EBV infection is associated with histone bivalent switch modifications in squamous epithelial cells.

Epstein-Barr virus (EBV) induces histone modifications to regulate signaling pathways involved in EBV-driven tumorigenesis. To date, the regulatory mechanisms involved are poorly understood. In this study, we show that EBV infection of epithelial cells is associated with aberrant histone modification; specifically, aberrant histone bivalent switches by reducing the transcriptional activation histone mark (H3K4me3) and enhancing the suppressive mark (H3K27me3) at the promoter regions of a panel of DNA damage repair members in immortalized nasopharyngeal epithelial (NPE) cells. Sixteen DNA damage repair family members in base excision repair (BER), homologous recombination, nonhomologous end-joining, and mismatch repair (MMR) pathways showed aberrant histone bivalent switches. Among this panel of DNA repair members, MLH1, involved in MMR, was significantly down-regulated in EBV-infected NPE cells through aberrant histone bivalent switches in a promoter hypermethylation-independent manner. Functionally, expression of MLH1 correlated closely with cisplatin sensitivity both in vitro and in vivo. Moreover, seven BER members with aberrant histone bivalent switches in the EBV-positive NPE cell lines were significantly enriched in pathway analysis in a promoter hypermethylation-independent manner. This observation is further validated by their down-regulation in EBV-infected NPE cells. The in vitro comet and apurinic/apyrimidinic site assays further confirmed that EBV-infected NPE cells showed reduced DNA damage repair responsiveness. These findings suggest the importance of EBV-associated aberrant histone bivalent switch in host cells in subsequent suppression of DNA damage repair genes in a methylation-independent manner.

Activation of sterol regulatory element-binding protein 1 (SREBP1)-mediated lipogenesis by the Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) promotes cell proliferation and progression of nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. The EBV-encoded latent membrane protein 1 (LMP1), which is commonly expressed in NPC, engages multiple signaling pathways that promote cell growth, transformation, and metabolic reprogramming. Here, we report a novel function of LMP1 in promoting de novo lipogenesis. LMP1 increases the expression, maturation and activation of sterol regulatory element-binding protein 1 (SREBP1), a master regulator of lipogenesis, and its downstream target fatty acid synthase (FASN). LMP1 also induces de novo lipid synthesis and lipid droplet formation. In contrast, small interfering RNA (siRNA) knockdown of LMP1 in EBV-infected epithelial cells diminished SREBP1 activation and lipid biosynthesis. Furthermore, inhibition of the mammalian target of rapamycin (mTOR) pathway, through the use of either mTOR inhibitors or siRNAs, significantly reduced LMP1-mediated SREBP1 activity and lipogenesis, indicating that LMP1 activation of the mTOR pathway is required for SREBP1-mediated lipogenesis. In primary NPC tumors, FASN overexpression is common, with high levels correlating significantly with LMP1 expression. Moreover, elevated FASN expression was associated with aggressive disease and poor survival in NPC patients. Luteolin and fatostatin, two inhibitors of lipogenesis, suppressed lipogenesis and proliferation of nasopharyngeal epithelial cells, effects that were more profound in cells expressing LMP1. Luteolin and fatostatin also dramatically inhibited NPC tumor growth in vitro and in vivo. Our findings demonstrate that LMP1 activation of SREBP1-mediated lipogenesis promotes tumor cell growth and is involved in EBV-driven NPC pathogenesis. Our results also reveal the therapeutic potential of utilizing lipogenesis inhibitors in the treatment of locally advanced or metastatic NPC. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Oncogenic S1P signalling in EBV-associated nasopharyngeal carcinoma activates AKT and promotes cell migration through S1P receptor 3.

