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Epstein-Barr virus (EBV) is a ubiquitous, oncogenic virus that is associated with a number of different human malignancies as well as autoimmune disorders. The expression of EBV viral proteins and non-coding RNAs contribute to EBV-mediated disease pathologies. The virus establishes life-long latency in the human host and is adept at evading host innate and adaptive immune responses. In this review, we discuss the life cycle of EBV, the various functions of EBV-encoded proteins and RNAs, the ability of the virus to activate and evade immune responses, as well as the neoplastic and autoimmune diseases that are associated with EBV infection in the human population.

This study identified the total effects of EBV and a viral long noncoding RNA (BART lncRNA) on cellular RNA expression when grown as cells in culture and when grown as tumors in immunodeficient mice. The effects on cellular mRNA expression, lncRNA expression, and cellular and viral miR expression were determined using next-generation sequencing (NGS) and bioinformatics functional analysis.

The broad impact of Epstein-Barr Virus (EBV) infection on the immune system, joined with the ability to manipulate virus-cell interactions in vitro, make EBV one of the most important of viruses from an immunogenetic standpoint. Primary infection with EBV is usually silent, especially in infants and children; the classic syndrome of infectious mononucleosis is the most characteristic in adolescent and young adults. The sequence of viral-cellular junction fragments has been analyzed in the Namalwa cell line, which contains a single intact copy of the EBV genome integrated into chromo-some 1, with both duplication and deletion of cellular sequences at the site of integration. Superinfection of Raji cells is the only experimental EBV system that approximates a lytic permissive infection. Primary infection with EBV occurs early in life in most populations and is almost always subclinical. Infection is delayed in some Western societies, and then appears as the clinical syndrome of infectious mononucleosis.

The release of membrane-bound vesicles from cells is being increasingly recognized as a mechanism of intercellular communication. Extracellular vesicles (EVs) or exosomes are produced by virus-infected cells and are thought to be involved in intercellular communication between infected and uninfected cells. Viruses, in particular oncogenic viruses and viruses that establish chronic infections, have been shown to modulate the production and content of EVs. Viral microRNAs, proteins and even entire virions can be incorporated into EVs, which can affect the immune recognition of viruses or modulate neighbouring cells. In this Review, we discuss the roles that EVs have during viral infection to either promote or restrict viral replication in target cells. We will also discuss our current understanding of the molecular mechanisms that underlie these roles, the potential consequences for the infected host and possible future diagnostic applications.

Primary effusion lymphoma (PEL) is a B cell lymphoma that is always associated with Kaposi’s sarcoma-associated herpesvirus (KSHV) and in many cases also with Epstein–Barr virus (EBV); however, the requirement for EBV coinfection is not clear. Here, we demonstrate that adding exogenous EBV to KSHV(+) single-positive PEL leads to increased KSHV genome maintenance and KSHV latency-associated nuclear antigen (LANA) expression. To show that EBV was necessary for naturally coinfected PEL, we nucleofected KSHV(+)/EBV(+) PEL cell lines with an EBV-specific CRISPR/Cas9 plasmid to delete EBV and observed a dramatic decrease in cell viability, KSHV genome copy number, and LANA expression. This phenotype was reversed by expressing Epstein–Barr nuclear antigen 1 (EBNA-1) in trans, even though EBNA-1 and LANA do not colocalize in infected cells. This work reveals that EBV EBNA-1 plays an essential role in the pathogenesis of PEL by increasing KSHV viral load and LANA expression.

The Epstein-Barr virus (EBV) oncoproteins latent membrane protein 1 (LMP1) and LMP2A constitutively activate multiple signaling pathways, and both have been shown to interact with cellular ubiquitin ligases and affect cellular ubiquitination. To detect the LMP1- and LMP2A-mediated effects on the global cellular proteome, epithelial cell lines expressing LMP1 or LMP2A were analyzed using label-free quantitative proteomics. To identify proteins whose ubiquitination is affected by the viral proteins, the cells were cultured in the presence and absence of deubiquitinase (DUB) and proteasome inhibitors. More than 7,700 proteins were identified with high confidence and considerably more proteins showed significant differences in expression in the presence of inhibitors. Few of the differentially expressed proteins with or without inhibitors were common between LMP1 and LMP2A, confirming that the viral proteins induce unique changes in cell expression and function. However, ingenuity pathway analysis (IPA) of the data indicated that LMP1 and LMP2A modulate many of the same cellular regulatory pathways, including cell death and survival, cell movement, and actin filament dynamics. In addition, various proteasome subunits, ubiquitin-specific peptidases and conjugating enzymes, vesicle trafficking proteins, and NF-κB and mitogen-activated protein kinase signaling proteins were affected by LMP1 or LMP2A. These findings suggest that LMP1 and LMP2A may commonly target critical cell pathways through effects on distinct genes, with many cellular proteins modified by ubiquitination and/or degradation., IMPORTANCE The Epstein-Barr virus proteins latent membrane protein 1 and 2 have potent effects on cell growth and signaling. Both proteins bind to specific ubiquitin ligases and likely modulate the cellular proteome through ubiquitin-mediated effects on stability and intracellular location. In this study, a comprehensive proteomic analysis of the effects of LMP1 and LMP2A revealed that both proteins affected proteasome subunits, ubiquitin-specific conjugases and peptidases, and vesical trafficking proteins. The data suggest that the effects of these proteins on the abundance and ubiquitination of cellular proteins are in part responsible for their effects on cell growth regulation.

Epstein-Barr virus latent membrane protein 2A (LMP2A) induces many characteristics of carcinoma, including transformation, migration, invasion, and impaired differentiation. The MCF10A cell line differentiates to form hollow acini when grown in Matrigel, and expression of LMP2A inhibited differentiation and anoikis induced by loss of matrix attachment. LMP2A-infected cells formed large, lobular structures rather than hollow acini. Autophagy inhibitors impaired this abnormal growth and induced caspase 3 activation and acinus formation. LMP2A also increased autophagosome formation and expression of proteins in the autophagosome pathway. These findings suggest that LMP2A may inhibit anoikis and luminal clearance in acini through induction of autophagy.

Latent infection with Epstein-Barr virus (EBV) is responsible for multiple types of malignancies, including 10% of all gastric carcinomas. The microRNA (miRNA) expression in several EBV-infected AGS gastric carcinoma cell lines was determined. Infected cells expressed the viral BamHI A rightward transcript (BART) miRNAs at high levels and had consistently decreased expression of a small fraction of cellular miRNAs with specific downregulation of tumor suppressor miRNAs. These changes likely reflect expression of the viral noncoding RNAs and not latent protein expression.

The Epstein Barr virus (EBV) is linked to the development of two major epithelial malignancies, gastric carcinoma and nasopharyngeal carcinoma. This study evaluates the effects of EBV on cellular expression in a gastric epithelial cell line infected with or without EBV and a nasopharyngeal carcinoma cell line containing EBV. The cells were grown in vitro and as tumors in vivo. The effects on cellular expression were determined using both 2D DIGE proteomics and high throughput RNA sequencing. The data identify multiple pathways that were uniquely activated in vitro. RNA sequences mapping to the mouse genome were identified in both the EBV positive and negative tumor samples in vivo, although, differences between the EBV positive and negative cells were not apparent. However, the tumors appeared to be grossly distinct. The majority of the identified canonical pathways based on two fold changes in expression had decreased activity within the tumors in vivo. Identification of the predicted upstream regulating factors revealed that in vitro the regulating factors were primarily protein transcriptional regulators. In contrast, in vivo the predicted regulators were frequently noncoding RNAs. Hierarchical clustering distinguished the cell lines and tumors, the EBV positive tumors from the EBV negative tumors, and the NPC tumors from the gastric tumors and cell lines. The delineating genes were changed greater than 4 fold and were frequently regulated by protein transcription factors. These data suggest that EBV distinctly affects cellular expression in gastric tumors and NPC and that growth in vivo requires activation of fewer cellular signaling pathways. It is likely that the broad changes in cellular expression that occur at low levels are controlled by regulatory viral and cellular RNAs while major changes are affected by induced protein regulators., Author summary This study analyzes the effects of the Epstein Barr virus on cellular RNA expression in epithelial cells that were grown in culture dishes or were inoculated into immunodeficient mice to form tumors. The presence of EBV induced many changes in expression and affected the appearance of the tumors which were very bloody with EBV and solid and white without EBV. Multiple cellular pathways that were activated during growth in culture were decreased when grown as tumors. Many of the cellular genes that were affected and were changed approximately two fold were predicted to be regulated by noncoding RNAs. The clustering expression analysis identified greater changes in expression and these changes distinguished tumors from the cell lines, the gastric cells from the nasopharyngeal cancer cells, and those that contained EBV from the EBV negative cells. These findings indicate that EBV alters cell growth through major effects on cellular RNA transcription and that these changes can be induced by both noncoding RNAs and cellular transcription factors.

