EBV) Nuclear Antigen 1 Binding Research Articles

Structure‐based design of antibodies targeting the EBNA1 DNA‐binding domain to block Epstein–Barr virus latent infection and tumor growth

AbstractThe Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is critically involved in maintaining episomes during latent infection and promoting tumorigenesis. The development of an epitope‐specific monoclonal antibody (mAb) for EBNA1 holds great promise due to its high affinity and specificity, offering a new and innovative approach for the treatment of EBV‐related diseases. In this proof‐of‐concept study, we employed a structure‐based design strategy to create three unique immunogens specifically targeting the DNA binding state of the EBNA1 DBD. By immunizing mice, we successfully generated a mAb, named 5E2‐12, which selectively targets the DNA binding interface of EBNA1. The 5E2‐12 mAb effectively disrupts the interaction between EBNA1 and DNA binding, resulting in reduced proliferation of EBV‐positive cells and inhibition of xenograft tumor growth in both cellular assays and mouse tumor models. These findings open up new avenues for the development of innovative biological macromolecular drugs that specifically target EBNA1 and provide potential for clinical therapy options for early‐stage EBV‐positive tumors. The epitope‐specific mAb approach demonstrates novelty and innovation in tackling EBV‐related diseases and may have broad implications for precision medicine strategies in the field of viral‐associated cancers.

Ribosome Protein L4 is essential for Epstein–Barr Virus Nuclear Antigen 1 function

Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1)-mediated origin of plasmid replication (oriP) DNA episome maintenance is essential for EBV-mediated tumorigenesis. We have now found that EBNA1 binds to Ribosome Protein L4 (RPL4). RPL4 shRNA knockdown decreased EBNA1 activation of an oriP luciferase reporter, EBNA1 DNA binding in lymphoblastoid cell lines, and EBV genome number per lymphoblastoid cell line. EBV infection increased RPL4 expression and redistributed RPL4 to cell nuclei. RPL4 and Nucleolin (NCL) were a scaffold for an EBNA1-induced oriP complex. The RPL4 N terminus cooperated with NCL-K429 to support EBNA1 and oriP-mediated episome binding and maintenance, whereas the NCL C-terminal K380 and K393 induced oriP DNA H3K4me2 modification and promoted EBNA1 activation of oriP-dependent transcription. These observations provide new insights into the mechanisms by which EBV uses NCL and RPL4 to establish persistent B-lymphoblastoid cell infection.

α-1-Antitrypsin Gene Delivery Reduces Inflammation, Increases T-Regulatory Cell Population Size and Prevents Islet Allograft Rejection

Antiinflammatory clinical-grade, plasma-derived human α-1 antitrypsin (hAAT) protects islets from allorejection as well as from autoimmune destruction. hAAT also interferes with disease progression in experimental autoimmune encephalomyelitis (EAE) and in collagen-induced arthritis (CIA) mouse models. hAAT increases IL-1 receptor antagonist expression in human mononuclear cells and T-regulatory (Treg) cell population size in animal models. Clinical-grade hAAT contains plasma impurities, multiple hAAT isoforms and various states of inactive hAAT. We thus wished to establish islet-protective activities and effect on Treg cells of plasmid-derived circulating hAAT in whole animals. Islet function was assessed in mice that received allogeneic islet transplants after mice were given hydrodynamic tail-vein injection with pEF-hAAT, a previously described Epstein-Barr virus (EBV) plasmid construct containing the EBV nuclear antigen 1 (EBNA1) and the family of repeat EBNA1 binding site components (designated "EF") alongside the hAAT gene. Sera collected from hAAT-expressing mice were added to lipopolysaccharide (LPS)-stimulated macrophages to assess macrophage responsiveness. Also, maturation of peritoneal cells from hAAT-expressing mice was evaluated. hAAT-expressing mice accepted islet allografts (n = 11), whereas phosphate-buffered saline-injected animals (n = 11), as well as mice treated with truncated-hAAT-plasmid (n = 6) and untreated animals (n = 20) rapidly rejected islet allografts. In hAAT-expressing animals, local Treg cells were abundant at graft sites, and the IL-1 receptor antagonist was elevated in grafts and circulation. Sera from hAAT-expressing mice, but not control mice, inhibited macrophage responses. Finally, peritoneal cells from hAAT-expressing mice exhibited a semimature phenotype. We conclude that plasmid-derived circulating hAAT protects islet allografts from acute rejection, and human plasma impurities are unrelated to islet protection. Future studies may use this in vivo approach to examine the structure-function characteristics of the protective activities of AAT by manipulation of the hAAT plasmid.