Undifferentiated nasopharyngeal carcinoma (NPC) is a cancer with high metastatic potential that is consistently associated with Epstein-Barr virus (EBV) infection. In this study, we have investigated the functional contribution of sphingosine-1-phosphate (S1P) signalling to the pathogenesis of NPC. We show that EBV infection or ectopic expression of the EBV-encoded latent genes (EBNA1, LMP1, and LMP2A) can up-regulate sphingosine kinase 1 (SPHK1), the key enzyme that produces S1P, in NPC cell lines. Exogenous addition of S1P promotes the migration of NPC cells through the activation of AKT; shRNA knockdown of SPHK1 resulted in a reduction in the levels of activated AKT and inhibition of cell migration. We also show that S1P receptor 3 (S1PR3) mRNA is overexpressed in EBV-positive NPC patient-derived xenografts and a subset of primary NPC tissues, and that knockdown of S1PR3 suppressed the activation of AKT and the S1P-induced migration of NPC cells. Taken together, our data point to a central role for EBV in mediating the oncogenic effects of S1P in NPC and identify S1P signalling as a potential therapeutic target in this disease. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

The role of metabolic reprogramming in γ-herpesvirus-associated oncogenesis.

The γ-herpesviruses, EBV and KSHV, are closely associated with a number of human cancers. While the signal transduction pathways exploited by γ-herpesviruses to promote cell growth, survival and transformation have been reported, recent studies have uncovered the impact of γ-herpesvirus infection on host cell metabolism. Here, we review the mechanisms used by γ-herpesviruses to induce metabolic reprogramming in host cells, focusing on their ability to modulate the activity of metabolic regulators and manipulate metabolic pathways. While γ-herpesviruses alter metabolic phenotypes as a means to support viral infection and long-term persistence, this modulation can inadvertently contribute to cancer development. Strategies that target deregulated metabolic phenotypes induced by γ-herpesviruses provide new opportunities for therapeutic intervention.

Activation of the FGFR1 signalling pathway by the Epstein-Barr virus-encoded LMP1 promotes aerobic glycolysis and transformation of human nasopharyngeal epithelial cells.

Non-keratinizing nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. The EBV-encoded latent membrane protein 1 (LMP1) is believed to play an important role in NPC pathogenesis by virtue of its ability to activate multiple cell signalling pathways which collectively promote cell proliferation, transformation, angiogenesis, and invasiveness, as well as modulation of energy metabolism. In this study, we report that LMP1 increases cellular uptake of glucose and glutamine, enhances LDHA activity and lactate production, but reduces pyruvate kinase activity and pyruvate concentrations. LMP1 also increases the phosphorylation of PKM2, LDHA, and FGFR1, as well as the expression of PDHK1, FGFR1, c-Myc, and HIF-1α, regardless of oxygen availability. Collectively, these findings suggest that LMP1 promotes aerobic glycolysis. With respect to FGFR1 signalling, LMP1 not only increases FGFR1 expression, but also up-regulates FGF2, leading to constitutive activation of the FGFR1 signalling pathway. Furthermore, two inhibitors of FGFR1 (PD161570 and SU5402) attenuate LMP1-mediated aerobic glycolysis, cellular transformation (proliferation and anchorage-independent growth), cell migration, and invasion in nasopharyngeal epithelial cells, identifying FGFR1 signalling as a key pathway in LMP1-mediated growth transformation. Immunohistochemical staining revealed that high levels of phosphorylated FGFR1 are common in primary NPC specimens and that this correlated with the expression of LMP1. In addition, FGFR1 inhibitors suppress cell proliferation and anchorage-independent growth of NPC cells. Our current findings demonstrate that LMP1-mediated FGFR1 activation contributes to aerobic glycolysis and transformation of epithelial cells, thereby implicating FGF2/FGFR1 signalling activation in the EBV-driven pathogenesis of NPC.

Down-regulation of LPA receptor 5 contributes to aberrant LPA signalling in EBV-associated nasopharyngeal carcinoma.

