Expression Of Epstein-Barr Virus Nuclear Antigen Research Articles

Abstract 5786: Epstein-Barr virus nuclear antigen 1 (EBNA1)-targeting probes stimulate interferon-β production by nasopharyngeal carcinoma (NPC) cells to enhance the natural killer cell-mediated cytotoxicity against NPC

Abstract Introduction: The prevailing histologic subtype of nasopharyngeal carcinoma (NPC) in the endemic regions is non-keratinizing carcinoma which shows almost consistent association with latent infection by Epstein-Barr virus (EBV). Previously our group developed a series of Epstein-Barr virus nuclear antigen 1 (EBNA1)-targeting probes, which can disrupt the EBV latency maintained by EBNA1, and such lytic induction could alert the immune cells. Methods: Natural killer (NK) cells of the innate immunity play a significant role in controlling EBV infection. In this study, we investigated whether our EBNA1 probes (ZRL5P4 & L2P4) could activate NK cells to eliminate NPC. Results: Studies of clinical specimens indicated that tumor infiltration of the cytotoxic NK cell subset is a favorable prognostic marker for NPC patients. Furthermore, the cytotoxicity of circulating NK cells derived from NPC patients is equally well the cells from healthy individuals. If those patients’ NK cells can be attracted to infiltrate into the tumors, the tumor burden should be relieved. Indeed, the NPC xenograft with the ZRL5P4 treatment showed heavy mouse NK cell infiltration and tumor size reduction. The essential role of those mouse NK cells was demonstrated. Next, we showed that ZRL5P4 could induce the production of a soluble factor by NPC cells to stimulate the NK cell cytotoxicity. Such NK cell activity was verified in a metastasis NPC model. Mechanistically, the EBNA1 probe treatment could induce the expression of EBV early lytic gene Rta and EBNA1 itself. Both Rta and EBNA1 are known transcription factors to drive the expression of LMP1, the NF-κB signaling was then activated, which turned on the production of the well-known NK cell activation cytokine IFN-β. Conclusion: Taken together, our results demonstrated that our EBNA1 probes can mobilize NK cells to the tumor sites and activate their anti-tumor activity via the EBV reactivation and stimulation of IFN-β production. Citation Format: Luo Chen, Xiao Han Liu, Hei Tung Yuen, William C. Cho, Ka-Leung Wong, Hong Lok Lung. Epstein-Barr virus nuclear antigen 1 (EBNA1)-targeting probes stimulate interferon-β production by nasopharyngeal carcinoma (NPC) cells to enhance the natural killer cell-mediated cytotoxicity against NPC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5786.

Downregulation of MUS81 expression inhibits cell migration and maintains EBV latent infection through miR-BART9-5p in EBV-associated gastric cancer.

Epstein-Barr virus (EBV) infection is associated with the occurrence and development of gastric cancer (GC). Methyl methanesulfonate and ultraviolet-sensitive gene 81 (MUS81) is the catalytic component of a structure-specific endonuclease and plays an important role in chromosomal stability. However, the link between EBV infection and MUS81 remains unclear. In the present study, we found that MUS81 expression was much lower in EBV-associated GC cells than in EBV-negative GC. MUS81 acts as an oncogene in GC by inducing the cell migration and proliferation. Western blot and luciferase reporter assays revealed that miR-BART9-5p directly targeted MUS81 and downregulated its expression. Additionally, overexpression of MUS81 in EBV-positive GC cells inhibited the expression of EBV nuclear antigen 1 (EBNA1). EBNA1 is critical for the pathogenesis of EBV-associated tumors and the maintenance of a stable copy number of the viral genomes. Altogether, these results indicated that the lowering MUS81 expression might be a mechanism by EBV to maintain its latent infection.

