Sequence-specific DNA binding of the Epstein-Barr virus nuclear antigen (EBNA-1) to clustered sites in the plasmid maintenance region

doi:10.1016/0092-8674(85)90282-X

Cell

Volume 42, Issue 3, October 1985, Pages 859-868

https://doi.org/10.1016/0092-8674(85)90282-X Get rights and content

Abstract

Latently infected B lymphocytes continuously express an Epstein-Barr Virus nuclear antigen (EBNA-1) required in trans for maintenance of the plasmid state of the EBV genome. Filter binding assays and DNAase I footprinting analyses revealed that the carboxy-terminal domain of EBNA-1 protects binding sites at three different loci in the 172,000 bp EBV genome. Two of these loci correspond to essential elements within an 1800 bp segment defined as the minimal region required for plasmid maintenance (ori-P). Binding to each of 20 × 30 bp tandem repeats in the “sink” locus protects 25 bp centered over a 12 bp palindromic consensus sequence TAGCATATGCTA. The nearby dyad symmetry “origin” locus contains two 46 bp protected regions each encompassing two paired core binding sites. The demonstration of sequence-specific binding at multiple loci suggests that EBNA-1 has pleiotropic functions, which may include control of copy number and segregation of the EBV plasmids as well as initiation of replication.

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Cited by (392)

