scholarly journals Epstein-Barr Virus BGLF2 commandeers RISC to interfere with cellular miRNA function

2022 ◽  
Vol 18 (1) ◽  
pp. e1010235
Author(s):  
Ashley M. Campbell ◽  
Carlos F. De La Cruz Herrera ◽  
Edyta Marcon ◽  
Jack Greenblatt ◽  
Lori Frappier
Keyword(s):  
Epstein Barr Virus ◽  
Affinity Purification ◽  
Cell Types ◽  
Cellular Level ◽  
Processing Bodies ◽  
Barr Virus ◽  
Cytoplasmic Bodies ◽  
Cellular Environment ◽  
Multiple Cell ◽  
Epstein Barr

The Epstein-Barr virus (EBV) BGLF2 protein is a tegument protein with multiple effects on the cellular environment, including induction of SUMOylation of cellular proteins. Using affinity-purification coupled to mass-spectrometry, we identified the miRNA-Induced Silencing Complex (RISC), essential for miRNA function, as a top interactor of BGLF2. We confirmed BGLF2 interaction with the Ago2 and TNRC6 components of RISC in multiple cell lines and their co-localization in cytoplasmic bodies that also contain the stress granule marker G3BP1. In addition, BGLF2 expression led to the loss of processing bodies in multiple cell types, suggesting disruption of RISC function in mRNA regulation. Consistent with this observation, BGLF2 disrupted Ago2 association with multiple miRNAs. Using let-7 miRNAs as a model, we tested the hypothesis that BGLF2 interfered with the function of RISC in miRNA-mediated mRNA silencing. Using multiple reporter constructs with 3’UTRs containing let-7a regulated sites, we showed that BGLF2 inhibited let-7a miRNA activity dependent on these 3’UTRs, including those from SUMO transcripts which are known to be regulated by let-7 miRNAs. In keeping with these results, we showed that BGLF2 increased the cellular level of unconjugated SUMO proteins without affecting the level of SUMO transcripts. Such an increase in free SUMO is known to drive SUMOylation and would account for the effect of BGLF2 in inducing SUMOylation. We further showed that BGLF2 expression inhibited the loading of let-7 miRNAs into Ago2 proteins, and conversely, that lytic infection with EBV lacking BGLF2 resulted in increased interaction of let-7a and SUMO transcripts with Ago2, relative to WT EBV infection. Therefore, we have identified a novel role for BGLF2 as a miRNA regulator and shown that one outcome of this activity is the dysregulation of SUMO transcripts that leads to increased levels of free SUMO proteins and SUMOylation.

DNA Affinity Purification of Epstein-Barr Virus OriP-Binding Proteins

DNA Viruses ◽  
2004 ◽  
pp. 267-276
Author(s):  
Constandache Atanasiu ◽  
Larissa Lezina ◽  
Paul M. Lieberman
Keyword(s):  
Epstein Barr Virus ◽  
Binding Proteins ◽  
Affinity Purification ◽  
Barr Virus ◽  
Epstein Barr

scholarly journals Determination of Epstein-Barr Virus–Infected Lymphocyte Cell Types in Peripheral Blood Mononuclear Cells as a Valuable Diagnostic Tool in Hematological Diseases

2019 ◽  
Vol 6 (5) ◽  
Author(s):  
Peiling Zhang ◽  
Chen Zeng ◽  
Jiali Cheng ◽  
Jing Zhou ◽  
Jia Gu ◽  
...  
Keyword(s):  
Infected Cell ◽  
B Cell ◽  
Peripheral Blood Mononuclear Cells ◽  
Epstein Barr Virus ◽  
Nk Cell ◽  
Cell Types ◽  
Cell Type ◽  
Peripheral Blood Mononuclear ◽  
Barr Virus ◽  
Epstein Barr

Abstract Background High loads of Epstein-Barr virus (EBV) in peripheral blood mononuclear cells (PBMCs) can be indicative of a broad spectrum of diseases, ranging from asymptomatic infection to fatal cancers. Methods We retrospectively investigated the EBV-infected cell types in PBMCs among 291 patients. Based on EBV-infected cell types, the clinical features and prognoses of 93 patients with EBV-associated (EBV+) T/natural killer (NK)–cell lymphoproliferative diseases (LPDs) T/NK-LPDs) were investigated over a 5-year period. Results Although B-cell-type infection was found in immunocompromised patients and patients with asymptomatic high EBV carriage, infectious mononucleosis, EBV+ B-cell LPDs and B-cell lymphomas, T-cell, NK-cell or multiple-cell-type infection in immunocompetent hosts were highly suggestive of EBV+ T/NK-LPDs, EBV+ T/NK-cell lymphomas, and aggressive NK-cell leukemia. Patients with non–B-cell infection had a poorer prognosis than those with B-cell-type infection. In our cohort, 79.6% of patients with EBV+ T/NK-LPDs were >18 years old, and NK cells were identified as EBV-infected cell type in 54.8%. Nearly half of patients with EBV+ T/NK-LPDs had genetic defects associated with immunodeficiency. However, hemophagocytic lymphohistiocytosis, and not genetic defects, was the only parameter correlated with poor prognosis of EBV+ T/NK-LPDs. Conclusions Determination of EBV-infected cell types among PBMCs is a valuable tool for the differential diagnosis of EBV+ hematological diseases. In this study, determination of Epstein-Barr virus-infected cell types in peripheral blood mononuclear cells of 291 patients with high Epstein-Barr virus loads were retrospectively investigated, which indicate it is a valuable tool for Epstein-Barr virus-associated hematological diseases.