A New Insight Into p53-Inhibiting Genes in Epstein–Barr Virus-Associated Gastric Adenocarcinoma

The p53 mutation is uncommon in Epstein–Barr virus-linked gastric carcinoma, but its suppression occurs through mechanisms such as ubiquitin specific peptidase 7 (USP7) inhibitions via Epstein–Barr virus nuclear antigen-1 (EBNA1) activity. This study aimed to evaluate the effect of EBNA1 on p53-inhibiting gene expression and the impact of USP7 inhibition on p53 suppression. MKN-45 cells were transfected with the EBNA1 plasmid. A stable EBNA1 expression cell line was developed through selection based on hygromycin B resistance. Murine double minute (MDM)4, MDM2, sirtuin (SIRT)3, histone deacetylase (HDAC)1, proteasome 26S subunit, Non-ATPase (PSMD)10, USP7, and p53 expression were checked using real-time PCR. Also, cells containing EBNA1 or control plasmid were treated with GNE-6776, and the expression of the interested genes and cell survival were assessed. MDM4, MDM2, and PSMD10 were significantly upregulated in the MKN-45 cell line following EBNA1 transfection. Morphological changes were observed in the cells harboring EBNA1 after 20 days. In the control cells, USP7 inhibition significantly upregulated the HDAC1, PSMD10, MDM4, and MDM2 genes after 24 h, but downregulated these genes after four days. In the EBNA1-harboring cells, MDM2, MDM4, and PSMD10 genes were significantly upregulated after 24 h, and this effect was sustained for all genes except for MDM4, even after four days. Furthermore, USP7 inhibition induced apoptosis in both cell groups. EBNA1 enhances the expression of p53-inhibiting genes. Two events—p53 protein overexpression and apoptosis activation—followed the suppression of the USP7 protein and provided evidence for its possible function. The significance of the EBNA1-USP7 interaction in p53 suppression warrants additional investigation and possibly reconsideration.

Epstein-Barr Virus Nuclear Antigen 1 Increases the Expression of Viral Oncogenes and Cellular Genes in the HeLa Cell Line.

Epstein-Barr virus (EBV) represents one of the most important viral carcinogens. EBV nuclear antigen-1 (EBNA1) can induce the expression of different cellular and viral genes. In this study, we evaluated the EBNA1 effects on the expression patterns of human papillomavirus type 18 (HPV-18) E6 and E7 oncogenes and three cellular genes, including BIRC5, c-MYC, and STMN1, in a cervical adenocarcinoma cell line. HeLa cells were divided into three groups: one transfected with a plasmid containing the EBNA1 gene, one transfected with a control plasmid, and one without transfection.In all three groups, the expression levels of E6, E7, BIRC5, c-MYC, and STMN1 genes were checked using real-time PCR. Pathological staining was used to examine changes in cell morphology. Real-time PCR results showed that the expression level of HPV-18 E6 (P=0.02) and E7 (P=0.02) oncogenes significantly increased in HeLa cells transfected with the EBNA1 plasmid compared to cells transfected with control plasmid. Also, the presence of EBNA1 induced the expression of BIRC5 and c-MYC, which increased tenfold (P=0.03) and threefold (P=0.02), respectively. Regarding the STMN1 cellular gene, although the expression level in HeLa cells transfected with EBNA1 plasmid showed a twofold increase, this change was insignificant (P=0.11). Also, EBNA1 expression caused the creation of large HeLa cells with abundant cytoplasm and numerous nuclei. The EBV-EBNA1 could increase the expression levels of HPV-18 E6 and E7 viral oncogenes as well as c-MYC and BIRC5 cellular genes in the HeLa cell line. These findings indicate that the simultaneous infection of cervical cells with HPV-18 and EBV might accelerate the progression of cervical cancer.

Amplification of EBNA-1 through a single-plasmid vector-based gene amplification system in HEK293 cells as an efficient transient gene expression system.

Our previous work showed that there is a limitation in the use of dihydrofolate reductase (dhfr)/methotrexate (MTX)-mediated gene amplification systems in dhfr-non-deficient HEK293 cells, as endogenous dhfr may interfere with the amplification process. In the present study, we successfully generated Epstein-Barr virus nuclear antigen-1 (EBNA-1)-amplified HEK293 cells in a dhfr-non-deficient HEK293 cell background using a single-plasmid vector-based gene amplification system with shRNA targeting the 3'-UTR of endogenous dhfr. The introduction of this shRNA efficiently downregulated the expression of endogenous dhfr in the HEK293 cells without affecting exogenous dhfr expression. The downregulation of endogenous dhfr improved the efficiency of EBNA-1 amplification, as evidenced by a comparison with the amplification extent in cells lacking shRNA expression at the same MTX concentration. The EBNA-1 expression levels from the EBNA-1-amplified clones selected in this study were higher than those obtained from EBNA-1-amplified clones that were generated using the conventional amplification in our previous study. Consistent with previous studies, EBNA-1 amplification improved the production of the Fc-fusion protein through a specific protein productivity (qp)-enhancing effect, rather than by improving cell growth or transfection efficiency. In addition, the N-glycan profiles in the Fc-fusion protein produced using this transient gene expression (TGE) system were not affected by EBNA-1 amplification. These results indicate the potential utility of EBNA-1-amplified mammalian cells, developed using a single-plasmid vector-based gene amplification system, for efficient protein production. KEY POINTS: • EBNA-1-amplified HEK293 cells were established using gene amplification system. • EBNA-1 amplification in TGE system can increase the Fc-fusion protein productivity. • EBNA-1 amplification does not affect the N-glycan profile in the Fc-fusion protein.