Undifferentiated nasopharyngeal carcinoma (NPC) is a highly metastatic disease that is consistently associated with Epstein-Barr virus (EBV) infection. In this study, we have investigated the contribution of lysophosphatidic acid (LPA) signalling to the pathogenesis of NPC. Here we demonstrate two distinct functional roles for LPA in NPC. First, we show that LPA enhances the migration of NPC cells and second, that it can inhibit the activity of EBV-specific cytotoxic T cells. Focusing on the first of these phenotypes, we show that one of the LPA receptors, LPA receptor 5 (LPAR5), is down-regulated in primary NPC tissues and that this down-regulation promotes the LPA-induced migration of NPC cell lines. Furthermore, we found that EBV infection or ectopic expression of the EBV-encoded LMP2A was sufficient to down-regulate LPAR5 in NPC cell lines. Our data point to a central role for EBV in mediating the oncogenic effects of LPA in NPC and identify LPA signalling as a potential therapeutic target in this disease.

Inhibition of the LKB1-AMPK pathway by the Epstein-Barr virus-encoded LMP1 promotes proliferation and transformation of human nasopharyngeal epithelial cells.

The association of Epstein-Barr virus (EBV) infection with the development of nasopharyngeal carcinoma (NPC) is well established. Latent membrane protein 1 (LMP1), the major oncogene encoded by EBV, is believed to play a crucial role in NPC pathogenesis by virtue of its ability to constitutively activate multiple cell signalling pathways. The LKB1-AMPK pathway is a master regulator of cellular metabolism that, via modulation of energy metabolism, has tumour suppressor activity. In this study we identify a novel ability of LMP1 to inhibit the LKB1-AMPK pathway through phosphorylation of LKB1 at serine 428 with subsequent suppression of the phosphorylation of AMPK and its substrates, ACC and Raptor. We show that MEK/ERK-MAPK signalling, activated by the CTAR1 domain of LMP1, is responsible for LKB1-AMPK inactivation. In addition, reactivation of AMPK signalling by AMPK activator, AICAR, abolished LMP1-induced cellular transformation (proliferation and anchorage-independent growth) in nasopharyngeal epithelial cells. Immunohistochemical staining revealed that a low level of phosphorylated AMPK is common in primary NPC specimens, and that this correlated significantly with the expression of LMP1. AICAR treatment inhibited the proliferation and anchorage-independent growth of NPC cells as well as potentiating the cytotoxic effect of the chemotherapeutic drug 5-fluorouracil. The current findings demonstrate that LMP1-mediated AMPK inactivation contributes to the proliferation and transformation of epithelial cells, thereby implicating the LKB1-AMPK pathway in the EBV-driven pathogenesis of NPC. Our findings also suggest that AMPK activators could be used to enhance the efficacy of conventional chemotherapeutic agents in the treatment of local and metastatic NPC.

Epstein-Barr virus induction of the Hedgehog signalling pathway imposes a stem cell phenotype on human epithelial cells.

Nasopharyngeal carcinoma (NPC) is a cancer common in southern China and South East Asia that is causally linked to Epstein-Barr virus (EBV) infection. Here, we demonstrate that NPC displays frequent dysregulation of the Hedgehog (HH) pathway, a pathway implicated in the maintenance of stem cells, but whose aberrant activation in adult tissues can lead to cancer. Using authentic EBV-positive carcinoma-derived cell lines and nasopharyngeal epithelial cell lines latently infected with EBV as models for NPC in vitro, we show that EBV activates the HH signalling pathway through autocrine induction of SHH ligand. Moreover, we find that constitutive engagement of the HH pathway induces the expression of a number of stemness-associated genes and imposes stem-like characteristics on EBV-infected epithelial cells in vitro. Using epithelial cells expressing individual EBV latent genes detected in NPC, we show that EBNA1, LMP1, and LMP2A are all capable of inducing SHH ligand and activating the HH pathway, but only LMP1 and LMP2A are able to induce expression of stemness-associated marker genes. Our findings not only identify a role for dysregulated HH signalling in NPC oncogenesis, but also provide a novel rationale for therapeutic intervention.

Epstein-Barr virus and nasopharyngeal carcinoma.