scholarly journals The BRRF1 Early Gene of Epstein-Barr Virus Encodes a Transcription Factor That Enhances Induction of Lytic Infection by BRLF1

2004 ◽  
Vol 78 (10) ◽  
pp. 4983-4992 ◽  
Author(s):  
Gregory K. Hong ◽  
Henri-Jacques Delecluse ◽  
Henri Gruffat ◽  
Thomas E. Morrison ◽  
Wen-Hai Feng ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Epstein Barr Virus ◽  
Cell Types ◽  
Early Gene ◽  
Barr Virus ◽  
Ebv Infection ◽  
Early Protein ◽  
293 Cells ◽  
Latently Infected ◽  
Epstein Barr

ABSTRACT The switch from the latent to the lytic form of Epstein-Barr virus (EBV) infection is mediated by expression of the viral immediate-early (IE) proteins, BZLF1 (Z) and BRLF1 (R). An EBV early protein, BRRF1 (Na), is encoded by the opposite strand of the BRLF1 intron, but the function of this nuclear protein in the viral life cycle is unknown. Here we demonstrate that Na enhances the R-mediated induction of lytic EBV infection in 293 cells latently infected with a recombinant EBV (R-KO) defective for the expression of both R and Na. Na also enhances R-induced lytic infections in a gastric carcinoma line (AGS) carrying the R-KO virus, although it has no effect in a Burkitt lymphoma line (BL-30) stably infected with the same mutant virus. We show that Na is a transcription factor that increases the ability of R to activate Z expression from the R-KO viral genome in 293 cells and that Na by itself activates the Z promoter (Zp) in EBV-negative cells. Na activation of Zp requires a CRE motif (ZII), and a consensus CRE motif is sufficient to transfer Na responsiveness to the heterologous E1b promoter. Furthermore, we show that Na enhances the transactivator function of a Gal4-c-Jun fusion protein but does not increase the transactivator function of other transcription factors (including ATF-1, ATF-2, and CREB) known to bind CRE motifs. Na expression in cells results in increased levels of a hyperphosphorylated form of c-Jun, suggesting a mechanism by which Na activates c-Jun. Our results indicate that Na is a transcription factor that activates the EBV Zp IE promoter through its effects on c-Jun and suggest that Na cooperates with BRLF1 to induce the lytic form of EBV infection in certain cell types.

scholarly journals Cell Cycle Association of the Retinoblastoma Protein Rb and the Histone Demethylase LSD1 with the Epstein-Barr Virus Latency Promoter Cp

2008 ◽  
Vol 82 (7) ◽  
pp. 3428-3437 ◽  
Author(s):  
Charles M. Chau ◽  
Zhong Deng ◽  
Hyojueng Kang ◽  
Paul M. Lieberman
Keyword(s):  
Cell Cycle ◽  
Epstein Barr Virus ◽  
Affinity Purification ◽  
Histone H3 ◽  
Histone Demethylase ◽  
Stable Complex ◽  
Dependent Manner ◽  
Barr Virus ◽  
A Cell ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus C promoter (Cp) regulates the major multicistronic transcript encoding the EBNA-LP, 1, 2, and 3 genes required for B-cell proliferation during latency. The growth-transforming potential of these viral genes suggests that they must be tightly regulated with the host cell cycle and differentiation process. To better understand Cp regulation, we used DNA affinity purification to identify cellular and viral proteins that bind to Cp in latently infected cells. Several previously unknown factors were identified, including the cell cycle regulatory proteins E2F1 and Rb. E2F1 bound to a specific site in Cp located in the core Cp region 3′ of the known EBNA2-responsive RBP-Jk (CSL, CBF1) binding site. The histone H3 K4 demethylase LSD1 (BCC110) was also identified by DNA affinity and was shown to form a stable complex with Rb. Coimmunoprecipitation assays demonstrated that E2F1, Rb, and LSD1 bind to Cp in a cell cycle-dependent manner. Rb and LSD1 binding to Cp increased after the S phase, corresponding to a decrease in histone H3 K4 methylation and Cp transcription. Coimmunoprecipitation and immunofluorescence assays reveal that LSD1 interacts with Rb. Surprisingly, LSD1 did not coimmunoprecipitate with E2F1, suggesting that it associates with Rb independently of E2F1. Depletion of LSD1 by small interfering RNAs inhibited Cp basal transcription levels, and overexpression of LSD1 altered the cell cycle profile in p53-positive (p53+), but not p53-negative (p53−), HCT cells. These findings indicate that Cp is a cell cycle-regulated promoter that is under the control of Rb and the histone demethylase LSD1 in multiple latency types.