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.

DNA methylation enzymes and PRC1 restrict B-cell Epstein-Barr virus oncoprotein expression.

To accomplish the remarkable task of lifelong infection, Epstein-Barr virus (EBV) switches between four viral genome latency and lytic programs to navigate the B-cell compartment and evade immune responses. The transforming program, comprised of highly immunogenic EBV nuclear antigen (EBNA) and Latent Membrane Proteins (LMP), is expressed in newly infected B-lymphocytes and in post-transplant lymphomas. Upon memory cell differentiation and in most EBV-associated Burkitt lymphomas (BL), all but one viral antigen are repressed for immunoevasion. To gain insights into epigenetic mechanisms that restrict immunogenic oncoprotein expression, a genome-scale CRISPR/Cas9 screen was performed in EBV+ BL cells. Here we show that the ubiquitin ligase UHRF1 and its DNA methyltransferase partner DNMT1 were critical for restriction of EBNA and LMP expression. All UHRF1 reader and writer domains were necessary for silencing, and DNMT3B was identified as an upstream viral genome CpG methylation initiator. Polycomb repressive complex I exerted a further layer of control over LMP expression, suggesting a second mechanism for latency program switching. UHRF1, DNMT1 and DNMT3B are upregulated in germinal center B-cells, the BL cell of origin, providing a molecular link between B-cell state and EBV latency program. These results suggest rational therapeutic targets to manipulate EBV oncoprotein expression.

Epstein-Barr Virus Nuclear Antigen 1 Recruits Cyclophilin A to Facilitate the Replication of Viral DNA Genome

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1)-mediated DNA episomal genome replication and persistence are essential for the viral pathogenesis. Cyclophilin A (CYPA) is upregulated in EBV-associated nasopharyngeal carcinoma (NPC) with unknown roles. In the present approach, cytosolic CYPA was found to be bound with EBNA1 into the nucleus. The amino acid 376-459 of the EBNA1 domain was important for the binding. CYPA depletion attenuated and ectopic CYPA expression improved EBNA1 expression in EBV-positive cells. The loss of viral copy number was also accelerated by CYPA consumption in daughter cells during culture passages. Mechanistically, CYPA mediated the connection of EBNA1 with oriP (origin of EBV DNA replication) and subsequent oriP transcription, which is a key step for the initiation of EBV genome replication. Moreover, CYPA overexpression markedly antagonized the connection of EBNA1 to Ubiquitin-specific protease 7 (USP7), which is a strong host barrier with a role of inhibiting EBV genome replication. The PPIase activity of CYPA was required for the promotion of oriP transcription and antagonism with USP7. The results revealed a strategy that EBV recruited a host factor to counteract the host defense, thus facilitating its own latent genome replication. This study provides a new insight into EBV pathogenesis and potential virus-targeted therapeutics in EBV-associated NPC, in which CYPA is upregulated at all stages.

The Hsp70 inhibitor 2-phenylethynesulfonamide inhibits replication and carcinogenicity of Epstein\u2013Barr virus by inhibiting the molecular chaperone function of Hsp70

