Epstein-Barr Virus Nuclear Antigen 1 Protein Research Articles

USP7 Inhibitors Destabilize EBNA1 and Suppress Epstein-Barr Virus Tumorigenesis.

Epstein-Barr virus (EBV) is a ubiquitous human ɣ-herpesvirus implicated in various malignancies, including Burkitt's lymphoma and gastric carcinomas. In most EBV-associated cancers, the viral genome is maintained as an extrachromosomal episome by the EBV nuclear antigen-1 (EBNA1). EBNA1 is considered to be a highly stable protein that interacts with the ubiquitin-specific protease 7 (USP7). Here, we show that pharmacological inhibitors and small interfering RNA (siRNA) targeting USP7 reduce EBNA1 protein levels in a proteosome-dependent manner. Proteomic analysis revealed that USP7 inhibitor GNE6776 altered the EBNA1 protein interactome, including disrupting USP7 association with EBNA1. GNE6776 also inhibited EBNA1 binding to EBV oriP DNA and reduced viral episome copy number. Transcriptomic studies revealed that USP7 inhibition affected chromosome segregation and mitotic cell division pathways in EBV+ cells. Finally, we show that GNE6776 selectively inhibited EBV+ gastric and lymphoid cell proliferation in cell culture and slowed EBV+ tumor growth in mouse xenograft models. These findings suggest that USP7 inhibitors perturb EBNA1 stability and function and may be exploited to treat EBV latent infection and tumorigenesis.

An in-silico insight into the predictive interaction of Apolipoprotein-E with Epstein-Barr virus proteins and their probable role in mediating Alzheimer’s disease

Recent reports suggest that persistent Epstein-Barr virus (EBV) infection and its recurrent reactivation could instigate the formation of proteinaceous plaques in the brain: a hallmark of Alzheimer’s disease (AD). Interestingly, a major genetic risk factor of AD, the apolipoprotein E (ApoE), could also influence the outcome of EBV infection in an individual. The ApoE is believed to influence the proteinaceous plaque clearance from the brain, and its defective functioning could result in the aggregate deposition. The persistent presence of EBV infection in a genetically predisposed individual could create a perfect recipe for severe neurodegenerative consequences. Therefore, in the present study, we investigated the possible interactions between ApoE and various EBV proteins using computational tools. Our results showed possibly stable de-novo interactions between the C-terminal domain of ApoE3 and EBV proteins: EBV nuclear antigen-1 (EBNA1) and BamHI Z fragment leftward open reading frame-1 (BZLF1). The EBNA1 protein of EBV plays a crucial role in establishing latency and replication of the virus. Whereas BZLF1 is involved in the lytic replication cycle. The proposed interaction of EBV proteins at the ligand-binding site of ApoE3 on CTD could interfere with- its capability to sequester amyloid fragments and, hence their clearance from the brain giving rise to AD pathology. This study provides a new outlook on EBV’s underexplored role in AD development and paves the way for novel avenues of investigation which could further our understanding of AD pathogenesis. Communicated by Ramaswamy H. Sarma

Characterization of an immune-evading doxycycline-inducible lentiviral vector for gene therapy in the spinal cord

Gene therapy is a powerful approach to promote spinal cord regeneration. For a clinical application it is important to restrict therapeutic gene expression to the appropriate time window to limit unwanted side effects. The doxycycline (dox)-inducible system is a widely used regulatable gene expression platform, however, this system depends on a bacterial-derived immunogenic transactivator. The foreign origin of this transactivator prevents reliable regulation of therapeutic gene expression and currently limits clinical translation. The glycine-alanine repeat (GAR) of Epstein-Barr virus nuclear antigen-1 protein inhibits its presentation to cytotoxic T cells, allowing virus-infected cells to evade the host immune system. We developed a chimeric transactivator (GARrtTA) and show that GARrtTA has an immune-evading advantage over “classical” rtTA in vivo. Direct comparison of lentiviral vectors expressing rtTA and GARrtTA in the rat spinal cord shows that the GARrtTA system is inducible for 6 doxycycline-cycles over a 47 week period, whereas with the rtTA-based system luciferase reporter expression declines during the 3rd cycle and is no longer re-inducible, indicating that GARrtTA provides an immune-advantage over rtTA. Immunohistochemistry revealed that GARrtTA expressing cells in the spinal cord appear healthier and survive better than rtTA expressing cells. Characterization of the immune response shows that expression of GARrtTA, in contrast to rtTA, does not recruit cytotoxic T-cells to the transduced spinal cord. This study demonstrates that fusion of the GAR domain to rtTA results in a functional doxycycline-inducible transactivator with a clear immune-advantage over the classical rtTA in vivo.