The Epstein-Barr virus (EBV) is an important human pathogen that is associated with multiple cancers. The major oncoprotein of the virus, latent membrane protein 1 (LMP1), is essential for EBV B-cell immortalization and is sufficient to transform rodent fibroblasts. This viral transmembrane protein activates multiple cellular signaling pathways by engaging critical effector molecules and thus acts as a ligand-independent growth factor receptor. LMP1 is thought to signal from internal lipid raft containing membranes; however, the mechanisms through which these events occur remain largely unknown. Lipid rafts are microdomains within membranes that are rich in cholesterol and sphingolipids. Lipid rafts act as organization centers for biological processes, including signal transduction, protein trafficking, and pathogen entry and egress. In this study, the recruitment of key signaling components to lipid raft microdomains by LMP1 was analyzed. LMP1 increased the localization of phosphatidylinositol 3-kinase (PI3K) and its activated downstream target, Akt, to lipid rafts. In addition, mass spectrometry analyses identified elevated vimentin in rafts isolated from LMP1 expressing NPC cells. Disruption of lipid rafts through cholesterol depletion inhibited PI3K localization to membranes and decreased both Akt and ERK activation. Reduction of vimentin levels or disruption of its organization also decreased LMP1-mediated Akt and ERK activation and inhibited transformation of rodent fibroblasts. These findings indicate that LMP1 reorganizes membrane and cytoskeleton microdomains to modulate signal transduction.

Summary Kaposi's sarcoma-associated herpesvirus (KSHV) is linked to human malignancies. The majority of tumor cells harbor latent virus, and a small percentage undergo spontaneous lytic replication. Both latency and lytic replication are important for viral pathogenesis and spread, but the cellular players involved in the switch between the two viral life-cycle phases are not clearly understood. We conducted a small interfering RNA (siRNA) screen targeting the cellular kinome and identified Tousled-like kinases (TLKs) as cellular kinases that control KSHV reactivation from latency. Upon treatment of latent KSHV-infected cells with siRNAs targeting TLKs, we saw robust viral reactivation. Knockdown of TLKs in latent KSHV-infected cells induced expression of viral lytic proteins and production of infectious virus. TLKs were also found to play a role in regulating reactivation from latency of another related oncogenic gammaherpesvirus, Epstein-Barr virus. Our results establish the TLKs as cellular repressors of gammaherpesvirus reactivation.

Epstein-Barr virus (EBV) is a highly prevalent herpesvirus associated with epithelial cancers, including nasopharyngeal carcinoma (NPC). The EBV protein latent membrane protein 2 (LMP2) is expressed in NPC tumor tissue and has been shown to induce transformation, inhibit differentiation, and promote migration of epithelial cells. In this study, the effect of LMP2A on migration of human epithelial cells was further analyzed. LMP2A expression induced migration in human foreskin keratinocytes (HFK) and HaCaT keratinocytes measured by wound healing scratch assay and chemoattractant-induced Transwell migration assay. The induction of migration by LMP2A required the ITAM signaling domain of LMP2A and activation of the Syk tyrosine kinase. LMP2A-induced Transwell migration required the Akt signaling pathway, and activation of Akt by LMP2A required the ITAM signaling domain of LMP2A. LMP2A also induced phosphorylation of the Akt target GSK3β, a Wnt signaling mediator that has been shown to regulate the activity of focal adhesion kinase (FAK), a tyrosine kinase activated by clustering and ligand interaction of integrins. Inhibition of either FAK or its signaling mediator Src kinase inhibited LMP2A-induced migration. Interestingly, αV-integrin was greatly increased in membrane-enriched fractions by LMP2A, and a neutralizing antibody to αV-integrin blocked migration, suggesting that the effects of LMP2A on membrane-localized αV-integrin promoted migration. The results of this study indicate that LMP2A expression in human epithelial cells induces αV-integrin-dependent migration through a mechanism requiring ITAM-mediated Syk and Akt activation and inducing membrane translocation or stabilization of αV-integrin and FAK activation. The specific effects of LMP2A on an integrin with a diverse repertoire of ligand specificities could promote migration of different cell types and be initiated by multiple chemoattractants.

Epstein-Barr virus is an etiologic factor in multiple types of cancer that primarily develop in lymphocytes and epithelial cells. The tumors are latently infected yet express distinct subsets of viral proteins that are essential for transformation. The viral oncogenes may be expressed in a subset of cells and are transferred through exosomes to many cells to induce growth and alter the tumor environment. In some of the viral cancers, viral proteins are not expressed, however, the viral miRNAs can alter growth by decreasing expression of negative regulators of cell growth such as tumor suppressors and cellular proteins that induce apoptosis.

Cells secrete various membrane-enclosed microvesicles from their cell surface (shedding microvesicles) and from internal, endosome-derived membranes (exosomes). Intriguingly, these vesicles have many characteristics in common with enveloped viruses, including biophysical properties, biogenesis, and uptake by cells. Recent discoveries describing the microvesicle-mediated intercellular transfer of functional cellular proteins, RNAs, and mRNAs have revealed additional similarities between viruses and cellular microvesicles. Apparent differences include the complexity of viral entry, temporally regulated viral expression, and self-replication proceeding to infection of new cells. Interestingly, many virally infected cells secrete microvesicles that differ in content from their virion counterparts but may contain various viral proteins and RNAs. For the most part, these particles have not been analyzed for their content or functions during viral infection. However, early studies of microvesicles (L-particles) secreted from herpes simplex virus-infected cells provided the first evidence of microvesicle-mediated intercellular communication. In the case of Epstein-Barr virus, recent evidence suggests that this tumorigenic herpesvirus also utilizes exosomes as a mechanism of cell-to-cell communication through the transfer of signaling competent proteins and functional microRNAs to uninfected cells. This review focuses on aspects of the biology of microvesicles with an emphasis on their potential contributions to viral infection and pathogenesis.

In Epstein-Barr Virus infected epithelial cancers, the alternatively spliced BamHI A rightward transcripts (BARTs) are abundantly expressed and are the template for two large clusters of miRNAs. This study indicates that both of these clusters independently can inhibit apoptosis in response to etoposide in an epithelial cell line. The Bcl-2 interacting mediator of cell death (Bim) was identified using gene expression microarrays and bioinformatic analysis indicated multiple potential binding sites for several BART miRNAs in the Bim 3'UTR. Bim protein was reduced by Cluster I and the individual expression of several miRNAs, while mRNA levels were unaffected. In reporter assays, the Bim 3' untranslated region (UTR) was inhibited by both clusters but not by any individual miRNAs. These results are consistent with the BART miRNAs downregulating Bim post-transcriptionally in part through the 3'UTR and suggest that there are miRNA recognition sites within other areas of the Bim mRNA.