scholarly journals A potent and protective human neutralizing antibody targeting a novel vulnerable site of Epstein-Barr virus

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Qian-Ying Zhu ◽  
Sisi Shan ◽  
Jinfang Yu ◽  
Si-Ying Peng ◽  
Cong Sun ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Infected Individual ◽  
Neutralizing Antibody ◽  
Cell Types ◽  
Crystal Structure Analysis ◽  
Target Cells ◽  
High Dose ◽  
Barr Virus ◽  
Ebv Infection ◽  
Epstein Barr

AbstractEpstein-Barr virus (EBV) is associated with a range of epithelial and B cell malignancies as well as autoimmune disorders, for which there are still no specific treatments or effective vaccines. Here, we isolate EBV gH/gL-specific antibodies from an EBV-infected individual. One antibody, 1D8, efficiently neutralizes EBV infection of two major target cell types, B cells and epithelial cells. In humanized mice, 1D8 provides protection against a high-dose EBV challenge by substantially reducing viral loads and associated tumor burden. Crystal structure analysis reveals that 1D8 binds to a key vulnerable interface between the D-I/D-II domains of the viral gH/gL protein, especially the D-II of the gH, thereby interfering with the gH/gL-mediated membrane fusion and binding to target cells. Overall, we identify a potent and protective neutralizing antibody capable of reducing the EBV load. The novel vulnerable site represents an attractive target that is potentially important for antibody and vaccine intervention against EBV infection.

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 Lytic Replication Elicits ATM Checkpoint Signal Transduction While Providing an S-phase-like Cellular Environment

2004 ◽  
Vol 280 (9) ◽  
pp. 8156-8163 ◽  
Author(s):  
Ayumi Kudoh ◽  
Masatoshi Fujita ◽  
Lumin Zhang ◽  
Noriko Shirata ◽  
Tohru Daikoku ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Epstein Barr Virus ◽  
S Phase ◽  
Lytic Replication ◽  
Barr Virus ◽  
Cellular Environment ◽  
Epstein Barr ◽  
Checkpoint Signal

scholarly journals E2F1, ARID3A/Bright and Oct-2 factors bind to the Epstein–Barr virus C promoter, EBNA1 and oriP, participating in long-distance promoter–enhancer interactions

2012 ◽  
Vol 93 (5) ◽  
pp. 1065-1075 ◽  
Author(s):  
Cecilia Boreström ◽  
Alma Forsman ◽  
Ulla Rüetschi ◽  
Lars Rymo
Keyword(s):  
B Cell ◽  
Protein Complex ◽  
Epstein Barr Virus ◽  
Affinity Purification ◽  
Regulatory Protein ◽  
Immunoblot Analysis ◽  
Nuclear Antigen ◽  
Long Distance ◽  
Barr Virus ◽  
Epstein Barr

The Epstein–Barr virus (EBV) C promoter (Cp) regulates several genes required for B-cell proliferation in latent EBV infection. The family of repeats (FR) region of the latent origin of plasmid replication (oriP) functions as an Epstein–Barr nuclear antigen 1 (EBNA1)-dependent distant enhancer of Cp activity, and the enhancer–promoter interaction is mediated by a higher-order multi-protein complex containing several copies of EBNA1. Using DNA-affinity purification with a 170 bp region of the Cp in combination with mass spectrometry, we identified the cell cycle-regulatory protein E2F1, the E2F-binding protein ARID3A, and the B-cell-specific transcription factor Oct-2 as components of this multi-protein complex. Binding of the three factors to the FR region of oriP was determined by DNA-affinity and immunoblot analysis. Co-immunoprecipitation and proximity ligation analysis revealed that the three factors, E2F1, ARID3A and Oct-2, interact with each other as well as with EBNA1 in the nuclei of EBV-positive cells. Using the chromatin immunoprecipitation assay, we showed that E2F1 and Oct-2 interacted with the FR part of oriP and the Cp, but the ARID3A interaction was, however, only detected at the Cp. Our findings support the hypothesis that EBNA1 initiates transcription at the Cp via interactions between multiple EBNA1 homodimers and cellular transcription factors in a large molecular machinery that forms a dynamic interaction between Cp and FR.

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