Epstein-Barr Virus, But Not Human Papillomavirus, Is Associated With Preinvasive and Invasive Ocular Surface Squamous Neoplasias in Zambian Patients.

BackgroundThe etiopathogenesis of ocular surface squamous neoplasia (OSSN) is not fully understood. We assessed the frequency of oncogenic viruses in OSSN by immunohistochemistry (IHC) and polymerase chain reaction (PCR) for human papillomavirus (HPV), Epstein–Barr virus (EBV), Merkel cell polyomavirus (MCPyV), Kaposi sarcoma virus, and adenovirus. Cases from Zambia were prospectively enrolled using a cross-sectional study design between November 2017 and March 2020.MethodsDemographic and clinical data [age, sex, HIV status, antiretroviral therapy (ART) history, CD4 count, plasma viral load] and tumor biopsies were collected from 243 consenting patients. Tumor samples were bisected, and half was used for DNA isolation, while the other half was formalin fixed and paraffin embedded (FFPE) for histopathology analysis. The expressions of latent EBV nuclear antigen 1 (EBNA1), CDKN2A/p16INK4A (p16), and MCPyV large T-antigen (LT) were tested by IHC. Multiplex PCR was used to detect 16 HPV genotypes and four other DNA tumor viruses [Kaposi’s sarcoma-associated herpesvirus (KSHV), EBV, MCPyV, and adenovirus]. Relationships between HIV status, viral DNA and protein expression, and tumor grades were determined by statistical analysis.ResultsOSSN tumors from patients were 29.6% preinvasive and 70.4% invasive. Patients presented with unilateral tumors that were 70.4% late stage (T3/T4). OSSN patients were HIV positive (72.8%). IHC on 243 FFPE biopsies resulted in the detection of EBNA1 (EBV), p16 high-risk HPV (HR-HPV), and MCPyV LT expression in 89.0%, 4.9%, and 0.0%, respectively. EBNA1 was expressed in all grades of preinvasive [cornea–conjunctiva intraepithelial neoplasia (CIN)1, 100%; CIN2, 85.7%; CIN3, 95.8%; and carcinoma in situ (CIS), 83.8%] and in invasive (89.2%) OSSN. PCR on 178 samples detected EBV, HR-HPV, and MCPyV in 80.3%, 9.0%, and 13.5% of tumors, respectively. EBV was detected in all grades of preinvasive and invasive OSSN. EBV detection was associated with high HIV viral loads (p = 0.022). HR-HPV was detected in 0.0% CIN1, 0.0% CIN2, 5.6% CIN3, 13.0% CIS, and 7.0% invasive OSSN.ConclusionsOur findings of EBV DNA and EBNA1 protein in all the grades of preinvasive and especially invasive OSSN are consistent with a potential causal role for EBV in OSSN. A role of HPV in OSSN was not clearly established in this study.

Mechanisms of Epstein-Barr virus nuclear antigen 1 favor Tregs accumulation in nasopharyngeal carcinoma.

BackgroundDocumented reports proved that Epstein‐Barr virus (EBV) infection increased infiltration of Tregs in malignancy. However, the mechanism of EBV recruitment Tregs into nasopharyngeal carcinoma (NPC) tissues has not been detailed discussion.MethodsExpression of EBV nuclear antigen 1 (EBNA1) and Foxp3 in NPC tissue samples was detected by immunohistochemistry. EBNA1+ NPC cell lines were used to coculture with PBMC, naïve T cells, Tregs, and monocytes. Percent of Treg was detected by flow cytometry.ResultsEBNA1 protein was overexpressed in NPC tissues, and was associated with a number of infiltrated Tregs. EBNA1+ NPC cells converted naïve T cells into Tregs by up‐regulated TGF‐β1. Enhanced CCL20 production in EBNA1‐expressed tumor cells increased Tregs migration. Polarized‐M2 macrophages by EBNA1 expression cells converted naïve T cells into Tregs.ConclusionsEBNA1 favors accumulation of Tregs in NPC through: (a) upregulated TGF‐β1 converted naïve T cell into Treg; (b) upregulated CCL20 increased Treg migration; and (c) polarized‐M2 macrophage converted naïve T cell into Treg.