Epstein–Barr virus (EBV) can infect cells in latent and lytic period and cause serious disease. Epstein–Barr virus nuclear antigen 1 (EBNA1) is essential for the maintenance of the EBV DNA episome, replication and transcription. 2-phenylethynesulfonamide (PES) is a small molecular inhibitor of Heat shock protein 70 (Hsp70), which can interact with Hsp70 and disrupts its association with co-chaperones and substrate proteins of Hsp70. In our study, we found that PES could decrease the expression of EBNA1, which is independent of effects on EBNA1 transcription or proteasomal degradation pathway. The central glycine–alanine repeats domain was not required for inhibition of EBNA1 expression by PES. Also, PES could reduce the amount of intracellular EBV genomic DNA. PES inhibited proliferation and migration but induced cell cycle arrest and apoptosis of EBV positive cells. In addition, silencing of Hsp70 decreased expression of EBNA1 and the amounts of intracellular EBV genomic DNA, and PES increased this effect on a dose-dependent manner. On the contrast, over-expression of Hsp70 enhanced the expression of EBNA1 and the amounts of intracellular EBV genomic DNA, but PES inhibited this effect on a dose-dependent manner. Furthermore, Hsp70 interacted with EBNA1 but PES interfered this interaction. Our results indicate that PES suppresses replication and carcinogenicity of Epstein–Barr virus via inhibiting the molecular chaperone function of Hsp70.

Lymphomas driven by Epstein-Barr virus nuclear antigen-1 (EBNA1) are dependant upon Mdm2.

Epstein-Barr virus (EBV) associated Burkitt’s lymphoma is characterised by the deregulation of c-Myc expression and a restricted viral gene expression pattern in which the EBV nuclear antigen-1 (EBNA1) is the only viral protein to be consistently expressed. EBNA1 is required for viral genome propagation and segregation during latency. However, it has been much debated whether the protein plays a role in viral-associated tumourigenesis. We show that the lymphomas which arise in EµEBNA1 transgenic mice are unequivocally linked to EBNA1 expression and that both C-Myc and Mdm2 deregulation are central to this process. Tumour cell survival is supported by IL-2 and there is a skew towards CD8 positive T-cells in the tumour environment, while the immune check-point protein PD-L1 is upregulated in the tumours. Additionally, several isoforms of Mdm2 are upregulated in the EµEBNA1 tumours, with increased phosphorylation at ser166, an expression pattern not seen in Eµc-Myc transgenic tumours. Concomitantly, E2F1, Xiap, Mta1, C-Fos and Stat1 are upregulated in the tumours. Using four independent inhibitors of Mdm2 we demonstrate that the EµEBNA1 tumour cells are dependant upon Mdm2 for survival (as they are upon c-Myc) and that Mdm2 inhibition is not accompanied by upregulation of p53, instead cell death is linked to loss of E2F1 expression, providing new insight into the underlying tumourigenic mechanism. This opens a new path to combat EBV-associated disease.

NF-κB Signaling Regulates Epstein-Barr Virus BamHI-Q-Driven EBNA1 Expression.

Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is one of the few viral proteins expressed by EBV in nasopharyngeal carcinoma (NPC), most likely because of its essential role in maintaining the viral genome in EBV-infected cells. In NPC, EBNA1 expression is driven by the BamHI-Q promoter (Qp), which is regulated by both cellular and viral factors. We previously determined that the expression of another group of EBV transcripts, BamHI-A rightward transcripts (BARTs), is associated with constitutively activated nuclear factor-κB (NF-κB) signaling in NPC cells. Here, we show that, like the EBV BART promoter, the EBV Qp also responds to NF-κB signaling. NF-κB p65, but not p50, can activate Qp in vitro, and NF-κB signaling regulates Qp-EBNA1 expression in NPC cells, as well as in other EBV-infected epithelial cells. The introduction of mutations in the putative NF-κB site reduced Qp activation by the NF-κB p65 subunit. Binding of p65 to Qp was shown by chromatin immunoprecipitation (ChIP) analysis, while electrophoretic mobility shift assays (EMSAs) demonstrated that p50 can also bind to Qp. Inhibition of NF-κB signaling by the IκB kinase inhibitor PS-1145 resulted in the downregulation of Qp-EBNA1 expression in C666-1 NPC cells. Since EBNA1 has been reported to block p65 activation by inhibiting IKKα/β through an unknown mechanism, we suggest that, in NPC, NF-κB signaling and EBNA1 may form a regulatory loop which supports EBV latent gene expression, while also limiting NF-κB activity. These findings emphasize the role of NF-κB signaling in the regulation of EBV latency in EBV-associated tumors.

Co-amplification of EBNA-1 and PyLT through dhfr-mediated gene amplification for improving foreign protein production in transient gene expression in CHO cells.