Since its discovery 50 years ago, Epstein-Barr virus (EBV) has been linked to the development of cancers originating from both lymphoid and epithelial cells. Approximately 95% of the world's population sustains an asymptomatic, life-long infection with EBV. The virus persists in the memory B-cell pool of normal healthy individuals, and any disruption of this interaction results in virus-associated B-cell tumors. The association of EBV with epithelial cell tumors, specifically nasopharyngeal carcinoma (NPC) and EBV-positive gastric carcinoma (EBV-GC), is less clear and is currently thought to be caused by the aberrant establishment of virus latency in epithelial cells that display premalignant genetic changes. Although the precise role of EBV in the carcinogenic process is currently poorly understood, the presence of the virus in all tumor cells provides opportunities for developing novel therapeutic and diagnostic approaches. The study of EBV and its role in carcinomas continues to provide insight into the carcinogenic process that is relevant to a broader understanding of tumor pathogenesis and to the development of targeted cancer therapies.

The role of the EBV-encoded latent membrane proteins LMP1 and LMP2 in the pathogenesis of nasopharyngeal carcinoma (NPC).

Although frequently expressed in EBV-positive malignancies, the contribution of the oncogenic latent membrane proteins, LMP1 and LMP2, to the pathogenesis of nasopharyngeal carcinoma (NPC) is not fully defined. As a key effector in EBV-driven B cell transformation and an established "transforming" gene, LMP1 displays oncogenic properties in rodent fibroblasts and induces profound morphological and phenotypic effects in epithelial cells. LMP1 functions as a viral mimic of the TNFR family member, CD40, engaging a number of signalling pathways that induce morphological and phenotypic alterations in epithelial cells. Although LMP2A plays an essential role in maintaining viral latency in EBV infected B cells, its role in epithelial cells is less clear. Unlike LMP1, LMP2A does not display "classical" transforming functions in rodent fibroblasts but its ability to engage a number of potentially oncogenic cell signalling pathways suggests that LMP2A can also participate in EBV-induced epithelial cell growth transformation. Here we review the effects of LMP1 and LMP2 on various aspects of epithelial cell behaviour highlighting key aspects that may contribute to the pathogenesis of NPC.

Contributions of the Epstein-Barr virus EBNA1 protein to gastric carcinoma.

Approximately 10% of gastric carcinomas (GC) are comprised of cells latently infected with Epstein-Barr virus (EBV); however, the mechanism by which EBV contributes to the development of this malignancy is unclear. We have investigated the cellular effects of the only EBV nuclear protein expressed in GC, EBNA1, focusing on promyelocytic leukemia (PML) nuclear bodies (NBs), which play important roles in apoptosis, p53 activation, and tumor suppression. AGS GC cells infected with EBV were found to contain fewer PML NBs and less PML protein than the parental EBV-negative AGS cells, and these levels were restored by silencing EBNA1. Conversely, EBNA1 expression was sufficient to induce the loss of PML NBs and proteins in AGS cells. Consistent with PML functions, EBNA1 expression decreased p53 activation and apoptosis in response to DNA damage and resulted in increased cell survival. In addition, EBNA1 mutants unable to bind CK2 kinase or ubiquitin-specific protease 7 had decreased ability to induce PML loss and to interfere with p53 activation. PML levels in EBV-positive and EBV-negative GC biopsy specimens were then compared by immunohistochemistry. Consistent with the results in the AGS cells, EBV-positive tumors had significantly lower PML levels than EBV-negative tumors. The results indicate that EBV infection of GC cells leads to loss of PML NBs through the action of EBNA1, resulting in impaired responses to DNA damage and promotion of cell survival. Therefore, PML disruption by EBNA1 is one mechanism by which EBV may contribute to the development of gastric cancer.