The hide-and-seek game of the oncogenic Epstein-Barr virus-encoded EBNA1 protein with the immune system: An RNA G-quadruplex tale
2023, Biochimie
The Epstein-Barr virus (EBV) is the first oncogenic virus described in human. EBV infects more than 90% of the human population worldwide, but most EBV infections are asymptomatic. After the primary infection, the virus persists lifelong in the memory B cells of the infected individuals. Under certain conditions the virus can cause several human cancers, that include lymphoproliferative disorders such as Burkitt and Hodgkin lymphomas and non-lymphoid malignancies such as 100% of nasopharyngeal carcinoma and 10% of gastric cancers. Each year, about 200,000 EBV-related cancers emerge, hence accounting for at least 1% of worldwide cancers. Like all gammaherpesviruses, EBV has evolved a strategy to escape the host immune system. This strategy is mainly based on the tight control of the expression of its Epstein-Barr nuclear antigen-1 (EBNA1) protein, the EBV-encoded genome maintenance protein. Indeed, EBNA1 is essential for viral genome replication and maintenance but, at the same time, is also highly antigenic and T cells raised against EBNA1 exist in infected individuals. For this reason, EBNA1 is considered as the Achilles heel of EBV and the virus has seemingly evolved a strategy that employs the binding of nucleolin, a host cell factor, to RNA G-quadruplex (rG4) within EBNA1 mRNA to limit its expression to the minimal level required for function while minimizing immune recognition. This review recapitulates in a historical way the knowledge accumulated on EBNA1 immune evasion and discusses how this rG4-dependent mechanism can be exploited as an intervention point to unveil EBV-related cancers to the immune system.
Specific antibodies to EBNA1 epitopes as risk factors for multiple sclerosis, a systematic review
2023, Journal of Neuroimmunology
Seroprevalence of anti-EBV antibodies was found to be almost 100% and 90% for multiple sclerosis patients and normal people, respectively. Furthermore, anti EBNA1 antibody which is an indicator of past EBV infection has a higher titer in the serum of Persons with MS (pwMS) compared to the EBV-infected subjects without MS. Though, this difference in anti-EBNA1 antibody titer between pwMS and non-MS controls is not a reliable marker to be used for discriminating pwMS and non-MS individuals. Some Studies have revealed specific epitopes on EBNA1 as the target for anti-EBNA1 antibodies in pwMS. Measuring antibody response against such specific epitopes can help better discriminate pwMS and non-MS individuals. This systematic review aims to obtain conclusive data from the studies which have sought to identify and map such epitopes on EBNA1. Five databases, including PubMed, Google Scholar, web of Science, Scopus, and Elsevier were searched for this purpose. Overall, 12 articles were finally included. Despite different articles describing not exactly the same epitopes, most of the epitopes described are within the amino acid sequence 385–420 of EBNA1. Among these epitopes, most of the epitopes have overlapping amino acid sequences with one another. The most highly overlapping sequence is RRPFF, which encompasses the amino acid 402 to 406 of EBNA1.
Cell-cycle-dependent EBNA1-DNA crosslinking promotes replication termination at oriP and viral episome maintenance
2021, Cell
Epstein-Barr virus (EBV) is an oncogenic human herpesvirus that persists as a multicopy episome in proliferating host cells. Episome maintenance is strictly dependent on EBNA1, a sequence-specific DNA-binding protein with no known enzymatic activities. Here, we show that EBNA1 forms a cell cycle-dependent DNA crosslink with the EBV origin of plasmid replication oriP. EBNA1 tyrosine 518 (Y518) is essential for crosslinking to oriP and functionally required for episome maintenance and generation of EBV-transformed lymphoblastoid cell lines (LCLs). Mechanistically, Y518 is required for replication fork termination at oriP in vivo and for formation of SDS-resistant complexes in vitro. EBNA1-DNA crosslinking corresponds to single-strand endonuclease activity specific to DNA structures enriched at replication-termination sites, such as 4-way junctions. These findings reveal that EBNA1 forms tyrosine-dependent DNA-protein crosslinks and single-strand cleavage at oriP required for replication termination and viral episome maintenance.
Prolonged siRNA expression in mammalian cells using an Epstein–Barr virus-based plasmid expression system
2020, Biochemical and Biophysical Research Communications
RNA interference (RNAi) is a powerful tool in gene function analysis and disease treatment, especially diseases that are ‘undruggable’ by classical small molecules. However, the RNAi applications are limited due to some defects, such as short duration and toxic side effects. New strategies are still needed to improve RNAi applications. Previous studies have illustrated that Epstein-Barr virus nuclear antigen 1 (EBNA-1) and the origin of plasmid replication (oriP) are critical factors for EBV latent gene expression, which can keep the replication of the EBV genome as an extrachromosomal element for a relatively long time. Here we report a plasmid expression system on the base of oriP and EBNA-1, which could produce protein as well as short interfering RNAs(siRNAs) for a long time in mammalian cells. siRNA expression mediated by this system causes efficient and specific down-regulation of gene expression. Except for analyzing gene function, this study also provided a new optional and practical way for protein and/or RNAi-based therapies that require enduring effect.
Epstein-Barr virus-derived vector suitable for long-term expression in neurons
2020, Heliyon
Exogenous gene expression is a fundamental and indispensable technique for testing gene function in neurons. Several ways to express exogenous genes in neurons are available, but each method has pros and cons. The lentivirus vector is useful for high efficiency gene transfer to neurons and stabilizes gene expression via genome integration, but this integration may destroy the host genome. The Epstein-Barr virus (EBV)-derived vector (EB vector) is an accessible and useful vector in human cell lines because the vector is not integrated into the host genome but stays in the nucleus as an episome. However, there has been no report on this process in rodent neurons.
We examined the usefulness of the EB vector for testing gene function in neurons. We found that EB vector-derived exogenous proteins such as green fluorescent protein (GFP) and GFP-tagged actin were easily detectable even after three weeks of transfection. Second, a tetracycline-induced gene expression system in the EB vector was active after three weeks of transfection, indicating that plasmids were retained in neurons for up to three weeks. Third, we determined that only Family of repeat element of the plasmid vector is essential for its long-term presence in neurons. These results show that the modified EB vector is a useful tool for examining gene function in neurons.
Cellular-based immunotherapy in Epstein-Barr virus induced nasopharyngeal cancer
2018, Oral Oncology
Undifferentiated Nasopharyngeal carcinoma (NPC) is ubiquitously identified with the Epstein-Barr virus (EBV), making this cancer a suitable candidate for cellular-based immunotherapy (CBI) due to its expression of potentially targetable tumor-associated viral antigens. Various preclinical and clinical studies have explored the use of cytotoxic T cells (CTLs), tumor-infiltrating lymphocytes (TILs), natural killer (NK) cells, and dendritic cells (DCs) in the treatment of both refractory and locally advanced NPC with some success. Notably, immune-mediated antitumor effects were observed even in heavily pre-treated NPC patients, suggesting potential clinical benefit of CBI in this group of patients. These immune anti-tumor effects may be even more clinically evident when used as a first-line treatment, since there may not be an intense immunosuppressive environment which is typically encountered in refractory cancer patients. Additionally, CBI may exert an effect in priming the immune system and diminishing the cancer’s acquired resistance to exert a more robust response to previously failed chemotherapy. Although these results are encouraging, further refinements of clinical protocols to boost anti-tumor response and benefit a larger subset of patients proved necessary. Herein, we aim to review the rational of developing CBI in EBV-induced NPC and summarize its current applications in clinical studies.

View all citing articles on Scopus

^‡: Present address: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, Georgia 30322.

