Autorepression of Epstein-Barr Virus Nuclear Antigen 1 Expression by Inhibition of Pre-mRNA Processing

ABSTRACT Epstein-Barr virus (EBV) latent infection, and its associated oncogenic potential, is dependent on genome maintenance functions of EBV nuclear antigen 1 (EBNA-1), one of six EBNAs expressed from a common promoter (Wp and then Cp) upon infection of naive B cells. Subsequent host-mediated silencing, however, necessitates the expression of EBNA-1 from the EBNA-1-specific promoter Qp to ensure against genome loss during cell division, including EBV-associated malignancy. Here we addressed the mechanism by which EBNA-1 represses Qp through binding downstream of the transcription start site and the role of this autoregulatory function in EBV latency. Our results revealed that EBNA-1 does not inhibit transcription from Qp, as previously predicted, but acts post- or cotranscriptionally to block the processing of primary transcripts. This does not, however, require the RGG motifs responsible for strong but nonspecific RNA binding by EBNA-1. Within isogenic B-cell lines using either Cp/Wp or Qp, EBNA-1 occupancy of Qp is equivalent, suggesting that autoregulation occurs, albeit to different degrees, during full and restricted EBV latency programs. Finally, in cell lines using Cp or Wp for EBNA expression, unprocessed transcripts from Qp are detectable in the absence of corresponding mRNAs, providing further evidence that this novel mechanism of EBNA-1 action functions during latency. This posttranscriptional mechanism of regulation would provide an efficient means to monitor and regulate EBNA-1 expression from Qp, ensuring levels adequate for genome maintenance but, perhaps more importantly, below an immunogenic threshold above which latently infected cells may be at risk for elimination by EBNA-1-specific cytotoxic T cells.

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Epstein–Barr virus nuclear antigen 1 is a DNA-binding protein with strong RNA-binding activity

Journal of General Virology ◽

10.1099/vir.0.80239-0 ◽

2004 ◽

Vol 85 (10) ◽

pp. 2755-2765 ◽

Cited By ~ 19

Author(s):

Chih-Chung Lu ◽

Chia-Wei Wu ◽

Shin C. Chang ◽

Tzu-Yi Chen ◽

Chwan-Ren Hu ◽

...

Keyword(s):

Dna Binding ◽

Epstein Barr Virus ◽

Rna Binding ◽

Binding Protein ◽

Dna Binding Protein ◽

Binding Activity ◽

Nuclear Antigen ◽

Barr Virus ◽

Infected Cells ◽

Epstein Barr

Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA-1) plays key roles in both the regulation of gene expression and the replication of the EBV genome in latently infected cells. To characterize the RNA-binding activity of EBNA-1, it was demonstrated that EBNA-1 binds efficiently to RNA homopolymers that are composed of poly(G) and weakly to those composed of poly(U). All three RGG boxes of EBNA-1 contributed additively to poly(G)-binding activity and could mediate RNA binding when attached to a heterologous protein in an RNA gel mobility-shift assay. In vitro-transcribed EBV and non-EBV RNA probes revealed that EBNA-1 bound to most RNAs examined and the affinity increased as the content of G and U increased, as demonstrated in competition assays. Among these probes, the 5′ non-coding region (NCR) (nt 131–278) of hepatitis C virus RNA appeared to be the strongest competitor for EBNA-1 binding to the EBV-encoded small nuclear RNA 1 (EBER1) probe, whereas a mutant 5′ NCR RNA with partially disrupted secondary structure was a weak competitor. Furthermore, the interaction of endogenous EBNA-1 and EBER1 in EBV-infected cells was demonstrated by a ribonucleoprotein immunoprecipitation assay. These results revealed that EBNA-1 is a DNA-binding protein with strong binding activity to a relatively broad spectrum of RNA and suggested an additional biological impact of EBNA-1 through its ability to bind to RNA.

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Epstein–Barr virus enhances genome maintenance of Kaposi sarcoma-associated herpesvirus

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1810128115 ◽

2018 ◽

Vol 115 (48) ◽

pp. E11379-E11387 ◽

Cited By ~ 17

Author(s):

Rachele Bigi ◽

Justin T. Landis ◽

Hyowon An ◽

Carolina Caro-Vegas ◽

Nancy Raab-Traub ◽

...