Despite the relatively low transfection efficiency and low specific foreign protein productivity (qp) of Chinese hamster ovary (CHO) cell-based transient gene expression (TGE) systems, TGE-based recombinant protein production technology predominantly employs CHO cells for pre-clinical research and development purposes. To improve TGE in CHO cells, Epstein-Barr virus nuclear antigen-1 (EBNA-1)/polyoma virus large T antigen (PyLT)-co-amplified recombinant CHO (rCHO) cells stably expressing EBNA-1 and PyLT were established using dihydrofolate reductase/methotrexate-mediated gene amplification. The level of transiently expressed Fc-fusion protein was significantly higher in the EBNA-1/PyLT-co-amplified pools compared to control cultures. Increased Fc-fusion protein production by EBNA-1/PyLT-co-amplification resulted from a higher qp attributable to EBNA-1 but not PyLT expression. The qp for TGE-based production with EBNA-1/PyLT-co-amplified rCHO cells (EP-amp-20) was approximately 22.9-fold that of the control culture with CHO-DG44 cells. Rather than improved transfection efficiency, this cell line demonstrated increased levels of mRNA expression and replicated DNA, contributing to an increased qp. Furthermore, there was no significant difference in N-glycan profiles in Fc-fusion proteins produced in the TGE system. Taken together, these results showed that the use of rCHO cells with co-amplified expression of the viral elements EBNA-1 and PyLT improves TGE-based therapeutic protein production dramatically. Therefore, EBNA-1/PyLT-co-amplified rCHO cells will likely be useful as host cells in CHO cell-based TGE systems.

Baicalein inhibits growth of Epstein-Barr virus-positive nasopharyngeal carcinoma by repressing the activity of EBNA1 Q-promoter

Epstein-Barr virus (EBV) can establish a life-long latent infection in the host and is associated with various human malignancies, including nasopharyngeal carcinoma (NPC), the most common cancer originated from nasopharynx. EBV nuclear antigen 1 (EBNA1) is the only viral protein absolutely demanded for segregation, replication, transcription and maintenance of EBV viral genome in host cells. Baicalein, a bioactive flavonoid compound purified from the root of Scutellariae baicaleinsis, displays anti-inflammatory, immunosuppressive, and anti-tumor properties. In this study, the therapeutic effects and functional mechanism of baicalein on EBV-positive human NPC were determined. Cell Counting Kit-8 assays and cell formation colony were performed to investigate that baicalein can suppress proliferation of EBV-infected human NPC cells. Flow cytometric and hoechst 33258 staining results indicated that baicalein induced cell cycle arrest and apoptosis. Western blotting results demonstrated that baicalein down-regulates EBNA1 expression but not reduces the stability and half-life of EBNA1 in EBV-infected NPC cells. Additionally, the mRNA level of EBNA1 was examined by real time-PCR, the activity of EBNA1 Q promoter (Qp) was determined by dual luciferase reporter assay. Considering that transcription factor specificity protein 1 (Sp1) can maintain EBNA1 Qp active. Further analyses also elucidated that baicalein inhibits the expression of Sp1 while knock-down Sp1 by specific shRNAs decreases the expression and transcription levels of EBNA1. Therefore, the results suggested that baicalein may decrease EBNA1 expression level in EBV-positive NPC cells via inhibiting the activity of EBNA1 Q-promoter while over-expression of EBNA1 attenuate the inhibitory effect of baicalein. Finally, it was found that baicalein may strongly reduce growth of tumor in the mouse xenograft model of EBV-positive NPC. These results indicated that baicalein inhibits growth of EBV-positive NPC by repressing the activity of EBNA1 Q-promoter. Baicalein may be used as a therapeutic agent to treat EBV-positive NPC.