Targeting Epstein-Barr virus nuclear antigen 1 (EBNA-1) with Murraya koengii bio-compounds: An in-silico approach.

Epstein-Barr virus (EBV), a B lymphotrophic herpesvirus associated with various forms of tumors, exhibits several latency phases with expressed EBV nuclear antigen 1 (EBNA-1). In the search of novel EBV-inhibiting targets, to curb the menace of EBV-borne lymphotropic transformations, EBNA-1 protein might serve as a best target for novel antiviral natural compounds. This study is thus aimed to explore the inhibitory potential of Muuraya koengii bioactive compounds isomahanine, murrayanol and mahanimbine against the EBNA-1 of EBV. 3D structure of EBNA-1 was retrieved from the PDB data bank with further optimization of both the protein and ligands. In-silico inhibitory potential of the selected M. koengii bio-compounds against EBNA-1 as well as the molecular properties of the derivatives against EBNA-1 were assessed. Murrayanol seems to be a potent inhibitory drug to target EBNA-1 with a promising binding energy of -7.21 with two hydrogen bonds. Drug likeliness parameters recorded murrayanol to be the most promising of the tested compounds, followed by isomahanine. Molecular docking evaluations show that EBNA-1 might be inhibited with M. koengii biocompounds. Keywords: EBV; EBNA; M. koengii; in-silico.

Developing a potentially immunologically inert tetracycline-regulatable viral vector for gene therapy in the peripheral nerve

Viral vector-mediated gene transfer of neurotrophic factors is an emerging and promising strategy to promote the regeneration of injured peripheral nerves. Unfortunately, the chronic exposure to neurotrophic factors results in local trapping of regenerating axons or other unwanted side effects. Therefore, tight control of therapeutic gene expression is required. The tetracycline/doxycycline-inducible system is considered to be one of the most promising systems for regulating heterologous gene expression. However, an immune response directed against the transactivator protein rtTA hampers further translational studies. Immunogenic proteins fused with the Gly-Ala repeat of the Epstein-Barr virus Nuclear Antigen-1 protein have been shown to successfully evade the immune system. In this article, we used this strategy to demonstrate that a chimeric transactivator, created by fusing the Gly-Ala repeat with rtTA and embedded in a lentiviral vector (i) retained its transactivator function in vitro, in muscle explants, and in vivo following injection into the rat peripheral nerve, (ii) exhibited a reduced leaky expression, and (iii) had an immune-evasive advantage over rtTA as shown in a novel bioassay for human antigen presentation. The current findings are an important step toward creating a clinically applicable potentially immune-evasive tetracycline-regulatable viral vector system.

Slippery Substrates Impair ATP-dependent Protease Function by Slowing Unfolding

ATP-dependent proteases are responsible for most energy-dependent protein degradation across all species. Proteases initially bind an unstructured region on a substrate and then translocate along the polypeptide chain, unfolding and degrading protein domains as they are encountered. Although this process is normally processive, resulting in the complete degradation of substrate proteins to small peptides, some substrates are released prematurely. Regions of low sequence complexity within the substrate such as the glycine-rich region (GRR) from p105 or glycine-alanine repeats (GAr) from the EBNA1 (Epstein-Barr virus nuclear antigen-1) protein, can trigger partial degradation and fragment release. Loss of processivity could be due to inability to hold on to the substrate (faster release) or inability to unfold and degrade a substrate domain (slower unfolding). I previously showed that the GRR slows domain unfolding by the proteasome (Kraut, D. A., Israeli, E., Schrader, E. K., Patil, A., Nakai, K., Nanavati, D., Inobe, T., and Matouschek, A. (2012) ACS Chem. Biol. 7, 1444-1453). In contrast, a recently published study concluded that GArs increase the rate of substrate release from ClpXP, a bacterial ATP-dependent protease (Too, P. H., Erales, J., Simen, J. D., Marjanovic, A., and Coffino, P. (2013) J. Biol. Chem. 288, 13243-13257). Here, I show that these apparently contradictory results can be reconciled through a reanalysis of the ClpXP GAr data. This reanalysis shows that, as with the proteasome, low complexity sequences in substrates slow their unfolding and degradation by ClpXP, with little effect on release rates. Thus, despite their evolutionary distance and limited sequence identity, both ClpXP and the proteasome share a common mechanism by which substrate sequences regulate the processivity of degradation.