Keyword(s):

Epstein Barr Virus ◽

Primary Effusion Lymphoma ◽

Kaposi Sarcoma ◽

B Cell Lymphoma ◽

Nuclear Antigen ◽

Genome Maintenance ◽

Barr Virus ◽

Single Positive ◽

Infected Cells ◽

Epstein Barr

Primary effusion lymphoma (PEL) is a B cell lymphoma that is always associated with Kaposi’s sarcoma-associated herpesvirus (KSHV) and in many cases also with Epstein–Barr virus (EBV); however, the requirement for EBV coinfection is not clear. Here, we demonstrate that adding exogenous EBV to KSHV+single-positive PEL leads to increased KSHV genome maintenance and KSHV latency-associated nuclear antigen (LANA) expression. To show that EBV was necessary for naturally coinfected PEL, we nucleofected KSHV+/EBV+PEL cell lines with an EBV-specific CRISPR/Cas9 plasmid to delete EBV and observed a dramatic decrease in cell viability, KSHV genome copy number, and LANA expression. This phenotype was reversed by expressing Epstein–Barr nuclear antigen 1 (EBNA-1)in trans, even though EBNA-1 and LANA do not colocalize in infected cells. This work reveals that EBV EBNA-1 plays an essential role in the pathogenesis of PEL by increasing KSHV viral load and LANA expression.

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Epstein-Barr Virus Nuclear Antigen 1 Interacts with Nm23-H1 in Lymphoblastoid Cell Lines and Inhibits Its Ability To Suppress Cell Migration

Journal of Virology ◽

10.1128/jvi.79.3.1559-1568.2005 ◽

2005 ◽

Vol 79 (3) ◽

pp. 1559-1568 ◽

Cited By ~ 53

Author(s):

Masanao Murakami ◽

Ke Lan ◽

Chitra Subramanian ◽

Erle S. Robertson

Keyword(s):

Cell Migration ◽

Cell Lines ◽

Epstein Barr Virus ◽

Critical Role ◽

Nuclear Antigen ◽

Lymphoblastoid Cell Lines ◽

Barr Virus ◽

Infected Cells ◽

Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is expressed in the majority of latency programs in EBV-infected cells and is critical for the maintenance of EBV episomes in the infected cells. EBNA1 is also known to be involved in transcriptional activation and regulates expression of the EBV latent genes, including the EBNAs and LMP1. Thus, EBNA1 is a multifunctional protein with critical functions required for the persistence of the viral genome over successive generations, producing new daughter cells from the infected cell. We identify EBNA1 here as an interacting EBNA with the known suppressor of metastasis and cell migration, Nm23-H1. Nm23-H1 inhibits cell migration when expressed in cancer cells. We show that EBNA1 associates with Nm23-H1 in EBV-infected cells in vitro, as well as in lymphoblastoid cell lines (LCLs). Nm23-H1 predominantly localizes to the cytoplasm in BJAB and 293T cells; however, upon expression of EBNA1, Nm23-H1 is translocated to the nucleus in similar compartments to EBNA1, suggesting a potential functional role that is linked to EBNA1. Convincingly, in EBV-transformed LCLs Nm23-H1 is localized predominantly to the nucleus and colocalizes to similar compartment as EBNA1. Further, we tested the effects of EBNA1 on Nm23-H1-mediated suppression of cell migration and showed that EBNA1 rescues the suppression of cell migration mediated by Nm23-H1. These in vitro studies suggest that EBNA1 plays a critical role in regulating the activities of Nm23-H1, including cell migration, through a mechanism which involves direct interaction of this major regulator in EBV-infected cells.