Molecular Grading of Oral Squamous Cell Carcinomas Infected with EBV

BackgroundSquamous Cell Carcinoma (SCC) is a type of cancer that is often found in oral cavity and areas of the head and neck. Viruses are major etiological factors through production of factors that can disturb proliferation and apoptosis regulators such as p53i, c-myc and bcl-2. This study aimed to determine the molecular grading of oral squamous cell carcinoma (OSCCs) infected with the Epstein-Barr Virus (EBV).MethodsTwenty-seven OSCC patients underwent biopsy to detect EBV infection through in situ hybridization for RNA EBV (EBER) and immunohistochemical analysis of latent membrane protein-1 (LMP-1) and EBV nuclear antigen-1 (EBNA-1). To assess molecular grades, cell proliferation and apoptosis regulator expression i.e. inactive p53 (p53i), c-myc and bcl-2, were immunohistochemically analysed.ResultsThe cases were divided into two groups; infected and non-infected by EBV. Regression analysis showed that only EBNA-1 expression could affect p53i expression. Based on regression equations molecular grading of OSCCs infected by EBV was divided into three: Grade I (low), EBNA-1 expression was 7.60, and p53i expression was 9.74-17.5; Grade II (medium), EBNA-1 expression was 7.61-19.7, and p53i 17.5-30.1; Grade III (high), EBNA-1 expression was 19.71, and p53i ≥ 30.1.ConclusionIn OSCC infected with EBV, only EBNA-1 expression can influence p53i expression.

EBV-encoded EBNA1 regulates cell viability by modulating miR34a-NOX2-ROS signaling in gastric cancer cells

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is a viral protein expressed in all EBV-infected cells that induces malignant transformation. EBNA1 is reported to contribute to tumor progression through an increase in reactive oxygen species via nicotinamide adenine dinucleotide phosphate oxidase. However, the underlying molecular mechanism of EBNA1-induced ROS accumulation in gastric cancer is poorly understood. Here, we demonstrated that miR34a regulation by EBNA1 determined cell fate in EBV-infected gastric cancer cells. ROS content and NOX2 expression were higher in EBNA1-expressing SNU719 cells than in EBNA1-nonexpressing SNU638 cells. Downregulation of NOX2 using siRNA technology in SNU719 cells decreased cell viability and ROS content. Regulation of EBNA1 expression in EBV-associated gastric cancers modulated NOX2 expression, ROS content and cell viability. We also showed that upregulation of NOX2 by EBNA1 was mediated by downregulating miRNA34a. Finally, overexpression of miR34a in EBNA1-expressing SNU719 cells induced typical apoptosis, suggesting that reactivation of miR34a in EBNA1-expressing gastric cancer cells could be a strategy for treatment of EBV-infected gastric cancer cells.

Investigation of relationship between EBNA-1 expression level and specific foreign protein productivity in transient gene expression of HEK293 cells

Abstract In an attempt to determine the relationship between the Epstein–Barr virus nuclear antigen-1 (EBNA-1) expression level and specific foreign protein productivity (qp), EBNA-1-amplifed HEK293 cells, which achieved a higher EBNA-1 expression level than that achieved by HEK293E cells, were established using dihydrofolate reductase (dhfr)-mediated gene amplification. Compared with a control culture in a null pool, Fc-fusion protein production by transient transfection in the EBNA-1-amplified pool showed a significant improvement. qp was linearly correlated with the EBNA-1 expression level in the transient transfection of EBNA-1-amplified clones, as indicated by the correlation coefficient (R2 = 0.7407). The Fc-fusion protein production and qp in a transient gene expression-based culture with EBNA-1-amplified HEK293 cells, E-amp-68, were approximately 2.0 and 3.2 times, respectively, higher than those in a culture with HEK293E cells. The increase in qp by EBNA-1 amplification mainly resulted from an enhancement in the amount of replicated DNA and level of mRNA expression but not an improved transfection efficiency. Taken together, it was found that EBNA-1 amplification could improve the therapeutic protein production in an HEK293 cell-based transient gene expression system.

Epstein–Barr virus infection is equally distributed across the invasive ductal and invasive lobular forms of breast cancer

The role of Epstein–Barr virus (EBV) in the pathogenesis of breast cancer is still unclear, although a growing body of evidence supports a link. The aim of this study was to investigate if EBV infection was more prevalent in invasive ductal carcinoma or invasive lobular carcinoma.An immunohistochemical marker for EBV (Epstein–Barr virus nuclear antigen 1 (EBNA1) clone E1-2.5) was applied to a tissue micro array section. The tissue micro array contained 80 cases of invasive ductal carcinoma, and 80 cases of invasive lobular carcinoma. Each case was scored as positive or negative for nuclear expression of EBNA1 in tumor cells using standard light microscopy.EBNA1 staining was evident in the tumor cells of 63 cases (39.4% of tumor cases). By tumor type (ductal/lobular) EBV infection was noted in 34 (42.5%) cases of invasive ductal carcinoma and 29 (36.2%) cases of invasive lobular carcinoma, this difference was not found to be significant (P=0.518).This study indicates that EBV infection is equally distributed across the ductal and lobular tumor types.