Genome-wide analysis of host-chromosome binding sites for Epstein-Barr Virus Nuclear Antigen 1 (EBNA1)

The Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1) protein is required for the establishment of EBV latent infection in proliferating B-lymphocytes. EBNA1 is a multifunctional DNA-binding protein that stimulates DNA replication at the viral origin of plasmid replication (OriP), regulates transcription of viral and cellular genes, and tethers the viral episome to the cellular chromosome. EBNA1 also provides a survival function to B-lymphocytes, potentially through its ability to alter cellular gene expression. To better understand these various functions of EBNA1, we performed a genome-wide analysis of the viral and cellular DNA sites associated with EBNA1 protein in a latently infected Burkitt lymphoma B-cell line. Chromatin-immunoprecipitation (ChIP) combined with massively parallel deep-sequencing (ChIP-Seq) was used to identify cellular sites bound by EBNA1. Sites identified by ChIP-Seq were validated by conventional real-time PCR, and ChIP-Seq provided quantitative, high-resolution detection of the known EBNA1 binding sites on the EBV genome at OriP and Qp. We identified at least one cluster of unusually high-affinity EBNA1 binding sites on chromosome 11, between the divergent FAM55 D and FAM55B genes. A consensus for all cellular EBNA1 binding sites is distinct from those derived from the known viral binding sites, suggesting that some of these sites are indirectly bound by EBNA1. EBNA1 also bound close to the transcriptional start sites of a large number of cellular genes, including HDAC3, CDC7, and MAP3K1, which we show are positively regulated by EBNA1. EBNA1 binding sites were enriched in some repetitive elements, especially LINE 1 retrotransposons, and had weak correlations with histone modifications and ORC binding. We conclude that EBNA1 can interact with a large number of cellular genes and chromosomal loci in latently infected cells, but that these sites are likely to represent a complex ensemble of direct and indirect EBNA1 binding sites.

FR-like EBNA1 binding repeats in the human genome

Epstein–Barr virus (EBV) is widely spread in the human population. EBV nuclear antigen 1 (EBNA1) is a transcription factor that activates viral genes and is necessary for viral replication and partitioning, which binds the EBV genome cooperatively. We identify similar EBNA1 repeat binding sites in the human genome using a nearest-neighbor positional weight matrix. Previously experimentally verified EBNA1 sites in the human genome are successfully recovered by our approach. Most importantly, 40 novel regions are identified in the human genome, constituted of tandemly repeated binding sites for EBNA1. Genes located in the vicinity of these regions are presented as possible targets for EBNA1-mediated regulation. Among these, four are discussed in more detail: IQCB1, IMPG1, IRF2BP2 and TPO. Incorporating the cooperative actions of EBNA1 is essential when identifying regulatory regions in the human genome and we believe the findings presented here are highly valuable for the understanding of EBV-induced phenotypic changes.

EBNA1 regulates cellular gene expression by binding cellular promoters

Epstein-Barr virus (EBV) is associated with several types of lymphomas and epithelial tumors including Burkitt's lymphoma (BL), HIV-associated lymphoma, posttransplant lymphoproliferative disorder, and nasopharyngeal carcinoma. EBV nuclear antigen 1 (EBNA1) is expressed in all EBV associated tumors and is required for latency and transformation. EBNA1 initiates latent viral replication in B cells, maintains the viral genome copy number, and regulates transcription of other EBV-encoded latent genes. These activities are mediated through the ability of EBNA1 to bind viral-DNA. To further elucidate the role of EBNA1 in the host cell, we have examined the effect of EBNA1 on cellular gene expression by microarray analysis using the B cell BJAB and the epithelial 293 cell lines transfected with EBNA1. Analysis of the data revealed distinct profiles of cellular gene changes in BJAB and 293 cell lines. Subsequently, chromatin immune-precipitation revealed a direct binding of EBNA1 to cellular promoters. We have correlated EBNA1 bound promoters with changes in gene expression. Sequence analysis of the 100 promoters most enriched revealed a DNA motif that differs from the EBNA1 binding site in the EBV genome.