About making a CHO production cell line “research-friendly” by genetic engineering

The use of Chinese Hamster Ovary (CHO) cells for transient gene expression is gaining importance steadily, as it has been shown that both quality and quantity of proteins derived from CHO cells differs from and can even be superior to material derived from Human Embryonic Kidney (HEK293) cells [1-4]. In order to augment yields HEK cell lines have been genetically modified, by e.g. stable integration of an expression cassette coding for the Epstein-Barr Virus Nuclear Antigen 1 (EBNA1) in conjunction with expression plasmid vectors containing the EBNA1 interaction site oriP. Here we describe the generation of a CHO cell line stably expressing the EBNA1 gene to enhance yields after large scale transient transfection with polyethylenimine (PEI). An expression plasmid featuring the EBNA1 gene was transfected into an in-house available CHO wild type cell line by nucleofection and several stable pools were established by antibiotic selection. In a first approach, 133 clones were then isolated by limiting dilution cloning and subsequently expanded for further analysis. The approach aimed at identifying clones showing both the presence of EBNA1 protein and enhanced yields after PEI-mediated transient expression of a reporter protein at the same time. Cell lysates or nuclear and cytoplasmic protein extracts from these clones were tested for presence of EBNA1 protein by Western Blot. An in-licensed cell line, CHO EBNALT85 (Icosagen AS, Tartu, Estonia) expressing the full length EBNA1 gene, and the HEK293-6E cell line (from the group of Y. Durocher, NRC, Canada) expressing a truncated EBNA1 gene served as positive controls. A number of commercially available anti-EBNA1 antibodies were tested in Western blotting, but most antibodies failed to detect the EBNA1 protein produced by these positive control cells. Only one antibody (Antibody 1EB12, Santa Cruz Biotechnology) was identified that detected the EBNA1 proteins in both positive control cell lines. However, by applying this antibody on the 133 generated clones it became obvious that in most of them the EBNA1 protein appeared to be largely degraded. The functionality of the EBNA1 protein in the cells was probed by PEI-mediated transient transfection of expression plasmids encoding the gene for the reporter protein Secreted Alkaline Phosphatase (SeAP). SeAP expression levels were compared with respect to presence or absence of the EBNA1 interaction sequence oriP on the SeAP expression plasmid. Only one clone showed a twofold enhanced production level of the reporter protein compared to the non-engineered parental CHO cell line. However, in this specific clone no EBNA1 protein could be detected by Western Blot, and the enhanced expression level was independent from the presence or absence of the oriP sequence on the SeAP expression plasmid. We therefore conclude that this clone does not possess a functional EBNA1 protein and an EBNA1/oriP based enhancement of productivity. The mechanism underlying the enhanced yield of the reporter protein in this particular clone remains currently elusive. In an alternative approach flow cytometric cell sorting was applied to generate CHO clones producing a functional EBNA1 protein. Using the EBNA1 positive CHO cell line CHO EBNALT85 it could be shown that expressing eGFP from a transiently transfected expression plasmid with oriP sequence resulted in a 30- to 40-fold increase of highly fluorescent cells in comparison to an expression plasmid lacking the oriP sequence. Thus, the already generated CHO pools stably transfected with the EBNA1 gene were transiently supertransfected with an eGFP expression plasmid containing the oriP sequence. Highly fluorescent cells were collected by cell sorting and expanded. Analysis by Western Blot revealed that this sub-pool was enriched for cells exhibiting the presence of a full length EBNA1 protein besides a major breakdown product. Transient transfection of this sub-pool with SeAP expression vectors led to the conclusion that the pool contained cells with a functional EBNA1 protein capable of enhancing SeAP yields in dependence on the presence of the oriP sequence on the SeAP expression plasmid. Clones were again generated from this sub-pool by limiting dilution cloning and characterized in analogy to the first approach. 9 out of 12 clones that were analyzed further showed increased levels of reporter protein production in dependence on the presence of the oriP element on the expression plasmid. The series of clones exhibiting highest expression levels will now be further evaluated by expression of other protein candidates.