Epstein-Barr Virus (EBV) latent membrane protein 1 (LMP1) is required for EBV B-lymphocyte transformation, transforms rodent fibroblasts, and can induce lymphoma and epithelial hyperplasia in transgenic mice. Two domains have been identified within the intracellular carboxy terminus that can activate NF-κB, C-terminus-activating region 1 (CTAR1) and CTAR2, through interactions with tumor necrosis receptor-associated factors (TRAFs). CTAR1 can activate both the canonical and noncanonical NF-κB pathways and has unique effects on cellular gene expression. The epidermal growth factor receptor (EGFR) is highly induced by LMP1-CTAR1 in epithelial cells through activation of a novel NF-κB form containing p50 homodimers and Bcl-3. To further understand the regulation of NF-κB in CTAR1-induced EGFR expression, we evaluated the ability of CTAR1 to induce EGFR in mouse embryonic fibroblasts (MEFs) defective for different NF-κB effectors. CTAR1-mediated EGFR induction required the N F-κB- i nducing k inase (NIK) but not the IκB kinase (IKK) complex components that regulate canonical or noncanonical NF-κB pathways. CTAR1-mediated induction of nuclear p50 occurred in IKKβ-, IKKγ-, and NIK-defective MEFs, indicating that this induction is not dependent on the canonical or noncanonical NF-κB pathways. EGFR and nuclear p50 were expressed at high levels in TRAF2 −/− fibroblasts and were not induced by CTAR1. In TRAF3 −/− MEFs, CTAR1 induced nuclear p50 but did not affect basal levels of STAT3 serine phosphorylation or induce EGFR expression. EGFR was induced by LMP1 in TRAF6 −/− MEFs. These findings suggest that this novel NF-κB pathway is differentially regulated by TRAF2 and TRAF3, and that distinct interactions of LMP1 and its effectors regulate LMP1-mediated gene expression.

LMP1 induces the phenotypic transformation of fibroblasts and affects regulators of the cell cycle during this process. LMP1 decreases expression of the cyclin-dependent kinase inhibitor p27 and increases the levels and phosphorylation of cyclin-dependent kinase 2 and the retinoblastoma protein. In the present study, the effects of LMP1 on cell cycle progression and the mechanism of p27 downregulation by LMP1 were determined. Although p27 is frequently regulated at the posttranscriptional level during cell cycle progression and in cancer, LMP1 did not decrease ectopically expressed p27. However, LMP1 did decrease p27 RNA levels and inhibited the activity of p27 promoter reporters. The LMP1-regulated promoter element was mapped to a region containing two E2F sites. Electrophoretic mobility shift assays determined that the regulated cis element bound an inhibitory E2F complex containing E2F4 and p130. These findings indicate that LMP1 decreases p27 transcription through effects on E2F family transcription factors. This property likely contributes to the ability of LMP1 to stimulate cell cycle progression.

Latent Epstein-Barr virus (EBV) infection is associated with several lymphoproliferative disorders, including posttransplant lymphoma, Hodgkin's disease, and Burkitt's lymphoma, as well as nasopharyngeal carcinoma (NPC). Twenty-nine microRNAs (miRNAs) have been identified that are transcribed during latent infection from three clusters in the EBV genome. Two of the three clusters of miRNAs are made from the BamHI A rightward transcripts (BARTs), a set of alternatively spliced transcripts that are highly abundant in NPC but have not been shown to produce a detectable protein. This study indicates that while the BART miRNAs are located in the first four introns of the transcripts, processing of the pre-miRNAs from the primary transcript occurs prior to completion of the splicing reaction. Additionally, production of the BART miRNAs correlates with accumulation of a spliced mRNA in which exon 1 is joined directly to exon 3, suggesting that this form of the transcript may favor production of miRNAs. Sequence variations and processing of pre-miRNAs to the mature form also may account for various differences in miRNA abundance. Importantly, residual intronic pieces that result from processing of the pre-miRNAs were detected in the nucleus. The predicted structures of these pieces suggest there is a bias or temporal pattern to the production of the individual pre-miRNAs. These findings indicate that multiple factors contribute to the production of the BART miRNAs and to the apparent differences in abundance between the individual miRNAs of the cluster.

Latent membrane protein 1 (LMP1), the major oncoprotein of EBV, is likely responsible for many of the altered cellular growth properties in EBV-associated cancers, including nasopharyngeal carcinoma (NPC). In this study, the effects of LMP1 on cell growth and migration were studied in the context of the EBV-positive C666-1 NPC cell line. In the soft agar transformation and Transwell metastasis assays, LMP1 enhanced cell growth and migration through activation of phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor-κB (NF-κB) signaling. Inhibitors of PI3K, Akt, and NF-κB signaling dramatically reduced these enhanced properties. An IκBα super-repressor also blocked these effects. However, constitutive activation of Akt alone did not alter cell growth, suggesting that both PI3K/Akt and NF-κB activation are required by LMP1. These enhanced effects required the full-length LMP1 encompassing both the PI3K/Akt-activating COOH-terminal activation region (CTAR) 1 and the nonredundant NF-κB–activating regions CTAR1 and CTAR2. LMP2A, a latent protein that is also frequently expressed in NPC, similarly activates the PI3K/Akt pathway; however, its overexpression in C666-1 cells did not affect cell growth or migration. LMP1 also decreased expression of the junctional protein plakoglobin, which was shown to be partially responsible for enhanced migration induced by LMP1. This study reveals that in epithelial cells the transforming properties of LMP1 require activation of both PI3K/Akt and NF-κB and shows that the loss of plakoglobin expression by LMP1 is a significant factor in the enhanced migration. [Cancer Res 2008;68(17):6997–7005]

LMP1 induces the expression of two members of the family of Id proteins, Id1 and Id3, and affects cell cycle regulation by decreasing the expression of the cyclin dependent kinase inhibitor, p27, and increasing levels and phosphorylation of cdk2 and Rb. In the present study, the contribution of the Id proteins to LMP1-mediated transformation was determined. Although LMP1 effectively inhibited p27 expression, the Id proteins alone did not affect expression of p27, cdk2, and Rb. Neither Id1 nor Id3 was sufficient to transform Rat-1 cells and inhibition of Id1 expression did not affect LMP1-induced morphologic transformation of Rat-1 cells or reduction of p27. However, reduced Id expression resulted in smaller foci and impaired the growth rate of Rat-1 cells. These data indicate that overexpression of the Id proteins is not sufficient for the effects of LMP1 on the cell cycle but that inhibition of Id expression does affect the growth of LMP1-transformed and parental Rat1 cells.

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) activates multiple signaling pathways. Two regions, C-terminal-activating region 1 (CTAR1) and CTAR2, have been identified within the cytoplasmic carboxy terminal domain that activates NF-κB. CTAR2 activates the canonical NF-κB pathway, which includes p50/p65 complexes. CTAR1 can activate both the canonical and noncanonical pathways to produce multiple distinct NF-κB dimers, including p52/p50, p52/p65, and p50/p50. CTAR1 also uniquely upregulates the epidermal growth factor receptor (EGFR) in epithelial cells. Increased p50-Bcl-3 complexes have been detected by chromatin precipitation on the NF-κB consensus motifs within theegfrpromoter in CTAR1-expressing epithelial cells and nasopharyngeal carcinoma cells. In this study, the mechanism responsible for the increase in Bcl-3 has been further investigated. The data indicate that LMP1-CTAR1 induces Bcl-3 mRNA and increases the nuclear translocation of both Bcl-3 and p50. LMP1-CTAR1 constitutively activates STAT3, and this activation was not due to the induction of interleukin 6 (IL-6). In LMP1-CTAR1-expressing cells, increased levels of activated STAT3 were detected by chromatin immunoprecipitation on STAT-binding sites located within both the promoter and the second intron of Bcl-3. A STAT3 inhibitor significantly reduced the activation of STAT3, as well as the CTAR1-mediated upregulation of Bcl-3 and EGFR. These data suggest that LMP1 activates distinct forms of NF-κB through multiple pathways. In addition to activating the canonical and noncanonical pathways, LMP1-CTAR1 constitutively activates STAT3 and increases Bcl-3. The increased nuclear Bcl-3 and p50 homodimer complexes positively regulate EGFR expression. These results indicate that LMP1 likely regulates distinct cellular genes by activating specific NF-κB pathways.