Robust In Vivo Transduction of a Genetically Stable Epstein-Barr Virus Episome to Hepatocytes in Mice by a Hybrid Viral Vector

To make a safe, long-lasting gene delivery vehicle, we developed a hybrid vector that leverages the relative strengths of adenovirus and Epstein-Barr virus (EBV). A fully gene-deleted helper-dependent adenovirus (HDAd) is used as the delivery vehicle for its scalability and high transduction efficiency. Upon delivery, a portion of the HDAd vector is recombined to form a circular plasmid. This episome includes two elements from EBV: an EBV nuclear antigen 1 (EBNA1) expression cassette and an EBNA1 binding region. Along with a human replication origin, these elements provide considerable genetic stability to the episome in replicating cells while avoiding insertional mutagenesis. Here, we demonstrate that this hybrid approach is highly efficient at delivering EBV episomes to target cells in vivo. We achieved nearly 100% transduction of hepatocytes after a single intravenous injection in mice. This is a substantial improvement over the transduction efficiency of previously available physical and viral methods. Bioluminescent imaging of vector-transduced mice demonstrated that luciferase transgene expression from the hybrid was robust and compared well to a traditional HDAd vector. Quantitative PCR analysis confirmed that the EBV episome was stable at approximately 30 copies per cell for up to 50 weeks and that it remained circular and extrachromosomal. Approaches for adapting the HDAd-EBV hybrid to a variety of disease targets and the potential benefits of this approach are discussed.

Identifying Sites Bound by Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) in the Human Genome: Defining a Position-Weighted Matrix To Predict Sites Bound by EBNA1 in Viral Genomes

We identified binding sites for Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) in the human genome using chromatin immunoprecipitation and microarrays. The sequences for these newly identified sites were used to generate a position-weighted matrix (PWM) for EBNA1's DNA-binding sites. This PWM helped identify additional DNA-binding sites for EBNA1 in the genomes of EBV, Kaposi's sarcoma-associated herpesvirus, and cercopithecine herpesvirus 15 (CeHV-15) (also called herpesvirus papio 15). In particular, a homologue of the Rep* locus in EBV was predicted in the genome of CeHV-15, which is notable because Rep* of EBV was not predicted by the previously developed consensus sequence for EBNA1's binding DNA. The Rep* of CeHV-15 functions as an origin of DNA synthesis in the EBV-positive cell line Raji; this finding thus builds on a set of DNA-binding sites for EBNA1 predicted in silico.

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

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.

The replicator of the Epstein-Barr virus latent cycle origin of DNA replication, oriP, is composed of multiple functional elements.

Replication of the Epstein-Barr virus genome initiates at one of several sites in latently infected, dividing cells. One of these replication origins is close to the viral DNA maintenance element, and, together, this replication origin and the maintenance element are referred to as oriP. The replicator of oriP contains four binding sites for Epstein-Barr virus nuclear antigen 1 (EBNA-1), the sole viral protein required for the replication and maintenance of oriP plasmids. We showed previously that these EBNA-1 sites function in pairs and that mutational inactivation of one pair does not eliminate replicator function. In this study we characterized the contribution of each EBNA-1 site within the replicator and flanking sequences through the use of an internally controlled replication assay. We present evidence that shows that all four EBNA-1 sites are required for an oriP plasmid to be replicated in every cell cycle. Results from these experiments also show that the paired EBNA-1 binding sites are not functionally equivalent and that the low affinity of sites 2 and 3 compared to that of sites 1 and 4 is not essential for replicator function. Our results suggest that a host cell protein(s) binds sequences flanking the EBNA-1 sites and that interactions between EBNA-1 and this protein(s) are critical for replicator function. Finally, we present evidence that shows that the minimal replicator of oriP consists of EBNA-1 sites 3 and 4 and two copies of a 14-bp repeat that is present in inverse orientation flanking these EBNA-1 sites. EBNA-1 sites 1 and 2, together with an element(s) within nucleotides 9138 to 9516, are ancillary elements required for full replicator activity.