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Relationship between Epstein--Barr Virus Nuclear Antigen and DNA Genome Number in Superinfected and Induced Lymphoblastoid Cell Lines

Journal of General Virology ◽

10.1099/0022-1317-64-4-887 ◽

1983 ◽

Vol 64 (4) ◽

pp. 887-894 ◽

Cited By ~ 6

Author(s):

L. Boguszakova ◽

I. Hirsch ◽

B. Brichacek ◽

V. Vonka

Keyword(s):

Cell Lines ◽

Epstein Barr Virus ◽

Nuclear Antigen ◽

Lymphoblastoid Cell Lines ◽

Barr Virus ◽

Epstein Barr

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Infection of Epstein–Barr Virus in Type III Latency Modulates Biogenesis of Exosomes and the Expression Profile of Exosomal miRNAs in the Burkitt Lymphoma Mutu Cell Lines

Cancers ◽

10.3390/cancers10070237 ◽

2018 ◽

Vol 10 (7) ◽

pp. 237 ◽

Cited By ~ 13

Author(s):

Asuka Nanbo ◽

Harutaka Katano ◽

Michiyo Kataoka ◽

Shiho Hoshina ◽

Tsuyoshi Sekizuka ◽

...

Keyword(s):

Epithelial Cells ◽

Burkitt Lymphoma ◽

Epstein Barr Virus ◽

Type I ◽

Type Iii ◽

Barr Virus ◽

Ebv Infection ◽

Latently Infected ◽

Infected Cells ◽

Epstein Barr

Infection of Epstein–Barr virus (EBV), a ubiquitous human gamma herpesvirus, is associated with various malignancies in B lymphocytes and epithelial cells. EBV encodes 49 microRNAs in two separated regions, termed the BART and BHRF1 loci. Although accumulating evidence demonstrates that EBV infection regulates the profile of microRNAs in the cells, little is known about the microRNAs in exosomes released from infected cells. Here, we characterized the expression profile of intracellular and exosomal microRNAs in EBV-negative, and two related EBV-infected Burkitt lymphoma cell lines having type I and type III latency by next-generation sequencing. We found that the biogenesis of exosomes is upregulated in type III latently infected cells compared with EBV-negative and type I latently infected cells. We also observed that viral and several specific host microRNAs were predominantly incorporated in the exosomes released from the cells in type III latency. We confirmed that multiple viral microRNAs were transferred to the epithelial cells cocultured with EBV-infected B cells. Our findings indicate that EBV infection, in particular in type III latency, modulates the biogenesis of exosomes and the profile of exosomal microRNAs, potentially contributing to phenotypic changes in cells receiving these exosomes.

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Nascent Transcriptomics Reveal Cellular Prolytic Factors Upregulated Upstream of the Latent-to-Lytic Switch Protein of Epstein-Barr Virus

Journal of Virology ◽

10.1128/jvi.01966-19 ◽

2020 ◽

Vol 94 (7) ◽

Author(s):

Tiffany R. Frey ◽

Jozan Brathwaite ◽

Xiaofan Li ◽

Sandeepta Burgula ◽

Ibukun A. Akinyemi ◽

...

Keyword(s):