The Epstein-Barr virus (EBV) is associated with the development of numerous malignancies, including the epithelial malignancy nasopharyngeal carcinoma (NPC). The viral oncoprotein latent membrane protein 1 (LMP1) is expressed in almost all EBV-associated malignancies and has profound effects on gene expression. LMP1 acts as a constitutively active tumor necrosis factor receptor and activates multiple forms of the NF-κB family of transcription factors. LMP1 has two domains that both activate NF-κB. In epithelial cells, LMP1 C-terminal activating region 1 (CTAR1) uniquely activates p50/p50-, p50/p52-, and p65-containing complexes while CTAR2 activates canonical p50/p65 complexes. CTAR1 also uniquely upregulates the epidermal growth factor receptor (EGFR). In NPC, NF-κB p50/p50 homodimers and the transactivator Bcl-3 were detected on the EGFR promoter. In this study, the role of NF-κB p50 and Bcl-3 in LMP1-mediated upregulation of EGFR was analyzed. In LMP1-CTAR1-expressing cells, chromatin immunoprecipitation detected p50 and Bcl-3 on the NF-κB consensus sites within the egfr promoter. Transient overexpression of p50 and Bcl-3 increased EGFR expression, confirming the regulation of EGFR by these factors. Treatment with p105/p50 siRNA effectively reduced p105/p50 levels but unexpectedly increased Bcl-3 expression and levels of p50/Bcl-3 complexes, resulting in increased EGFR expression. These data suggest that induction of p50/p50/Bcl-3 complexes by LMP1 CTAR1 mediates LMP1-induced EGFR upregulation and that formation of the p50/p50/Bcl-3 complex is negatively regulated by the p105 precursor. The distinct forms of NF-κB that are induced by LMP1 CTAR1 likely activate distinct cellular genes.

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) gene is considered the EBV oncogene as it is necessary for EBV-mediated transformation of B lymphocytes and itself transforms rodent fibroblasts. LMP1 activates the NF-κB, phosphatidylinositol 3-kinase (PI3K)-Akt, mitogen-activated protein kinase, and Jun N-terminal protein kinase signaling pathways through its two signaling domains, carboxyl-terminal activating regions 1 and 2 (CTAR1 and CTAR2). CTAR1 and CTAR2 induce signal transduction pathways through their direct (CTAR1) or indirect (CTAR2) recruitment of tumor necrosis factor receptor-associated factors (TRAFs). CTAR1 is necessary for LMP1-mediated transformation as well as activation of PI3K signaling and induction of cell cycle markers associated with G 1 /S transition. In this study, activation of PI3K-Akt signaling and deregulation of cell cycle markers were mapped to the TRAF-binding domain within CTAR1 and to the residues between CTAR1 and CTAR2. LMP1 CTAR1 also activated the MEK1/2-extracellular signal-regulated kinase 1/2 signaling pathway, and this activation was necessary for LMP1-induced transformation of Rat-1 fibroblasts. Dominant-negative forms of TRAF2 and TRAF3 inhibited but did not fully block LMP1-mediated transformation. These findings identify a new signaling pathway that is uniquely activated by the TRAF-binding domain of LMP1 and is required for transformation.

The Epstein-Barr herpesvirus (EBV) is an important human pathogen that is closely linked to several major malignancies including the major epithelial tumor, undifferentiated nasopharyngeal carcinoma (NPC). This important tumor occurs with elevated incidence in specific areas, particularly in southern China but also in Mediterranean Africa and some regions of the Middle East. Regardless of tumor prevalence, undifferentiated NPC is consistently associated with EBV. The consistent detection of EBV in all cases of NPC, the maintenance of the viral genome in every cell, and the continued expression of viral gene products suggest that EBV is a necessary factor for the malignant growth in vivo. However, the molecular characterization of the infection and identification of critical events have been hampered by the difficulty in developing in vitro models of NPC. Epithelial cell infection is difficult in vitro and in contrast to B-cell infection does not result in immortalization and transformation. Cell lines established from NPC usually do not retain the genome, and the successful establishment of tumor xenografts is difficult. However, critical genetic changes that contribute to the onset and progression of NPC and key molecular properties of the viral genes expressed in NPC have been identified. In some cases, viral expression becomes increasingly restricted during tumor progression and tumor cells may express only the viral nuclear antigen EBNA1 and viral noncoding RNAs. As NPC develops in the immunocompetent, the continued progression of deregulated growth likely reflects the combination of expression of viral oncogenes in some cells and viral noncoding RNAs that likely function synergistically with changes in cellular RNA and miRNA expression.

The Epstein-Barr virus protein latent membrane protein 1 (LMP1) has two NF-κB activating domains within its intracellular carboxy terminus (carboxy-terminal activating region 1 [CTAR1] and CTAR2). LMP1-CTAR1 is required for B-lymphocyte transformation, is capable of transforming rodent fibroblasts, and uniquely activates phosphoinositol (PI3) kinase, the noncanonical NF-κB pathway, and expression of the epidermal growth factor receptor (EGFR). In this study, the effects of LMP1-CTAR1 on cellular gene expression were determined by high-throughput sequencing. Additionally, the binding of bcl3 was determined using chromatin immunoprecipitation (ChIP) and sequencing. LMP1-CTAR1 induced few changes in transcription with more genes showing decreased expression. Ingenuity pathway analysis indicated significant enrichment for genes involved in cancer and cellular movement, survival, growth, and proliferation pathways. ChIP in combination with high-throughput sequencing (ChIP-Seq) identified bcl3 binding for more than 2,000 genes in LMP1-CTAR1-expressing cells with more than 90% of the peaks at genes detected within the probable promoter region. Only a small subset of the genes with significant changes in expression had corresponding peaks in the bcl3 ChIP. However, both NFKB2 and PI3 kinase were identified in the bcl3 ChIP. Additionally, many of the predicted upstream regulators for the changes in expression were identified in the bcl3 ChIP. Analysis of the proteins in the NF-κB pathway revealed many changes identified by the high-throughput RNA sequencing (RNA-Seq) and bcl3 ChIP that would likely activate noncanonical NF-κB signaling and possibly inhibit canonical NF-κB signaling. These findings suggest that the two LMP1 signaling domains modulate their combined activity and that the bcl3 transcription factor is likely responsible for some of the unique effects of CTAR1 on cellular expression., IMPORTANCE The Epstein-Barr virus protein latent membrane protein 1 (LMP1) has potent effects on cell growth. LMP1 has two regions, carboxy-terminal activating region 1 (CTAR1) and CTAR2, that distinctly activate NF-κB, a transcription factor complex involved in activation of important host genes. In this study, analysis of the effects on cellular gene expression revealed that CTAR1 significantly affected cellular expression in part through effects on a specific form of NF-κB. The data suggest that LMP1 can activate a distinct subset of host gene expression through its CTAR1 domain which in combination with other signaling effects induced by the CTAR2 domain likely affects cell movement, survival, and growth.