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.

Expression of Epstein-Barr virus EBNA1 protein in Escherichia coli: Purification under nondenaturing conditions and use in DNA-binding studies

Epstein-Barr virus nuclear antigen 1 (EBNA1) is a viral protein required for stable replication and segregation of DNA episomes containing the Epstein-Barr virus (EBV) origin of replication, OriP. Overproduction of EBNA1 protein in Escherichia coli has previously been shown to be difficult due to the large number of codons in EBNA1 gene that are infrequently used in E. coli. Here we changed the 26 rare codons that are found among the first 78 codons of EBNA1 gene, and replaced them with codons that encode the same amino-acids but are abundant in E. coli. This led to a significant improvement of EBNA1 expression in a standard E. coli strain. Partial EBNA1 polypeptides of 11.5–16 kDa extending from the N-terminus to the second arginine and glycine-rich region were extremely abundant in the extract, however, resulting in a second limitation of the level of EBNA1 expression. EBNA1 was expressed as a fusion with a C-terminal six-histidine tag in order to get rid of the short polypeptides by Ni–NTA affinity purification, and salt conditions were used that allowed us to extract and purify EBNA1 without resorting to protein denaturing reagents. The purified protein was used in DNA-binding experiments with DNA fragments containing specific EBNA1 sites. The E. coli-expressed protein formed specific DNA–protein complexes that could be analyzed in polyacrylamide gels without showing the aggregation, or linking, phenomenon that is usually observed with EBNA1 expressed in eukaryotic cells. EBNA1 protein expressed in E. coli should therefore prove useful to further study the biochemical properties of this crucial Epstein-Barr virus protein.

Antigen-Binding Properties of Monoclonal Antibodies Reactive with EBNA1 and Use in Immunoaffinity Chromatography

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) was overexpressed and purified from Escherichia coli. Mouse monoclonal antibodies (mAbs) were prepared that react with EBNA1. Eleven high affinity mAbs were recovered. Nine mAbs are isotype IgG (all subisotype IgG1) and two mAbs are isotype IgM. All mAbs react strongly with EBNA1 in an ELISA assay while only one mAb (designated 1EB6) fails to react in a Western blot assay. The epitopes for these mAbs were mapped to seven different regions, providing good coverage of the entire EBNA1 protein. The mAbs had differing affinity for an EBNA1/DNA complex with four mAbs able to supershift the complex completely. All mAbs can immunoprecipitate EBNA1 from E. coli overexpressing EBNA1. A modified ELISA assay, termed ELISA-elution assay, was used to screen for mAbs that release EBNA1 in the presence of a low molecular weight polyhydroxylated compound (polyol) and a nonchaotropic salt. MAbs with this property, termed polyol-responsive (PR)-mAbs, allow gentle elution of labile proteins and protein complexes. Four mAbs are polyol-responsive with two showing usefulness in gentle immunoaffinity chromatography. Purification with these PR-mAbs may be useful in purifying EBNA1 complexes and elucidating EBNA1-associated proteins. This panel of anti-EBNA1 mAbs will advance the study of EBV by providing new tools to detect and purify EBNA1.