The pathological roles of BART miRNAs in nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is a distinct type of head and neck cancer prevalent in south-east Asia and southern China, where it constitutes a significant health burden. Although the close association of NPC with Epstein-Barr virus (EBV) infection has been known for more than four decades, the exact role that EBV plays in the pathogenesis of this malignancy is still unclear. While NPC tumours are known to express a number of EBV-encoded proteins, they also express a large number of virus-encoded microRNAs (miRNAs), the most abundant of which are those encoded from the BamHI-A region of the viral genome: the so-called BART miRNAs. miRNAs are small non-coding mRNAs that negatively regulate the expression of various genes at the post-transcriptional level. Accumulating evidence suggests that miRNAs play important roles in tumourigenesis. Here, we review the role of EBV-encoded BART miRNAs in modulating apoptosis and host innate defence mechanisms and their contribution to NPC pathogenesis. The rationale and strategies for therapeutic targeting of BART miRNAs in EBV-infected NPC are also discussed.

Immune evasion strategies of the human gamma-herpesviruses: implications for viral tumorigenesis.

Two human gamma-herpesviruses, Epstein-Barr virus and Kaposi's sarcoma associated herpesvirus/human herpesvirus 8 display oncogenic potential, causing benign and malignant lymphoproliferative disorders in genetically susceptible or immunosuppressed individuals. As a family of viruses that establish persistent life-long infections, herpesviruses have evolved strategies to limit innate antiviral responses and evade host immune surveillance. Herpesviruses have developed mechanisms to disrupt antigen presentation, pirate the production of immune regulating cytokines, and inhibit pro-apoptotic signaling pathways. Although these strategies are designed to facilitate the long-term persistence of herpesviruses, in certain circumstances they can contribute to viral-driven carcinogenesis.

The Major Constituent of Green Tea, Epigallocatechin-3-Gallate (EGCG), Inhibits the Growth of HPV18-Infected Keratinocytes by Stimulating Proteasomal Turnover of the E6 and E7 Oncoproteins.

Epigallocatechin-3-gallate (EGCG), the primary bioactive polyphenol in green tea, has been shown to inhibit the growth of human papilloma virus (HPV)-transformed keratinocytes. Here, we set out to examine the consequences of EGCG treatment on the growth of HPV18-immortalised foreskin keratinocytes (HFK-HPV18) and an authentic HPV18-positive vulvar intraepithelial neoplasia (VIN) clone, focusing on its ability to influence cell proliferation and differentiation and to impact on viral oncogene expression and virus replication. EGCG treatment was associated with degradation of the E6 and E7 oncoproteins and an upregulation of their associated tumour suppressor genes; consequently, keratinocyte proliferation was inhibited in both monolayer and organotypic raft culture. While EGCG exerted a profound effect on cell proliferation, it had little impact on keratinocyte differentiation. Expression of the late viral protein E4 was suppressed in the presence of EGCG, suggesting that EGCG was able to block productive viral replication in differentiating keratinocytes. Although EGCG did not alter the levels of E6 and E7 mRNA, it enhanced the turnover of the E6 and E7 proteins. The addition of MG132, a proteasome inhibitor, to EGCG-treated keratinocytes led to the accumulation of the E6/E7 proteins, showing that EGCG acts as an anti-viral, targeting the E6 and E7 proteins for proteasome-mediated degradation.

The EBV-Encoded Oncoprotein, LMP1, Recruits and Transforms Fibroblasts via an ERK-MAPK-Dependent Mechanism.

Latent membrane protein 1 (LMP1), the major oncoprotein encoded by Epstein-Barr virus (EBV), is expressed at widely variable levels in undifferentiated nasopharyngeal carcinoma (NPC) biopsies, fueling intense debate in the field as to the importance of this oncogenic protein in disease pathogenesis. LMP1-positive NPCs are reportedly more aggressive, and in a similar vein, the presence of cancer-associated fibroblasts (CAFs) surrounding "nests" of tumour cells in NPC serve as indicators of poor prognosis. However, there is currently no evidence linking LMP1 expression and the presence of CAFs in NPC. In this study, we demonstrate the ability of LMP1 to recruit fibroblasts in vitro in an ERK-MAPK-dependent mechanism, along with enhanced viability, invasiveness and transformation to a myofibroblast-like phenotype. Taken together, these findings support a putative role for LMP1 in recruiting CAFs to the tumour microenvironment in NPC, ultimately contributing to metastatic disease.

