-resolution structures obtained using small-angle X-ray scattering showed that three BS69CC-MYNDdimers bound two molecules of type 2 EBNA2TAD, in line with the dimeric state of full-length EBNA2in vivo. Importantly, mutation of the third BS69 binding motif in type 2 EBNA2 improved B-cell growth maintenance. Our data indicate that increased association with BS69 restricts growth promotion by EBNA2 and may contribute to reduced B-cell transformation by type 2 EBV.Author summaryEpstein-Barr virus (EBV) drives the development of many human cancers worldwide including specific types of lymphoma and carcinoma. EBV infects B lymphocytes and immortalises them, thus contributing to lymphoma development. The virus promotes B lymphocyte growth and survival by altering the level at which hundreds of genes are expressed. The EBV protein EBNA2 is known to activate many growth-promoting genes. Natural variation in the sequence of EBNA2 defines the two main EBV strains: type 1 and type 2. Type 2 strains immortalise B lymphocytes less efficiency and activate some growth genes poorly, although the mechanism of this difference is unclear. We now show that sequence variation in type 2 EBNA2 creates a third site of interaction for the repressor protein (BS69, ZMYND11). We have characterised the complex formed between type 2 EBNA2 and BS69 and show that three dimers of BS69 form a bridged complex with two molecules of type 2 EBNA2. We demonstrate that mutation of the additional BS69 interaction site in type 2 EBNA2 improves its growth-promoting function. Our results therefore provide a molecular explanation for the different B lymphocyte growth promoting activities of type 1 and type 2 EBV. This aids our understanding of immortalisation by EBV.

scholarly journals First Days in the Life of Naive Human B Lymphocytes Infected with Epstein-Barr Virus

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Dagmar Pich ◽  
Paulina Mrozek-Gorska ◽  
Mickaël Bouvet ◽  
Atsuko Sugimoto ◽  
Ezgi Akidil ◽  
...  
Keyword(s):  
B Cell ◽  
B Lymphocytes ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Cell Reprogramming ◽  
Barr Virus ◽  
Infected Host Cell ◽  
Cell Divisions ◽  
Infected Cells ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) infects and activates resting human B lymphocytes, reprograms them, induces their proliferation, and establishes a latent infection in them. In established EBV-infected cell lines, many viral latent genes are expressed. Their roles in supporting the continuous proliferation of EBV-infected B cells in vitro are known, but their functions in the early, prelatent phase of infection have not been investigated systematically. In studies during the first 8 days of infection using derivatives of EBV with mutations in single genes of EBVs, we found only Epstein-Barr nuclear antigen 2 (EBNA2) to be essential for activating naive human B lymphocytes, inducing their growth in cell volume, driving them into rapid cell divisions, and preventing cell death in a subset of infected cells. EBNA-LP, latent membrane protein 2A (LMP2A), and the viral microRNAs have supportive, auxiliary functions, but mutants of LMP1, EBNA3A, EBNA3C, and the noncoding Epstein-Barr virus with small RNA (EBERs) had no discernible phenotype compared with wild-type EBV. B cells infected with a double mutant of EBNA3A and 3C had an unexpected proliferative advantage and did not regulate the DNA damage response (DDR) of the infected host cell in the prelatent phase. Even EBNA1, which has very critical long-term functions in maintaining and replicating the viral genomic DNA in established cell lines, was dispensable for the early activation of infected cells. Our findings document that the virus dose is a decisive parameter and indicate that EBNA2 governs the infected cells initially and implements a strictly controlled temporal program independent of other viral latent genes. It thus appears that EBNA2 is sufficient to control all requirements for clonal cellular expansion and to reprogram human B lymphocytes from energetically quiescent to activated cells. IMPORTANCE The preferred target of Epstein-Barr virus (EBV) is human resting B lymphocytes. We found that their infection induces a well-coordinated, time-driven program that starts with a substantial increase in cell volume, followed by cellular DNA synthesis after 3 days and subsequent rapid rounds of cell divisions on the next day accompanied by some DNA replication stress (DRS). Two to 3 days later, the cells decelerate and turn into stably proliferating lymphoblast cell lines. With the aid of 16 different recombinant EBV strains, we investigated the individual contributions of EBV’s multiple latent genes during early B-cell infection and found that many do not exert a detectable phenotype or contribute little to EBV’s prelatent phase. The exception is EBNA2 that is essential in governing all aspects of B-cell reprogramming. EBV relies on EBNA2 to turn the infected B lymphocytes into proliferating lymphoblasts preparing the infected host cell for the ensuing stable, latent phase of viral infection. In the early steps of B-cell reprogramming, viral latent genes other than EBNA2 are dispensable, but some, EBNA-LP, for example, support the viral program and presumably stabilize the infected cells once viral latency is established.

scholarly journals Epstein-Barr Virus-Induced miR-155 Attenuates NF-κB Signaling and Stabilizes Latent Virus Persistence

2008 ◽  
Vol 82 (21) ◽  
pp. 10436-10443 ◽  
Author(s):  
Fang Lu ◽  
Andreas Weidmer ◽  
Chang-Gong Liu ◽  
Stefano Volinia ◽  
Carlo M. Croce ◽  
...  
Keyword(s):  
B Cell ◽  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
Cell Development ◽  
Barr Virus ◽  
Latent Viral Infection ◽  
Latently Infected ◽  
Infected Cells ◽  
Epstein Barr ◽  
Precursor Rna