Large-scale Epstein–Barr virus EBNA1 protein purification

The protein–DNA and protein–protein interactions of Epstein–Barr virus nuclear antigen 1 (EBNA1) are known to play an important role in the many functions of this viral protein. Large quantities of pure EBNA1 protein would be useful in biochemical assays to elucidate such interactions. In particular, the crystal structure of the full-length protein would be important to show possible regions of interaction and/or post-translational modification. Recently, we described a novel approach to overexpress and purify EBNA1 from Escherichia coli; however, it is not ideal for large-scale production of EBNA1. We were able to optimize this protocol by (1) adding a polyethyleneimine precipitation step prior to Ni–NTA chromatography to reduce complexity of the sample and remove nucleic acid, (2) optimizing the Ni–NTA gradient to further separate EBNA1 from impurities, and (3) concluding with a MonoS cation-exchange chromatography step to further purify and concentrate EBNA1. We were able to recover 10-mg quantities of pure EBNA1 protein.

Cell specific internal translation efficiency of Epstein–Barr virus present in solid organ transplant patients

The U leader exon in the 5' untranslated region of the Epstein-Barr virus nuclear antigen 1 (EBNA1) gene contains an internal ribosome entry site, the EBNA internal ribosome entry segment (IRES), which promotes cap-independent translation and increases the expression level of the EBNA1 protein. It was previously reported that immunosuppressed organ transplanted patients showed an alternatively spliced EBNA1 transcript, excluding the EBNA IRES element. To further investigate the function of the EBNA IRES, sequence analysis of the EBNA IRES mRNA was performed in samples from seven organ transplant patients. Two nucleotide changes, G --> A at position 67531 and C --> U at position 67585 were found in the EBNA IRES mRNA, relative to the corresponding genomic Epstein-Barr virus (EBV) sequence in all patients. Moreover, the patient derived EBNA IRES mRNA was shown to differ from the IRES mRNA derived from the cell line B95.8 at position 67531 and from the cell lines Rael and P3HR1 at positions 67531 and 67585. cDNA from the various EBNA IRES sequences were cloned into bicistronic vectors, respectively, and used in transient transfection experiments in six human cell lines. The patient specific sequence significantly decreased the IRES activity in T-cells, while the base changes had no significant impact on the activity in B- or in epithelial cells. The genetic mechanisms behind EBV-associated diseases are complex, involving gene regulation by alternative promoters, alternative splicing, and translational control. The nucleotide changes in the patient specific EBNA IRES transcript and its influence on the translational activity, might illustrate new strategies utilised by the EBV to adapt to the immune control in patients with EBV associated diseases.

Overproduction in Escherichia coli and purification of Epstein–Barr virus EBNA-1

Epstein–Barr virus nuclear antigen 1 (EBNA-1) is a multi-functional protein of the Epstein–Barr virus (EBV). Due to its low abundance in EBV-transformed cells, overproduction in a foreign host is preferred to obtain purified EBNA-1 protein. The EBNA-1 gene possesses a large number of Escherichia coli rare codons (23%). By using E. coli BL21(DE3)Rosetta2 cells that augment the low-abundance tRNA genes, the expression level of EBNA-1 in E. coli was greatly enhanced. EBNA-1 was then purified by applying the whole cell extract soluble fraction to a Ni–NTA Superflow column and eluting with an imidazole gradient. The improved overexpression in E. coli followed by a one-step Ni–NTA purification resulted in a sufficient amount of pure EBNA-1 protein to test DNA binding activity, and prepare and test EBNA-1-specific monoclonal antibodies (mAbs).

Epstein-Barr virus nuclear antigen 1 evades direct immune recognition by CD4+ T helper cells.

The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the only viral protein regularly expressed in EBV-associated malignancies. Immune recognition of EBNA1 by CD8+ T cells is prevented by an internal glycine-alanine repeat (GAr) which blocks proteasomal degradation. To test whether EBV-infected cells could be recognized by T helper cells, human CD4+ T cell clones specific for EBNA1 were isolated from latently EBV-infected individuals. These T cells, however, failed to recognize EBV-positive target cells. To investigate whether endogenous presentation of EBNA1 epitopes on MHC class II was prevented by the GAr domain, a mutant EBV strain with an EBNA1 lacking the GAr (EBNA1DeltaGA) was generated and used to establish an Epstein-Barr virus-immortalized lymphoblastoid B cell line (LCL). The EBNA1DeltaGA LCL were not recognized by the EBNA1-specific T cell clones either, indicating that the GAr domain does not mediate this effect. Immune recognition could be restored by overexpression of EBNA1, for which at least 60-fold higher levels of both EBNA1 or EBNA1DeltaGAr protein were required. These results demonstrate that EBNA1 evades direct recognition by CD4+ T helper cells, since its steady state level is below the threshold required for efficient presentation on MHC class II. These findings have important implications for the design of immunotherapeutic approaches to target EBV-positive malignancies.