The Epstein–Barr virus nuclear antigen-1 reprograms transcription by mimicry of high mobility group A proteins

Viral proteins reprogram their host cells by hijacking regulatory components of protein networks. Here we describe a novel property of the Epstein–Barr virus (EBV) nuclear antigen-1 (EBNA1) that may underlie the capacity of the virus to promote a global remodeling of chromatin architecture and cellular transcription. We found that the expression of EBNA1 in transfected human and mouse cells is associated with decreased prevalence of heterochromatin foci, enhanced accessibility of cellular DNA to micrococcal nuclease digestion and decreased average length of nucleosome repeats, suggesting de-protection of the nucleosome linker regions. This is a direct effect of EBNA1 because targeting the viral protein to heterochromatin promotes large-scale chromatin decondensation with slow kinetics and independent of the recruitment of adenosine triphosphate–dependent chromatin remodelers. The remodeling function is mediated by a bipartite Gly-Arg rich domain of EBNA1 that resembles the AT-hook of High Mobility Group A (HMGA) architectural transcription factors. Similar to HMGAs, EBNA1 is highly mobile in interphase nuclei and promotes the mobility of linker histone H1, which counteracts chromatin condensation and alters the transcription of numerous cellular genes. Thus, by regulating chromatin compaction, EBNA1 may reset cellular transcription during infection and prime the infected cells for malignant transformation.

Heat shock factor 1 upregulates transcription of Epstein–Barr Virus nuclear antigen 1 by binding to a heat shock element within the BamHI-Q promoter

Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA1) is essential for maintenance of the episome and establishment of latency. In this study, we observed that heat treatment effectively induced EBNA1 transcription in EBV-transformed B95-8 and human LCL cell lines. Although Cp is considered as the sole promoter used for the expression of EBNA1 transcripts in the lymphoblastoid cell lines, the RT-PCR results showed that the EBNA1 transcripts induced by heat treatment arise from Qp-initiated transcripts. Using bioinformatics, a high affinity and functional heat shock factor 1 (HSF1)-binding element within the − 17/+4 oligonucleotide of the Qp was found, and was determined by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Moreover, heat shock and exogenous HSF1 expression induced Qp activity in reporter assays. Further, RNA interference-mediated HSF1 gene silencing attenuated heat-induced EBNA1 expression in B95-8 cells. These results provide evidence that EBNA1 is a new target for the transcription factor HSF1.

Effect of Epstein-Barr virus nuclear antigen 1 on cell proliferation and cell cycle in nasopharyngeal carcinoma cells

To study the effect of Epstein-Barr virus nuclear antigen 1 (EBNA1) on cell proliferation and cell cycle in nasopharyngeal carcinoma (NPC) cells. Recombinant lentivirus that encoded EBNA1 short hairpin RNA (shRNA) was prepared. The C666-EBNA1 (CE) cells were transduced with lentivirus and selected by fluorescence activated cell sorting (FACS) to repress EBNA1 expression. The protein expression levels of EBNA1 were examined by Western blot. The effect of EBNA1 silence on cell proliferation was analyzed by MTT assay and cell growth assay, respectively. Cell cycle was assessed by flow cytometry. The mRNA and protein levels of cell cycle regulators were examined by real-time PCR and Western blot. Recombinant lentivirus encoded EBNA1 shRNA was successfully constructed. The EBNA1 expression in CE cells was significantly reduced by lentivirus-mediated RNA interference. The results of cell counting and MTT assay showed that EBNA1 down-regulation significantly inhibited cell growth in CE-shRNA EBNA1 cells (P < 0.05). Compared with the control group, the percentage of cells in G0-G1 phase was increased from (62.43 ± 6.62)% to (89.66 ± 0.64)% (t = -7.091, P = 0.002), and that in S phase was decreased from (34.93 ± 7.36)% to (7.82 ± 2.44)% (t = 6.095, P = 0.004). The mRNA expressions of c-myc, CDK4, CDK6 and pRb were decreased by 65.60%, 34.06%, 41.05% and 55.29% respectively with the similar results in protein expression levels. Suppression of EBNA1 may inhibit the growth of nasopharyngeal carcinoma cells in vitro and induce a G1-phase cell cycle arrest, which might be mediated by down-regulation of c-myc, CDK4, CDK6 and pRb.