In Epstein-Barr virus-infected epithelial cancers, the alternatively spliced BamHI A rightward transcripts (BARTs) are the most abundant viral polyadenylated RNA. The BART introns form the template for the production of 44 microRNAs (miRNAs), and the spliced and polyadenylated exons form nuclear non-protein-coding RNAs. Analysis of host cell transcription by RNA-seq during latency in AGS cells identified a large number of reproducibly changed genes. Genes that were downregulated were enriched for BART miRNA targets. Bioinformatics analysis predicted activation of the myc pathway and downregulation of XBP1 as likely mediators of the host transcriptional changes. Effects on XBP1 activity were not detected in these cells; however, myc activation was confirmed through use of a myc-responsive luciferase reporter. To identify potential regulatory properties of the spliced, polyadenylated BART RNAs, a full-length cDNA clone of one of the BART isoforms was obtained and expressed in the Epstein-Barr virus (EBV)-negative AGS cells. The BART cDNA transcript remained primarily nuclear yet induced considerable and consistent changes in cellular transcription, as profiled by RNA-seq. These transcriptional changes significantly overlapped the transcriptional changes induced during latent EBV infection of these same cells, where the BARTs are exclusively nuclear and do not encode proteins. These data suggest that the nuclear BART RNAs are functional long noncoding RNAs (lncRNAs). The abundant expression of multiple forms of noncoding RNAs that contribute to growth regulation without expression of immunogenic proteins would be an important mechanism for viral oncogenesis in the presence of a functional immune system. IMPORTANCE Infection with Epstein-Barr virus (EBV) is nearly ubiquitous in the human population; however, it does contribute to the formation of multiple types of cancer. In immunocompromised patients, EBV causes multiple types of lymphomas by expressing viral oncogenes that promote growth and survival of infected B lymphocytes. EBV-positive gastric carcinoma does not require immune suppression, and the viral oncoproteins that are frequent targets for an immunological response are not expressed. This study demonstrates using transcriptional analysis that the expression of various classes of viral non-protein-coding RNAs likely contribute to the considerable changes in the host transcriptional profile in the AGS gastric cancer cell line. This is the first report to show that the highly expressed polyadenylated BamHI A rightward transcripts (BART) viral transcript in gastric carcinoma is in fact a functional viral long noncoding RNA. These studies provide new insight into how EBV can promote transformation in the absence of viral protein expression.

The Epstein–Barr herpesvirus (EBV) is an important human pathogen that is closely linked to several major malignancies including the major epithelial tumor, undifferentiated nasopharyngeal carcinoma (NPC). This important tumor occurs with elevated incidence in specific areas, particularly in southern China but also in Mediterranean Africa and some regions of the Middle East. Regardless of tumor prevalence, undifferentiated NPC is consistently associated with EBV. The consistent detection of EBV in all cases of NPC, the maintenance of the viral genome in every cell, and the continued expression of viral gene products suggest that EBV is a necessary factor for the malignant growth in vivo. However, the molecular characterization of the infection and identification of critical events have been hampered by the difficulty in developing in vitro models of NPC. Epithelial cell infection is difficult in vitro and in contrast to B-cell infection does not result in immortalization and transformation. Cell lines established from NPC usually do not retain the genome, and the successful establishment of tumor xenografts is difficult. However, critical genetic changes that contribute to the onset and progression of NPC and key molecular properties of the viral genes expressed in NPC have been identified. In some cases, viral expression becomes increasingly restricted during tumor progression and tumor cells may express only the viral nuclear antigen EBNA1 and viral noncoding RNAs. As NPC develops in the immunocompetent, the continued progression of deregulated growth likely reflects the combination of expression of viral oncogenes in some cells and viral noncoding RNAs that likely function synergistically with changes in cellular RNA and miRNA expression.

Epstein-Barr Virus (EBV) is a gammaherpesvirus that infects the majority of the human population and is linked to the development of multiple cancers, including nasopharyngeal carcinoma. Latent membrane protein 1 (LMP1) is considered the primary oncoprotein of EBV, and in epithelial cells it induces the expression and activation, or phosphorylation, of the epidermal growth factor receptor kinase. To identify effects on additional kinases, an unbiased screen of receptor tyrosine kinases potentially activated by LMP1 was performed. Using a protein array, it was determined that LMP1 selectively activates insulin-like growth factor 1 receptor (IGF1R). This activation takes place in fibroblast, epithelial, and nasopharyngeal cell lines that express LMP1 stably and transiently. Of note, LMP1 altered the phosphorylation, but not the expression, of IGF1R. The use of LMP1 mutants with defective signaling domains revealed that the C-terminal activating region 2 domain of LMP1 increased the mRNA expression and the secretion of the ligand IGF1, which promoted phosphorylation of IGF1R. IGF1R phosphorylation was dependent upon activation of canonical NF-κB signaling and was suppressed by IκBα and a dominant negative form of TRAF6. Inhibition of IGF1R activation with two small-molecule inhibitors, AG1024 and picropodophyllin (PPP), or with short hairpin RNA (shRNA) directed against IGF1R selectively reduced proliferation, focus formation, and Akt activation in LMP1-positive cells but did not impair LMP1-induced cell migration. Expression of constitutively active Akt rescued cell proliferation in the presence of IGF1R inhibitors. These findings suggest that LMP1-mediated activation of IGF1R contributes to the ability of LMP1 to transform epithelial cells. IMPORTANCE EBV is linked to the development of multiple cancers in both lymphoid and epithelial cells, including nasopharyngeal carcinoma. Nasopharyngeal carcinoma is a major cancer that develops in specific populations, with nearly 80,000 new cases reported annually. LMP1 is consistently expressed in early lesions and continues to be detected within 50 to 80% of these cancers at later stages. It is therefore of paramount importance to understand the mechanisms through which LMP1 alters cell growth and contributes to tumorigenesis. This study is the first to determine that LMP1 activates the IGF1R tyrosine kinase by regulating expression of the ligand IGF1. Additionally, the data in this paper reveal that specific targeting of IGF1R selectively impacts LMP1-positive cells. These findings suggest that therapies directed against IGF1R may specifically impair the growth of EBV-infected cells.

The ubiquitous herpesvirus Epstein-Barr virus (EBV) is linked to the development of several malignancies, including nasopharyngeal carcinoma. Latent membrane protein 1 (LMP1) is considered the EBV oncogene as it is necessary for EBV-induced transformation of B lymphocytes and is able to transform Rat-1 fibroblasts. LMP1 can activate a wide array of signaling pathways, including phosphatidylinositol 3-kinase (PI3K)-Akt and NF-κB. Six sequence variants of LMP1, termed Alaskan, China 1, China 2, Med+, Med−, and NC, have been identified, and individuals can be infected with multiple variants. The frequencies of detection of these variants differ for various EBV-associated malignancies from different geographic regions. In this study, the biological and signaling properties of the LMP1 variants have been characterized. All of the LMP1 variants transformed Rat-1 fibroblasts, induced increased motility of HFK cells, and induced increased homotypic adhesion of BJAB cells. While all the variants activated the PI3K-Akt signaling pathway to similar extents, the Alaskan, China 1, and Med+ variants had limited binding to the E3 ubiquitin ligase component homologue of Slimb and had slightly enhanced NF-κB signaling. These findings indicate that the signature amino acid changes of the LMP1 variants do not hinder or enhance their in vitro transforming potentials or affect their signaling properties.

金沢大学医学部附属病院耳鼻咽喉科, In nasopharyngeal carcinoma (NPC), Epstein-Barr virus (EBV) infection is mainly latent, and the tumor cells contain episomal viral DNA. We have shown that the acyclic nucleoside phosphonate analog, cidofovir [(S)-1-(3-hydroxy-2- (phosphonylmethoxypropyl)cytosine] (HPMPC), inhibits growth of NPC xenografts in nude mice by causing apoptosis. The ribonucleotide reductase (RR) inhibitors, hydroxyurea and didox (3,4-dihydroxybenzohydroxamic acid), have been demonstrated to inhibit neoplastic growth and are used as antiviral and anticancer agents. Here we show that RR inhibitors enhance the antitumor effect of cidofovir in EBV-transformed epithelial cells. MTT assays indicate that hydroxyurea and didox enhance cidofovir-induced cell toxicity in NPC-KT cells, an EBV-positive epithelial cell line derived from NPC. The effect is due to enhancement of apoptosis through the caspase cascade as shown by pronounced cleavage of poly(ADP-ribose) polymerase. Finally, hydroxyurea strikingly enhanced the cidofovir-induced growth-inhibitory effect on NPC grown in athymic mice. The results suggest that RR inhibitors should enhance the antitumor effect of acyclic nucleoside phosphonate analogs on NPC. © 2005 Wiley-Liss, Inc.