The Role of EBV-Encoded LMP1 in the NPC Tumor Microenvironment: From Function to Therapy.

Nasopharyngeal carcinoma (NPC) is closely associated with Epstein-Barr virus (EBV) infection. It is also characterized by heavy infiltration with non-malignant leucocytes. The EBV-encoded latent membrane protein 1 (LMP1) is believed to play an important role in NPC pathogenesis by virtue of its ability to activate multiple cell signaling pathways which collectively promote cell proliferation and survival, angiogenesis, invasiveness, and aerobic glycolysis. LMP1 also affects cell-cell interactions, antigen presentation, and cytokine and chemokine production. Here, we discuss how LMP1 modulates local immune responses that contribute to the establishment of the NPC tumor microenvironment. We also discuss strategies for targeting the LMP1 protein as a novel therapy for EBV-driven malignancies.

Aberrant activation of the Hedgehog signalling pathway in squamous cell carcinoma of the vulva as a potential target for cancer therapy.

In a previous study, we showed that the Hedgehog (Hh) signalling pathway is aberrantly activated in vulval squamous cell carcinoma (VSCC). In this study, we further validated our findings on a prospective cohort of primary VSCC cases, where immunohistochemical staining confirmed that key Hh pathway components were overexpressed in VSCC compared to normal vulval epithelium. We also undertook a series of in vitro studies to determine the extent of Hh pathway activation in VSCC-derived cell lines, and examine the consequences of pathway inhibition on the growth of these cells. We found that of six cell lines tested, four displayed elevated baseline Hh pathway activity that was dependent on SHH ligand, or in one case, a PTCH1 gene mutation. Hh signalling appeared necessary to sustain cell growth, as SHH ligand depletion with Robotikinin or SMO inhibition, either with chemical inhibitors (Itraconazole or LDE-225) or SMO-specific siRNA, attenuated GLI1 activity and cell proliferation in both monolayer and organotypic raft culture. Furthermore, treatment of Hh-dependent cell lines with SMO inhibitors sensitised cells to Cisplatin. Findings from our study offer us the opportunity to explore further the development of targeted chemotherapy for women with VSCC driven by aberrant Hh activation.

Whole-genome profiling of nasopharyngeal carcinoma reveals viral-host co-operation in inflammatory NF-κB activation and immune escape.

Interplay between EBV infection and acquired genetic alterations during nasopharyngeal carcinoma (NPC) development remains vague. Here we report a comprehensive genomic analysis of 70 NPCs, combining whole-genome sequencing (WGS) of microdissected tumor cells with EBV oncogene expression to reveal multiple aspects of cellular-viral co-operation in tumorigenesis. Genomic aberrations along with EBV-encoded LMP1 expression underpin constitutive NF-κB activation in 90% of NPCs. A similar spectrum of somatic aberrations and viral gene expression undermine innate immunity in 79% of cases and adaptive immunity in 47% of cases; mechanisms by which NPC may evade immune surveillance despite its pro-inflammatory phenotype. Additionally, genomic changes impairing TGFBR2 promote oncogenesis and stabilize EBV infection in tumor cells. Fine-mapping of CDKN2A/CDKN2B deletion breakpoints reveals homozygous MTAP deletions in 32-34% of NPCs that confer marked sensitivity to MAT2A inhibition. Our work concludes that NPC is a homogeneously NF-κB-driven and immune-protected, yet potentially druggable, cancer.

Upregulation of Id1 by Epstein-Barr virus-encoded LMP1 confers resistance to TGFbeta-mediated growth inhibition.