ABSTRACT MicroRNAs have been implicated in the modulation of gene expression programs important for normal and cancer cell development. miR-155 is known to play a role in B-cell development and is upregulated in various B-cell lymphomas, including several that are latently infected with Epstein-Barr virus (EBV). We show here that EBV infection of primary human B lymphocytes leads to the sustained elevation of miR-155 and its precursor RNA, BIC. The EBV-encoded latency membrane protein 1 (LMP1) can partially reconstitute BIC activation in B lymphocytes but not in epithelial cell cultures. LMP1 is a potent activator of NF-κB signaling pathways and is essential for EBV immortalization of B lymphocytes. An inhibitor to miR-155 further stimulated NF-κB responsive gene transcription, and IKKε was identified as a potential target of miR-155 translational repression. Remarkably, miR-155 inhibitor reduced EBNA1 mRNA and the EBV copy number in latently infected cells. This suggests that miR-155 contributes to EBV immortalization by modulation of NF-κB signaling and the suppression of host innate immunity to latent viral infection.

scholarly journals Proteasomal Inhibition Triggers Viral Oncoprotein Degradation via Autophagy-Lysosomal Pathway

2019 ◽  
Author(s):  
Chandrima Gain ◽  
Samaresh Malik ◽  
Shaoni Bhattacharjee ◽  
Arijit Ghosh ◽  
Erle S. Robertson ◽  
...  
Keyword(s):  
B Cell ◽  
B Lymphocytes ◽  
Epstein Barr Virus ◽  
Viral Reactivation ◽  
B Cell Lymphomas ◽  
Viral Oncoprotein ◽  
Lytic Gene ◽  
Barr Virus ◽  
Proteasomal Inhibition ◽  
Epstein Barr

AbstractEpstein-Barr virus (EBV) nuclear oncoprotein EBNA3C is essential for B-cell transformation and development of several B-cell lymphomas particularly those are generated in an immuno-compromised background. EBNA3C recruits ubiquitin-proteasome machinery for deregulating multiple cellular oncoproteins and tumor suppressor proteins. Although EBNA3C is found to be ubiquitinated at its N-terminal region and interacts with 20S proteasome, the viral protein is surprisingly stable in growing B-lymphocytes. EBNA3C can also circumvent autophagy-lysosomal mediated protein degradation and subsequent antigen presentation for T-cell recognition. Recently, we have shown that EBNA3C enhances autophagy, which serve as a prerequisite for B-cell survival particularly under growth deprivation conditions. We now demonstrate that proteasomal inhibition by MG132 induces EBNA3C degradation both in EBV transformed B-lymphocytes and ectopic-expression systems. Interestingly, MG132 treatment promotes degradation of two EBNA3 family oncoproteins – EBNA3A and EBNA3C, but not the viral tumor suppressor protein EBNA3B. EBNA3C degradation induced by proteasomal inhibition is partially blocked when autophagy-lysosomal pathway is inhibited. In response to proteasomal inhibition, EBNA3C is predominantly K63-linked polyubiquitinated, colocalized with the autophagy-lsyosomal fraction in the cytoplasm and participated within p62-LC3B complex, which facilitates autophagy-mediated degradation. We further show that the degradation signal is present at the first 50 residues of the N-terminal region of EBNA3C. Proteasomal inhibition reduces the colony formation ability of this important viral oncoprotein, increases transcriptional activation of both latent and lytic gene expression and induces viral reactivation from EBV transformed B-lymphocytes. Altogether, this study offers rationale to use proteasome inhibitors as potential therapeutic strategy against multiple EBV associated B-cell lymphomas, where EBNA3C is expressed.Author SummaryEpstein-Barr virus (EBV) establishes latent infection in B-lymphocytes and is associated with a number of human malignancies, both of epithelial and lymphoid origin. EBV encoded EBNA3 family of nuclear latent antigens comprising of EBNA3A, EBNA3B, and EBNA3C are unique to immunoblastic lymphomas. While EBNA3A and EBNA3C are involved in blocking many important tumor suppressive mechanisms, EBNA3B exhibits tumor suppressive functions. Although EBNA3 proteins, in particular EBNA3C, interact with and employ different protein degradation machineries to induce B-cell lymphomagenesis, these viral proteins are extremely stable in growing B-lymphocytes. To this end, we now demonstrate that proteasomal inhibition leads to specifically degradation of oncogenic EBNA3A and EBNA3C proteins, whereas EBNA3B remains unaffected. Upon proteasomal inhibition, EBNA3C degradation occurs via autophagy-lysosomal pathway, through labeling with K63-linked polyubiquitination and participating in p62-LC3B complex involved in ubiquitin-mediated autophagy substrate selection and degradation through autolysosomal process. We also demonstrate that the N-terminal domain is responsible for autophgy-lysosomal mediated degradation, while the C-terminal domain plays a crucial role in cytoplasmic localization. Fascinatingly, while proteasomal inhibition reduces EBNA3C’s oncogenic property, it induces both latent and lytic gene expressions and promotes viral reactivation from EBV transformed B-lymphocytes. This is the first report which demonstrates a viral oncoprotein degrades through autophagy-lysosomal pathway upon proteasomal inhibition. In sum, the results promise development of novel strategies specifically targeting proteolytic pathway for the treatment of EBV associated B-cell lymphomas, particularly those are generated in immunocompromised individuals.

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