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus associated with a variety of malignancies including nasopharyngeal carcinoma. The EBV-encoded latent membrane protein 1 (LMP1) is considered the EBV oncogene as it is necessary for EBV-induced B-lymphocyte transformation and has been shown to transform rodent fibroblasts. LMP1 contains two signaling domains, the carboxy-terminal activating region 1 and 2 (CTAR1 and CTAR2), by which NF-kappaB, phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase, and c-Jun N-terminal kinase are activated. In this study, the role of CTAR1 and CTAR2 in LMP1-mediated transformation of rodent fibroblasts was analysed. CTAR1 was found to be necessary for rodent fibroblast transformation, whereas CTAR2 was dispensable. The activation of the PI3K pathway in Rat-1 cells by LMP1 and LMP1-CTAR1 in transformed cells resulted in phosphorylated Akt and phosphorylated glycogen synthase kinase 3beta. The role of PI3K and NF-kappaB activation in LMP1-mediated transformation was further analysed using the chemical inhibitors LY294002 and BAY 11-7085. LY294002 inhibited CTAR1-induced focus formation and anchorage-independent growth, whereas BAY 11-7085 did not inhibit focus formation or anchorage-independent growth. Similar studies in human fibroblasts confirmed that LMP1-CTAR1 also mediates aberrant growth, phosphorylation of Akt, and decreased levels of p27. These findings indicate that LMP1-mediated rodent fibroblast transformation is dependent upon activation of PI3K and Akt and is independent of activation of NF-kappaB.

Epstein-Barr virus (EBV) is the major cause of infectious mononucleosis and is associated with several malignancies including nasopharyngeal carcinoma, gastric carcinoma, Hodgkin lymphoma, Burkitt lymphoma, and lymphoma after organ or stem cell transplant. A candidate vaccine containing soluble EBV glycoprotein gp350 protected cottontop tamarins from EBV lymphoma after challenge with EBV. In the only phase 2 trial of an EBV vaccine in humans, soluble gp350 in alum and monophosphoryl lipid A adjuvant reduced the rate of infectious mononucleosis in EBV seronegative adults, but did not affect the rate of EBV infection. A peptide vaccine corresponding to EBV latency proteins has been tested in a small number of adults to prevent infectious mononucleosis. Some of the barriers to development of an EBV vaccine include (a) whether additional viral proteins in addition to gp350 would be more effective for preventing mononucleosis or EBV malignancies, (b) the difficulty of performing clinical trials to prevent EBV associated malignancies in the absence of good surrogate markers for tumor development, and the long period of time between primary EBV infection and development of many EBV tumors, (c) the lack of knowledge of immune correlates for protection against EBV infection and disease, (d) the limitations in animal models to study protection against EBV infection and disease, and (e) the need for additional information on the economic and societal burden of infectious mononucleosis to assess the cost-benefit of a prophylactic vaccine.

Latent membrane protein 1 (LMP1), the Epstein-Barr virus oncoprotein, activates NF-κB, phosphatidylinositol 3-kinase, mitogen-activated protein kinase, and c-Jun N-terminal kinase signaling. To determine global transcriptional changes induced by LMP1 in epithelial cells, genomic analysis of C33A cells stably expressing LMP1 was performed. Relatively few genes were induced by LMP1. Expression of two members of the Id (inhibitor of differentiation) family of proteins, Id1 and Id3, was induced in the presence of LMP1 and confirmed by mRNA and protein in C33A and Rat-1 cells. In Rat-1 foci transformed by LMP1, Id1 protein was also increased. Id proteins are known negative regulators of E-box proteins that positively regulate p16 and potentially other cyclin-dependent kinase inhibitors (cdki's). In LMP1-expressing Rat-1 cells, cdki p27 was specifically downregulated. Decreased p27 was correlated with increased levels of Cdk2 and increased levels of phosphorylated retinoblastoma protein. This study describes new properties of LMP1 that likely contribute to transformation and oncogenesis.

Infectious agents, mainly viruses, are among the few known causes of cancer and contribute to a variety of malignancies worldwide. The agents and cancers considered here are human papillomaviruses (cervical carcinoma); human polyomaviruses (mesotheliomas, brain tumors); Epstein-Barr virus (B-cell lymphoproliferative diseases and nasopharyngeal carcinoma); Kaposi’s Sarcoma Herpesvirus (Kaposi’s Sarcoma and primary effusion lymphomas); hepatitis B and hepatitis C viruses (hepatocellular carcinoma); Human T-cell Leukemia Virus-1 (T-cell leukemias); and helicobacter pylori(gastric carcinoma), which account for up to 20% of malignancies around the globe. The criteria most often used in determining causality are consistency of the association, either epidemiologic or on the molecular level, and oncogenicity of the agent in animal models or cell cultures. However use of these generally applied criteria in deciding on causality is selective, and the criteria may be weighted differently. Whereas for most of the tumor viruses the viral genome persists in an integrated or episomal form with a subset of viral genes expressed in the tumor cells, some agents (HBV, HCV, helicobacter) are not inherently oncogenic, but infection leads to transformation of cells by indirect means. For some malignancies the viral agent appears to serve as a cofactor (Burkitt’s lymphoma-EBV; mesothelioma - SV40). For others the association is inconsistent (Hodgkin’s Disease, gastric carcinomas, breast cancer-EBV) and may either define subsets of these malignancies, or the virus may act to modify phenotype of an established tumor, contributing to tumor progression rather than causing the tumor. In these cases and for the human polyomaviruses the association with malignancy is less consistent or still emerging. In contrast despite the potent oncogenic properties of some strains of human adenovirus in tissue culture and animals the virus has not been linked with any human cancers. Finally it is likely that more agents, most likely viruses, both known and unidentified, have yet to be implicated in human cancer. In the meantime study of tumorigenic infectious agents will continue to illuminate molecular oncogenic processes. © 2004 Elsevier Ltd. All rights reserved.

Background Patients with rheumatoid arthritis or polymyositis treated with methotrexate (MTX) develop Epstein-Barr virus (EBV)-positive lymphomas more frequently than patients treated with other, equally immunosuppressive regimens. Here we determined whether MTX, in contrast to other commonly used medications for rheumatoid arthritis or polymyositis, is unique in its ability to induce the release of infectious EBV from latently infected cells. Methods The effect of MTX and other immunosuppressant drugs on EBV replication in vitro was assessed using latently infected EBV-positive lymphoblastoid and gastric carcinoma cell lines. Inhibitors of signal transduction pathways were used to define requirements for induction of lytic infection. Drug effects on transcription of the two EBV immediate-early promoters (BRLF1 and BZLF1) and on promoter constructs lacking cis-acting sequences required for activation by other effectors was examined using reporter gene assays. EBV viral load in rheumatoid arthritis and polymyositis patients receiving MTX was compared with that in patients receiving other immunosuppressive medications. Statistical tests were two-sided. Results MTX activated the release of infectious EBV from latently infected cell lines in vitro, and MTX treatment was associated with activation of the two viral immediate-early promoters in reporter gene assays. Induction of lytic EBV infection by MTX required the p38 MAP kinase, PI3 kinase, and MEK pathways and specific cis-acting motifs in the two viral immediate-early promoters. Patients treated with MTX-containing regimens had statistically significantly higher mean EBV loads in their blood than patients treated with immunosuppressing regimens that did not include MTX (40 EBV copies per 10(6) cellular genomes versus 5.1 copies; geometric mean fold difference in copies = 10.8, 95%, confidence interval = 3.0 to 38; P = .011). Conclusion MTX may promote EBV-positive lymphomas in rheumatoid arthritis and polymyositis patients by its immunosuppressive properties as well as by reactivating latent EBV.