BACKGROUND: Epstein-Barr virus (EBV)-encoded LMP1 protein is commonly expressed in nasopharyngeal carcinoma (NPC). LMP1 is a prime candidate for driving tumourigenesis given its ability to activate multiple signalling pathways and to alter the expression and activity of variety of downstream targets. Resistance to TGFbeta-mediated cytostasis is one of the growth transforming effects of LMP1. Of the downstream targets manipulated by LMP1, the induction of Id1 and inactivation of Foxo3a appear particularly relevant to LMP1-mediated effects. Id1, a HLH protein is implicated in cell transformation and plays a role in cell proliferation, whilst Foxo3a, a transcription factor controls cell integrity and homeostasis by regulating apoptosis. The mechanism(s) by which LMP1 induces these effects have not been fully characterised. RESULTS: In this study, we demonstrate that the ability of LMP1 to induce the phosphorylation and inactivation of Foxo3a is linked to the upregulation of Id1. Furthermore, we show that the induction of Id1 is essential for the transforming function of LMP1 as over-expression of Id1 increases cell proliferation, attenuates TGFbeta-SMAD-mediated transcription and renders cells refractory to TGFbeta-mediated cytostasis. Id1 silencing in LMP1-expressing epithelial cells abolishes the inhibitory effect of LMP1 on TGFbeta-mediated cell growth arrest and reduces the ability of LMP1 to attenuate SMAD transcriptional activity. In response to TGFbeta stimulation, LMP1 does not abolish SMAD phosphorylation but inhibits p21 protein expression. In addition, we found the induction of Id1 in LMP1-expressing cells upon stimulation by TGFbeta. We provide evidence that LMP1 suppresses the transcriptional repressor ATF3, possibly leading to the TGFbeta-induced Id1 upregulation. CONCLUSION: The current data provide novel information regarding the mechanisms by which LMP1 suppresses TGFbeta-induced cytostasis, highlighting the importance of Id1 in LMP1 mediated cell transformation.

Epstein-Barr virus-encoded EBNA1 enhances RNA polymerase III-dependent EBER expression through induction of EBER-associated cellular transcription factors.

BACKGROUND: Epstein-Barr Virus (EBV)-encoded RNAs (EBERs) are non-polyadenylated RNA molecules transcribed from the EBV genome by RNA polymerase III (pol III). EBERs are the most abundant viral latent gene products, although the precise mechanisms by which EBV is able to achieve such high levels of EBER expression are not fully understood. Previously EBV has been demonstrated to induce transcription factors associated with EBER expression, including pol III transcription factors and ATF-2. We have recently demonstrated that EBV-encoded nuclear antigen-1 (EBNA1) induces cellular transcription factors, and given these findings, we investigated the role of EBNA1 in induction of EBER-associated transcription factors. RESULTS: Our data confirm that in epithelial cells EBNA1 can enhance cellular pol III transcription. Transient expression of EBNA1 in Ad/AH cells stably expressing the EBERs led to induction of both EBER1 and EBER2 and conversely, expression of a dominant negative EBNA1 led to reduced EBER expression in EBV-infected Ad/AH cells. EBNA1 can induce transcription factors used by EBER genes, including TFIIIC, ATF-2 and c-Myc. A variant chromatin precipitation procedure showed that EBNA1 is associated with the promoters of these genes but not with the promoters of pol III-transcribed genes, including the EBERs themselves. Using shRNA knock-down, we confirm the significance of both ATF-2 and c-Myc in EBER expression. Further, functional induction of a c-Myc fusion protein led to increased EBER expression, providing c-Myc binding sites upstream of EBER1 were intact. In vivo studies confirm elevated levels of the 102 kD subunit of TFIIIC in the tumour cells of EBV-positive nasopharyngeal carcinoma biopsies. CONCLUSIONS: Our findings reveal that EBNA1 is able to enhance EBER expression through induction of cellular transcription factors and add to the repertoire of EBNA1's transcription-regulatory properties.