Affinity Purification–Mass Spectroscopy Methods for Identifying Epstein–Barr Virus–Host Interactions

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.

Download Full-text

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

Journal of Virology ◽

10.1128/jvi.01637-08 ◽

2008 ◽

Vol 83 (3) ◽

pp. 1393-1401 ◽

Cited By ~ 29

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.

Download Full-text

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

Journal of General Virology ◽

10.1099/vir.0.038752-0 ◽

2012 ◽

Vol 93 (5) ◽

pp. 1065-1075 ◽

Cited By ~ 15

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.

Download Full-text

Counteracting Effects of Cellular Notch and Epstein-Barr Virus EBNA2: Implications for Stromal Effects on Virus-Host Interactions

Journal of Virology ◽

10.1128/jvi.01431-14 ◽

2014 ◽

Vol 88 (20) ◽

pp. 12065-12076 ◽

Cited By ~ 22

Author(s):

M. Rowe ◽

S. Raithatha ◽

C. Shannon-Lowe

Keyword(s):

Epstein Barr Virus ◽

Host Interactions ◽

Barr Virus ◽

Epstein Barr

Download Full-text

Epstein-Barr virus and gastric carcinoma: virus-host interactions leading to carcinoma

Cancer Science ◽

10.1111/j.1349-7006.2008.00888.x ◽

2008 ◽

Vol 99 (9) ◽

pp. 1726-1733 ◽

Cited By ~ 101

Author(s):

Masashi Fukayama ◽

Rumi Hino ◽

Hiroshi Uozaki

Keyword(s):

Gastric Carcinoma ◽

Epstein Barr Virus ◽

Host Interactions ◽

Barr Virus ◽

Epstein Barr

Download Full-text

Telomere Repeat Binding Factors TRF1, TRF2, and hRAP1 Modulate Replication of Epstein-Barr Virus OriP

Journal of Virology ◽

10.1128/jvi.77.22.11992-12001.2003 ◽

2003 ◽

Vol 77 (22) ◽

pp. 11992-12001 ◽

Cited By ~ 50

Author(s):

Zhong Deng ◽

Constandache Atanasiu ◽

John S. Burg ◽

Dominique Broccoli ◽

Paul M. Lieberman

Keyword(s):

Cell Cycle ◽

Dna Replication ◽

Epstein Barr Virus ◽

Affinity Purification ◽

Dominant Negative ◽

Full Length ◽

Barr Virus ◽

Amino Terminal ◽

Epstein Barr ◽

Dyad Symmetry

ABSTRACT Epstein-Barr virus OriP confers cell cycle-dependent DNA replication and stable maintenance on plasmids in EBNA1-positive cells. The dyad symmetry region of OriP contains four EBNA1 binding sites that are punctuated by 9-bp repeats referred to as nonamers. Previous work has shown that the nonamers bind to cellular factors associated with human telomeres and contribute to episomal maintenance of OriP. In this work, we show that substitution mutation of all three nonamer sites reduces both DNA replication and plasmid maintenance of OriP-containing plasmids by 2.5- to 5-fold. The nonamers were required for high-affinity binding of TRF1, TRF2, and hRap1 to the dyad symmetry element but were not essential for the binding of EBNA1 as determined by DNA affinity purification from nuclear extracts. Chromatin immunoprecipitation assays indicated that TRF1, TRF2, and hRap1 bound OriP in vivo. Cell cycle studies indicate that TRF2 binding to OriP peaks in G1/S while TRF1 binding peaks in G2/M. OriP replication was inhibited by transfection of full-length TRF1 but not by deletion mutants lacking the myb DNA binding domain. In contrast, OriP replication was not affected by transfection of full-length TRF2 or hRap1 but was potently inhibited by dominant-negative TRF2 or hRap1 amino-terminal truncation mutants. Knockdown experiments with short interfering RNAs (siRNAs) directed against TRF2 and hRap1 severely reduced OriP replication, while TRF1 siRNA had a modest stimulatory effect on OriP replication. These results indicate that TRF2 and hRap1 promote, while TRF1 antagonizes, OriP-dependent DNA replication and suggest that these telomeric factors contribute to the establishment of replication competence at OriP.

Download Full-text

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

PLoS Pathogens ◽

10.1371/journal.ppat.1010235 ◽

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.

Download Full-text

A Prospective Clinical Study of Epstein‐Barr Virus and Host Interactions during Acute Infectious Mononucleosis

The Journal of Infectious Diseases ◽

10.1086/491740 ◽

2005 ◽

Vol 192 (9) ◽

pp. 1505-1512 ◽

Cited By ~ 120

Author(s):

Henry H. Balfour, Jr. ◽

Carol J. Holman ◽

Kristin M. Hokanson ◽

Meghan M. Lelonek ◽

Jill E. Giesbrecht ◽

...

Keyword(s):

Clinical Study ◽

Infectious Mononucleosis ◽

Epstein Barr Virus ◽

Prospective Clinical Study ◽

Host Interactions ◽

Barr Virus ◽

Epstein Barr ◽

Acute Infectious Mononucleosis

Download Full-text

Virus–host interactions in carcinogenesis of Epstein-Barr virus-associated gastric carcinoma: Potential roles of lost ARID1A expression in its early stage

PLoS ONE ◽

10.1371/journal.pone.0256440 ◽

2021 ◽

Vol 16 (9) ◽

pp. e0256440

Author(s):

Hiroyuki Abe ◽

Akiko Kunita ◽

Yuya Otake ◽

Teru Kanda ◽

Atsushi Kaneda ◽

...

Keyword(s):

Gastric Mucosa ◽

Epithelial Cells ◽

Gastric Carcinoma ◽

Epstein Barr Virus ◽

Cancer Tissue ◽

Host Interactions ◽

Barr Virus ◽

Epstein Barr

Epstein–Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is a distinct molecular subtype of gastric cancer characterized by viral infection and cellular abnormalities, including loss of AT-rich interaction domain 1A (ARID1A) expression (lost ARID1A). To evaluate the significance of lost ARID1A in the development of EBVaGC, we performed in situ hybridization of EBV-encoded RNA (EBER) and immunohistochemistry of ARID1A in the non-neoplastic gastric mucosa and intramucosal cancer tissue of EBVaGC with in vitro infection analysis of ARID1A-knockdown and -knockout gastric cells. Screening of EBER by in situ hybridization revealed a frequency of approximately 0.2% EBER-positive epithelial cells in non-neoplastic gastric mucosa tissue samples. Six small foci of EBV-infected epithelial cells showed two types of histology: degenerated (n = 3) and metaplastic (n = 3) epithelial cells. ARID1A was lost in the former type. In intramucosal EBVaGC, there were ARID1A-lost (n = 5) and -preserved tumors (n = 7), suggesting that ARID1A-lost carcinomas are derived from ARID1A-lost precursor cells in the non-neoplastic mucosa. Lost ARID1A was also observed in non-neoplastic mucosa adjacent to an ARID1A-lost EBVaGC. In vitro experiments using siRNA knockdown and the CRISPR/Cas9-knockout system demonstrated that transient reduction or permanent loss of ARID1A expression markedly increased the efficiency of EBV infection to stomach epithelial cells. Taken together, lost ARID1A plays a role in initiating EBV-driven carcinogenesis in stomach epithelial cells, which develop to a distinct subtype of EBVaGC within the proper mucosal layer. Lost ARID1A is one of the constituents of virus–host interactions in the carcinogenesis of EBVaGC.

Download Full-text