Epstein Barr Virus ◽

Human Herpesvirus ◽

Lytic Cycle ◽

Barr Virus ◽

Viral Spread ◽

Cellular Genes ◽

Latently Infected ◽

Infected Cells ◽

Host Shutoff ◽

Epstein Barr

ABSTRACT Lytic activation from latency is a key transition point in the life cycle of herpesviruses. Epstein-Barr virus (EBV) is a human herpesvirus that can cause lymphomas, epithelial cancers, and other diseases, most of which require the lytic cycle. While the lytic cycle of EBV can be triggered by chemicals and immunologic ligands, the lytic cascade is activated only when expression of the EBV latent-to-lytic switch protein ZEBRA is turned on. ZEBRA then transcriptionally activates other EBV genes and, together with some of those gene products, ensures completion of the lytic cycle. However, not every latently infected cell exposed to a lytic trigger turns on the expression of ZEBRA, resulting in responsive and refractory subpopulations. What governs this dichotomy? By examining the nascent transcriptome following exposure to a lytic trigger, we find that several cellular genes are transcriptionally upregulated temporally upstream of ZEBRA. These genes regulate lytic susceptibility to various degrees in latently infected cells that respond to mechanistically distinct lytic triggers. While increased expression of these cellular genes defines a prolytic state, such upregulation also runs counter to the well-known mechanism of viral-nuclease-mediated host shutoff that is activated downstream of ZEBRA. Furthermore, a subset of upregulated cellular genes is transcriptionally repressed temporally downstream of ZEBRA, indicating an additional mode of virus-mediated host shutoff through transcriptional repression. Thus, increased transcription of a set of host genes contributes to a prolytic state that allows a subpopulation of cells to support the EBV lytic cycle. IMPORTANCE Transition from latency to the lytic phase is necessary for herpesvirus-mediated pathology as well as viral spread and persistence in the population at large. Yet, viral genomes in only some cells in a population of latently infected cells respond to lytic triggers, resulting in subpopulations of responsive/lytic and refractory cells. Our investigations into this partially permissive phenotype of the herpesvirus Epstein-Barr virus (EBV) indicate that upon exposure to lytic triggers, certain cellular genes are transcriptionally upregulated, while viral latency genes are downregulated ahead of expression of the viral latent-to-lytic switch protein. These cellular genes contribute to lytic susceptibility to various degrees. Apart from indicating that there may be a cellular “prolytic” state, our findings indicate that (i) early transcriptional upregulation of cellular genes counters the well-known viral-nuclease-mediated host shutoff and (ii) subsequent transcriptional downregulation of a subset of early upregulated cellular genes is a previously undescribed mode of host shutoff.

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Epstein-barr virus latently infected cells are selectively deleted in simulated-microgravity cultures

In Vitro Cellular & Developmental Biology - Animal ◽

10.1007/bf02577533 ◽

2001 ◽

Vol 37 (4) ◽

pp. 223-230 ◽

Cited By ~ 1

Author(s):

James P. Long ◽

John H. Hughes

Keyword(s):

Epstein Barr Virus ◽

Simulated Microgravity ◽

Barr Virus ◽

Latently Infected ◽

Infected Cells ◽

Epstein Barr

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Characterization of naturally Epstein–Barr virus-infected gastric carcinoma cell line YCCEL1

Journal of General Virology ◽

10.1099/vir.0.045237-0 ◽

2013 ◽

Vol 94 (3) ◽

pp. 497-506 ◽

Cited By ~ 32

Author(s):

Do Nyun Kim ◽

Min Koo Seo ◽

Hoyun Choi ◽

Su Yeon Kim ◽

Hee Jong Shin ◽

...

Keyword(s):

Cell Line ◽

Cell Lines ◽

Blot Analysis ◽

Epstein Barr Virus ◽

Model Systems ◽

Nuclear Antigen ◽

Gastric Carcinoma Cell ◽

Barr Virus ◽

Epstein Barr

Epstein–Barr virus (EBV) is a herpesvirus associated with lymphomas and carcinomas. While EBV-associated epithelial cell lines are good model systems to investigate the role of EBV in carcinoma, only a few cell lines are available as they are hard to acquire. A greater variety of naturally EBV-infected cell lines which are derived from tumour patients are needed to represent various features of EBVaGC. We characterized cell line YCCEL1, established from a Korean EBVaGC patient, to ascertain whether it can be used to study the roles of EBV in EBVaGC. The expression of EBV genes and cell surface markers was examined by in situ hybridization, RT-PCR, Western blot analysis, immunofluorescence assay and Northern blot analysis. EBV episomal status was analysed by Southern blotting and real-time PCR. This cell line expressed EBV nuclear antigen 1 (EBNA1) and latent membrane protein 2A (LMP2A), but not EBNA2, LMP2B nor LMP1. The majority of the lytic proteins were not detected in YCCEL1 cells either before or after treatment with 12-O-tetradecanoylphorbol-13-acetate. YCCEL1 cells expressed BART microRNAs (miRNAs) at high level but did not express BHRF1 miRNAs. YCCEL1 cells expressed cytokeratin, but not CD21 and CD19, suggesting CD21-independent EBV infection. The latent EBV gene and EBV miRNA expression pattern of YCCEL1 cells closely resembled that of general EBVaGC cases. Our results support the value of YCCEL1 cells as a good model system to study the role of EBV in gastric carcinogenesis.

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