Epstein-Barr Virus (EBV) infects nearly all humans and then persists for the life of the host. In some people who later develop cancer, EBV DNA is present within malignant cells and circulates at elevated levels in the plasma. In the current study, we validated five novel quantitative polymerase chain reaction (Q-PCR) assays targeting disparate but highly conserved segments of the EBV genome (BamH1W, EBNA1, LMP1, LMP2, and BZLF1). Each assay was sensitive to as few as 50 copies of EBV DNA per reaction and was linear across at least four orders of magnitude. When applied to paraffin-embedded tissues in concert with EBV-encoded RNA (EBER) in situ hybridization, the BamH1W and EBNA1 assays were the most informative, while use of the entire battery of EBV PCR assays may help identify genomic polymorphisms or deletions. Higher viral loads were found in the 17 EBER-positive compared with the 13 EBER-negative tumors (means 84,978 versus 22 copies of EBV per 100,000 cells, respectively). The five Q-PCR assays were also informative in plasma samples where EBV was measurable in all nine patients with lymphoma or infectious mononucleosis, whereas EBV was undetectable in all nine healthy controls. The findings suggest that Q-PCR is an effective method of distinguishing disease-associated virus from incidental virus in paraffin-embedded tissue and in plasma samples.

Epstein-Barr virus (EBV) causes infectious mononucleosis and is associated with cancers in immunocompromised populations. EBV establishes a latent infection and immortalizes and transforms B lymphocytes. Several latent proteins have profound effects on cellular growth, including activation of NF-κB, phosphatidylinositol 3′-OH kinase (PI3K) signaling, and notch signaling. Activation of PI3K can affect the activity of β-catenin, the target of the wnt signaling pathway. Deregulation of β-catenin is associated with a number of malignancies. To determine if β-catenin is regulated by EBV infection, EBV-infected cells were examined for β-catenin levels and localization. β-Catenin was increased in EBV-positive tumor cell lines compared to EBV-negative lines, in EBV-infected Burkitt's lymphoma cell lines, and in EBV-transformed lymphoblastoid cell lines (LCL). In contrast to wnt signaling, EBV consistently induced the accumulation of β-catenin in the cytoplasm but not the nucleus. The β-catenin regulating kinase, glycogen synthase kinase 3β (GSK3β), was shown to be phosphorylated and inactivated in EBV-infected lymphocytes. Inactivated GSK3β was localized to the nucleus of EBV-infected LCL. Neither the cytoplasmic accumulation of β-catenin nor the nuclear inactivation of GSK3β was affected by the inhibition of PI3K signaling. These data indicate that latent infection with EBV has unique effects on β-catenin signaling that are distinct from activation of wnt and independent of its effects on PI3K.

EBV is associated with the epithelial cancer, nasopharyngeal carcinoma (NPC), and the lymphoid malignancy, Hodgkin lymphoma (HL). The EBV latent membrane proteins 1 and 2A are expressed in these tumors. These proteins activate the phosphatidylinositol 3′-OH kinase (PI3K)/Akt pathway, which is commonly activated inappropriately in malignancy. In this study, the status of Akt activation and its targets, glycogen synthase kinase-3β (GSK-3β) and β-catenin, was investigated in NPC and HL clinical specimens. In the majority of HL and NPC specimens, Akt was activated, indicating an important role for this kinase in the development and/or progression of these tumors. Akt phosphorylates and inactivates GSK-3β, a negative regulator of the proto-oncoprotein β-catenin that is aberrantly activated in many cancers. GSK-3β was phosphorylated and inactivated with concomitant nuclear β-catenin accumulation in the majority of NPC specimens. The malignant cells of the majority of HL cases, however, did not have inactivated GSK-3β and lacked nuclear β-catenin expression. These data indicate that this signaling arm of PI3K/Akt is universal and important in NPC pathogenesis but is apparently not affected in HL. These findings point to a divergence in pathways activated by EBV in different cellular contexts.

Opportunistic DNA viruses, particularly members of the herpesvirus family, are frequently the aetiological agents of HIV-associated oral lesions. Oral lesions common to the early phase of the AIDS epidemic, including Kaposi's sarcoma (KS), oral aphthous ulceration, AIDS-associated oral lymphoma, and oral hairy leukoplakia (OHL), have been tested for the prevalence of Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV). While EBV DNA is detected by PCR in all of these lesions, abundant viral replication can only be detected in OHL. In OHL, a novel state of EBV infection has been discovered with concurrent expression of replicative and transforming proteins, with all of these proteins contributing to the development of the lesion. Activation of signalling pathways and up-regulation of the viral receptor, proliferative and antiapoptotic genes by these proteins induce several of the histological features common to OHL, such as acanthosis and hyperproliferation. In contrast to other permissive herpesvirus infections, expression of EBV transforming proteins within the permissively infected OHL tissue enables epithelial cell survival and may enhance viral replication. Detection of KSHV in these HIV-infected individuals has been localized only to their saliva. Replicative and latent KSHV gene products have been detected in association with the development of oral KS lesions. EBV, but not human cytomegalovirus (HCMV), has been detected by PCR in minor salivary gland biopsies of HIV-associated salivary gland disease. Human papillomaviruses (HPV) are associated with oral warts in HIV-positive individuals; a diagnosis that appears to be increasing in frequency in the era of highly active antiretroviral therapy. To date, there appears to be little increase in the incidence of HPV-associated oral cancer. The mechanisms of interaction between HIV and HPV are not fully understood. Expression of viral gene products is clearly important and necessary for the development of multiple AIDS-associated oral lesions.

Nasopharyngeal carcinoma (NPC) is endemic in parts of southern China and is etiologically associated with Epstein-Barr virus (EBV) infection as well as other dietary and environmental factors. Loss of heterozygosity (LOH) on chromosome 3p has been described in NPC from the endemic region. In this study, tumors originating from both the NPC nonendemic area of northern China and the endemic area in southern China were analyzed for LOH at 8 microsatellite markers on chromosome 3. Allele loss was detected at D3S1300 in 3p 14.2 in more than 50% of tumors from both the endemic and nonendemic areas, suggesting that LOH at this locus probably does not account for the endemicity of NPC in southern China. The 3p14.2 region encompasses FHIT, a candidate tumor suppressor gene previously shown to be rearranged in several NPC cell lines. In this study, analysis of FHIT gene structure and transcription in primary tumors did not support a role for FHIT in NPC. However, the high frequency of allele loss at 3p14.2 in NPC from endemic and nonendemic regions supports the possibility that an important tumor suppressor gene other than FHIT complements EBV transformation and resides in this region.

Air pollution is a serious health problem in major cities in Mexico. The concentrations of monitored criteria pollutants have been above the US National Ambient Air Quality Standards for the last decade. To determine whether the number of primary malignant nasal and paranasal neoplasms has increased, we surveyed 256 such cases admitted to a major adult oncology hospital located in metropolitan Mexico City (MMC) for the period from 1976-1997 and to a tertiary hospital in Monterrey, an industrial city, for the period from 1993-1998. The clinical histories and histopathologic material were reviewed, and a brief clinical summary was written for each case. In the MMC hospital the number of newly diagnosed nasal and paranasal neoplasms per year for the period from 1976-1986 averaged 5.1, whereas for the next 11 years it increased to 12.5. The maximal increase was observed in 1995-1997, with an average of 20.3 new cases per year (P = 0.0006). The predominant neoplasms in these series were non-Hodgkin's lymphoma, squamous cell carcinoma, melanoma, adenocarcinoma, Schneiderian carcinoma, and nasopharyngeal carcinoma. In the Monterrey hospital a 2-fold increase in the numbers of newly diagnosed nasal and paranasal neoplasms was recorded between 1993 and 1998. The predominant MMC neoplasm in this series, namely nasal T-cell/natural killer cell non-Hodgkin's lymphoma, is potentially Epstein-Barr virus related. Nasal and paranasal malignant neoplasms are generally rare. Environmental causative factors include exposure in industries such as nickel refining, leather, and wood furniture manufacturing. Although epidemiologic studies have not addressed the relationship between outdoor air pollution and sinonasal malignant neoplasms, there is strong evidence for the nasal and paranasal carcinogenic effect of occupational aerosol complex chemical mixtures. General practitioners and ear, nose, and throat physicians working in highly polluted cities should be aware of the clinical presentations of these patients. Identification of this apparent increase in sinonasal malignant neoplasms in two urban Mexican polluted cities warrants further mechanistic and epidemiologic studies.