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Abstract

References (34)

P1 Plasmid replication: replicon structure

J. Mol. Biol.

Characterization of the major Epstein-Barr virus-specific RNA in Burkitt lymphoma-derived cells

J. Virol.

Organization of the B95-8 Epstein-Barr Virus genome

Nature

Plasmid P1 replication: negative control by repeated DNA sequences

Localization of the coding region for an Epstein-Barr virus early antigen and inducible expression of this 45/60K nuclear protein in transfected fibroblast cell lines

J. Virol.

A second Epstein Barr virus early antigen gene in BamHI-M encodes a 48/50K nuclear protein

J. Virol.

U2 region of Epstein-Barr virus DNA may encode Epstein-Barr nuclear antigen 2

Identification of Epstein-Barr nuclear antigen polypeptide in mouse and monkey cells after gene transfer with a cloned 2.9 kilobase pair subfragment of the genome

Nucleotide sequence of an mRNA transcribed in latent growth-transforming virus infection indicates that it may encode a membrane protein

J. Virol.

The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites

Cell

Interaction between Epstein-Barr virus-determined nuclear antigen (EBNA) and the viral DNA

J. Gen. Virol.

Two Epstein-Barr viral nuclear neo antigens distinguishable by gene transfer, serology and chromosome binding

Organization of the Epstein-Barr Virus DNA molecule. II. Fine mapping of the boundaries of the interal repeat cluster of B95-8 and identification of additional small tandem repeats adjacent to the HR-1 deletion

J. Virol.

Identification of Epstein-Barr virus sequences that encode a nuclear antigen expressed in latently infected lymphocytes

Simple repeat sequence in Epstein-Barr virus DNA is transcribed in latent and productive infections

J. Virol.

One of two Epstein-Barr virus nuclear antigens contains a glycine-alanine copolymer domain

Simple repeat array in Epstein-Barr virus DNA encodes part of the Epstein-Barr nuclear antigen

Science

Cited by (392)

The hide-and-seek game of the oncogenic Epstein-Barr virus-encoded EBNA1 protein with the immune system: An RNA G-quadruplex tale

Specific antibodies to EBNA1 epitopes as risk factors for multiple sclerosis, a systematic review

Cell-cycle-dependent EBNA1-DNA crosslinking promotes replication termination at oriP and viral episome maintenance

Prolonged siRNA expression in mammalian cells using an Epstein–Barr virus-based plasmid expression system

Epstein-Barr virus-derived vector suitable for long-term expression in neurons

Cellular-based immunotherapy in Epstein-Barr virus induced nasopharyngeal cancer

Cell

ArticleSequence-specific DNA binding of the Epstein-Barr virus nuclear antigen (EBNA-1) to clustered sites in the plasmid maintenance region

Abstract

P1 Plasmid replication: replicon structure

J. Mol. Biol.

Characterization of the major Epstein-Barr virus-specific RNA in Burkitt lymphoma-derived cells

J. Virol.

Organization of the B95-8 Epstein-Barr Virus genome

Nature

Plasmid P1 replication: negative control by repeated DNA sequences

Localization of the coding region for an Epstein-Barr virus early antigen and inducible expression of this 45/60K nuclear protein in transfected fibroblast cell lines

J. Virol.

A second Epstein Barr virus early antigen gene in BamHI-M encodes a 48/50K nuclear protein

J. Virol.

U2 region of Epstein-Barr virus DNA may encode Epstein-Barr nuclear antigen 2

Identification of Epstein-Barr nuclear antigen polypeptide in mouse and monkey cells after gene transfer with a cloned 2.9 kilobase pair subfragment of the genome

Nucleotide sequence of an mRNA transcribed in latent growth-transforming virus infection indicates that it may encode a membrane protein

J. Virol.

The dnaA protein complex with the E. coli chromosomal replication origin (oriC) and other DNA sites

Cell

Interaction between Epstein-Barr virus-determined nuclear antigen (EBNA) and the viral DNA

J. Gen. Virol.

Two Epstein-Barr viral nuclear neo antigens distinguishable by gene transfer, serology and chromosome binding

Organization of the Epstein-Barr Virus DNA molecule. II. Fine mapping of the boundaries of the interal repeat cluster of B95-8 and identification of additional small tandem repeats adjacent to the HR-1 deletion

J. Virol.

Identification of Epstein-Barr virus sequences that encode a nuclear antigen expressed in latently infected lymphocytes

Simple repeat sequence in Epstein-Barr virus DNA is transcribed in latent and productive infections

J. Virol.

One of two Epstein-Barr virus nuclear antigens contains a glycine-alanine copolymer domain

Simple repeat array in Epstein-Barr virus DNA encodes part of the Epstein-Barr nuclear antigen

Science

Article
Sequence-specific DNA binding of the Epstein-Barr virus nuclear antigen (EBNA-1) to clustered sites in the plasmid maintenance region