Expression of the Epstein-Barr virus nuclear antigen-1 (EBNA-1) in the mouse can elicit the production of anti-dsDNA and anti-Sm antibodies

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder characterized by the production of anti-nuclear antibodies. The etiology of SLE is unknown, although several viruses including the Epstein-Barr virus (EBV) have been implicated. An increase in the frequency of EBV infection has been observed in SLE patients relative to normal individuals. Some patients with SLE develop antibodies that recognize a proline rich epitope in the ribonucleoprotein Sm B/B that is similar to an epitope in EBNA-1, a major nuclear antigen of EBV. In the present study we have cloned the EBNA-1 gene under the control of the CMV promoter in the vector pcDNA3. We now report for the first time that expression of the entire EBNA-1 protein in the mouse can elicit the production of IgG antibodies to Sm and to double-stranded DNA (dsDNA). Our data suggest that the anti-Sm response arises as a consequence of antigenic cross-reactivity by anti-EBNA-1 antibodies. These results support a possible association between EBV infection and SLE.

Infectious mononucleosis patients temporarily recognize a unique, cross-reactive epitope of Epstein-Barr virus nuclear antigen-1.

The spectrum of antibodies against Epstein-Barr virus nuclear antigen-1 (EBNA-1) in patients with a recent history of infectious mononucleosis and nonaffected EBV-positive individuals has been characterized by epitope mapping. Sera were evaluated for antibodies to all unique maximally overlapping octapeptides of EBNA-1. Both normal controls and patients with infectious mononucleosis produce IgG antibodies that recognize the glycine-alanine-rich portion of EBNA-1, as previously described. All EBNA-1 IgG-positive infectious mononucleosis patients tested, however, consistently produce IgG specific for an additional epitope (aa 398-412 PPPGRRPFFHPVGEA) near the middle of the EBNA-1 protein. This region was not found to be antigenic in healthy EBV-seropositive individuals. This region does, however, cross-react with the sequence PPPGMRPP from the common lupus spliceosomal autoantigen Sm B' in several infectious mononucleosis patient sera. Patients with recent clinical infectious mononucleosis temporarily recognize a unique cross-reactive epitope of EBNA-1 not bound by antibodies from non-infectious mononucleosis EBV-positive sera or those with a distant history of IM.

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.

Coupling of mitotic chromosome tethering and replication competence in epstein-barr virus-based plasmids.

The Epstein-Barr virus (EBV) replicates once per cell cycle and segregates with high efficiency yet does not encode the enzymes needed for DNA replication or the proteins required to contact mitotic spindles. The virus-encoded EBNA-1 (EBV nuclear antigen 1) and latent replication origin (oriP) are required for both replication and segregation. We developed a sensitive and specific fluorescent labeling strategy to analyze the interactions of both EBNA-1 with viral episomes and viral episomes with host chromosomes. This enabled investigation of the hypothesis that replication and chromosome tethering are linked through the EBNA-1 protein. We show that deleting EBNA-1 or oriP disrupts mitotic chromosome tethering but removing the dyad symmetry element of oriP does not. Microscopic and biochemical approaches demonstrated that an EBNA-1 mutant lacking residues 16 to 372 bound to oriP plasmids but did not support their mitotic chromosome association and that the mutant lost the ability of wild-type EBNA-1 to associate with interphase chromatin. Importantly, the transient-replication abilities of various mutant forms of EBV plasmids, including the mutant form with the EBNA-1 internal deletion, correlated directly with their chromosome-tethering abilities. These data lead us to propose that EBNA-1 recruits oriP-containing plasmids into chromatin subdomains in interphase nuclei to both engage the host replication machinery and enable the plasmids to adhere to host chromosomes to increase their segregation efficiency.