Epstein-Barr nuclear antigen 1 binds and destabilizes nucleosomes at the viral origin of latent DNA replication.

The EBNA1 protein of Epstein-Barr virus (EBV) activates latent-phase DNA replication by an unknown mechanism that involves binding to four recognition sites in the dyad symmetry (DS) element of the viral latent origin of DNA replication. Since EBV episomes are assembled into nucleosomes, we have examined the ability of Epstein-Barr virus nuclear antigen 1 (EBNA1) to interact with the DS element when it is assembled into a nucleosome core particle. EBNA1 bound to its recognition sites within this nucleosome, forming a ternary complex, and displaced the histone octamer upon competitor DNA challenge. The DNA binding and dimerization region of EBNA1 was sufficient for nucleosome binding and destabilization. Although EBNA1 was able to bind to nucleosomes containing two recognition sites from the DS element positioned at the edge of the nucleosome, nucleosome destabilization was only observed when all four sites of the DS element were present. Our results indicate that the presence of a nucleosome at the viral origin will not prevent EBNA1 binding to its recognition sites. In addition, since four EBNA1 recognition sites are required for both nucleosome destabilization and efficient origin activation, our findings also suggest that nucleosome destabilization by EBNA1 is important for origin activation.

EBNA1 distorts oriP, the Epstein-Barr virus latent replication origin.

The Epstein-Barr virus nuclear antigen 1 (EBNA1) protein binds and activates the latent replication origin (oriP) of the Epstein-Barr virus. We have been studying EBNA1 to determine how it activates replication at oriP. Here we demonstrate that upon binding of EBNA1 to oriP, two thymine residues become reactive to potassium permanganate (KMnO4), indicating a helical distortion at these sites. The KMnO4-reactive thymines are 64 bp apart in the region of dyad symmetry of oriP. Dimethyl sulfate protection studies indicated that EBNA1 binds on the opposite face of the helix from the reactive thymines. The nature of the helical distortion induced by EBNA1 and its possible significance to the initiation of replication are discussed.

Interaction of Epstein-Barr virus nuclear antigen 1 with the viral latent origin of replication.

The Epstein-Barr virus latent origin of replication (oriP) requires only one viral protein, the Epstein-Barr virus nuclear antigen 1 (EBNA-1), for activity. oriP consists of two spatially separated, essential sequence elements, regions I and II, both of which contain multiple EBNA-1-binding sites. Region II contains, or is close to, the site at which DNA synthesis initiates. The role of region I, a transcriptional enhancer in cells that express EBNA-1, in replication is not understood. To determine how the binding of EBNA-1 to sites in region II leads to the initiation of DNA synthesis and to investigate the role of region I, EBNA-1 has been overproduced in insect cells by using a baculovirus vector and purified to homogeneity, and the interaction of EBNA-1 with oriP has been examined. Footprinting experiments demonstrated that EBNA-1 binds to oriP in a sequence-specific manner and bends or untwists the DNA at two symmetry-related sites in region II. Distortion of region I by EBNA-1 was not detected, suggesting that differences in the spacing of binding sites in regions I and II and resulting protein-protein interactions underlie differences in their biological properties. KMnO4 footprinting experiments did not reveal significant single-stranded structures in region II, suggesting that cellular proteins may recognize the EBNA--region II complex and unwind the DNA duplex. Region I did not quantitatively or qualitatively alter the interaction of EBNA-1 with region II. The contribution of an A + T-rich sequence in region II to replication was investigated by a mutational analysis. The results indicated that the overall A + T-rich nature of this sequence is not essential for replication of oriP-bearing plasmids. Nuclease protection experiments performed with these mutagenized plasmids provided additional evidence for protein-protein interactions in region II.