The effect of the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) on the activation and differentiation of normal B cells was investigated. B cells of transgenic mice expressing LMP1 under the control of immunoglobulin promoter/enhancer displayed enhanced expression of activation antigens and spontaneously proliferated and produced antibody. Humoral immune responses of LMP1 transgenic mice in CD40-deficient or normal backgrounds revealed that LMP1 mimics CD40 signals to induce extrafollicular B cell differentiation but, unlike CD40, blocks germinal center formation. Thus, these specific properties of LMP1 may determine the site of primary B cell infection and the state of infection in the natural course of EBV infection, whereas subsequent loss of LMP1 expression may affect the site of persistent latent infection.

Nasopharyngeal carcinoma (NPC), which is closely associated with the Epstein-Barr virus (EBV), is a highly metastatic malignant tumor. An important activity in tumor invasion and metastasis is that of the 92-kDa type IV collagenase or gelatinase, matrix metalloproteinase 9 (MMP-9), which mediates the degradation of the basement membrane and extracellular matrix. The expression of MMP-9 has been shown to be enhanced by the EBV oncoprotein, latent membrane protein 1 (LMP-1). LMP-1, which is expressed in NPC, has two essential signaling domains within the carboxy terminus, termed C-terminal activation regions 1 (CTAR-1) and CTAR-2. This study reveals that either signaling domain can activate the MMP-9 promoter and induce MMP-9 activity; however, LMP-1 deletion mutants lacking either CTAR-1 or CTAR-2 had a decreased ability to induce MMP-9 expression. The deletion of both activation regions completely abolished the induction of MMP-9 activity, while the cotransfection of both the CTAR-1 and CTAR-2 deletion mutants restored MMP-9 activity to levels produced by wild-type LMP-1. The NF-κB and activator protein 1 (AP-1) binding sites in the MMP-9 promoter were essential for the activation of MMP-9 gene expression by both CTAR-1 and CTAR-2. The induction of MMP-9 expression by LMP-1 and both CTAR-1 and CTAR-2 mutants was blocked by the overexpression of IκB. The tumor necrosis factor receptor-associated factor (TRAF) pathway also contributed to the activation of the MMP-9 promoter as shown by the use of TRAF-2 and TRAF-3 dominant-negative constructs. These data indicate that the activation of both the NF-κB and AP-1 pathways by LMP-1, CTAR-1, and CTAR-2 is necessary for the activation of MMP-9 expression. In NPC, LMP-1 may contribute to invasiveness and metastasis through the induction of MMP-9 transcription and enzymatic activity.

The latent membrane protein 1 (LMP1) of the Epstein–Barr virus has transforming properties in rodent fibroblasts and is expressed in most of the cancers associated with Epstein–Barr virus (EBV) infection including posttransplant lymphomas, Hodgkin’s disease, nasopharyngeal carcinoma, and AIDS-related lymphomas. In this study, three lineages of LMP1 transgenic mice were established with LMP1 expressed under the control of the Ig heavy chain promoter and enhancer. Lymphoma developed in all three lineages, and the incidence of lymphoma increased significantly with age with lymphomas developing in 42% of transgenic mice over 18 months. The expression of LMP1 was detected at high levels in the lymphoma tissues but only at trace levels in normal lymphoid tissues. Gene rearrangement of the Ig heavy chain indicated monoclonality or oligoclonality in all lymphomas, some of the lymphoid hyperplastic spleens, and some histologically normal spleens. These data reveal that LMP1, without the expression of other EBV genes, is oncogenicin vivoand indicate that LMP1 is a major contributing factor to the development of EBV-associated lymphomas.

The hairy leukoplakia lesion (HLP) is a unique example of a permissive infection with Epstein–Barr virus (EBV) in the tongue epithelium. HLP contains abundant replicating viral DNA and may be coinfected with multiple EBV strains. In this study, characterization of viral gene transcription within HLP biopsy specimens revealed that several genes, usually expressed in latently infected lymphocytes, are also transcribed in the HLP lesion. The Bam HI W and C promoters, (Wp and Cp) are consistently active in the HLP lesion, resulting in transcription and processing of mRNAs that encode the Epstein–Barr nuclear antigens (EBNAs) EBNA-LP, EBNA1, EBNA2, EBNA3B, and EBNA3C. The EBNA2 protein has been shown to activate expression of the EBV receptor, CD21. In HLP, CD21 transcription is also detected, usually in samples that contain transcripts for EBNA2. Transcripts encoding the LMP1 gene, the LMP2 gene, and rightward transcripts from the Bam HI A fragment of the EBV genome are also detected in HLP. These gene products are invariably expressed in latently infected lymphocytes. This pattern of transcription suggests that genes characteristic of latent infection are also expressed in HLP. The activation of Wp and expression of EBNA2 and CD21 may contribute to the unique ability of the HLP lesion to permit superinfection and viral replication of multiple EBV strains.

Nasopharyngeal carcinoma (NPC) occurs with a striking geographic incidence and is endemic in parts of southern China, where it is the major cause of cancer death. Epstein-Barr virus (EBV) is detected in all cells of the majority of NPC cases regardless of geographic origin. A small subset of EBV genes is expressed in NPC, including the latent membrane protein (LMP-1). LMP-1 is essential for transformation of B lymphocytes and is considered to be the EBV oncogene. This analysis of the DNA sequence variation within the LMP-1 gene reveals a consensus sequence for a strain, denoted China1, which predominates in East Asia where NPC is endemic. The China1 strain is characterized by nucleotide changes at 13 loci in the amino terminal portion of the LMP-1 gene when compared with the B95-8 prototype, including a point mutation resulting in the loss of an Xho1 restriction site. This strain was present in 9 of 15 NPC biopsy specimens from the endemic region and in 7 of 13 from northern China, where NPC is non-endemic. A second strain, China2, was detected in 4 of 15 endemic isolates and in 2 of 13 non-endemic isolates; this strain was characterized by a cluster of 5 nucleotide changes in the amino terminal portion of LMP-1 in addition to those seen in China1. It was also marked by distinct changes in the carboxy terminal region of LMP-1 including the retention of amino acids 343-352. All China1 isolates were EBV type 1, whereas the China2 isolates did not correlate with EBV type. Phylogenetic relationships between these 2 strains were determined, as were signature amino acid alterations that discriminate between them.

The Epstein-Barr Virus (EBV) LMP1 protein is frequently expressed in nasopharyngeal carcinoma and is essential for the transforming effects of EBV. Analysis of LMP1 genes isolated from tumor biopsies has revealed considerable sequence variation including deletion of amino acids 343-352. Several studies have suggested that this sequence variation could enhance the transforming potential of LMP1. LMP1 has profound effects on cellular gene expression mediated in part through activation of the NF-kappa B transcription factor. In addition, LMP1 engages the TRAF signaling pathway resulting in the induction of EGFR expression. In this study, the LMP1 proteins derived from the laboratory strain B95-8 and the NPC strain C15 were analysed for their ability to activate NF-kappa B and also to induce expression of the EGFR. The data suggest that the C15-LMP1 protein activates NF-kappa B more efficiently and induces higher levels of the EGFR. Analysis of chimeric LMP1 proteins indicates that the amino terminal 181 amino acids of C15-LMP1 confer this increased signaling capability, and that deletion of amino acids 343-352 does not affect these properties. Finally, these data provide evidence that five amino acid changes within the transmembrane domain in the C15-LMP1 protein lead to enhanced NF-kappa B activation and EGFR induction.

The EBV latent infection transforming protein, LMP1, has six hydrophobic transmembrane domains that enable it to aggregate in the plasma membrane and a 200-amino acid carboxyl-terminal cytoplasmic domain (CT) that activates nuclear factor-κB and induces many of the phenotypic changes in B lymphocytes that accompany CD40 activation. Since the phenotypic effects of LMP1 are similar to those of activated CD40, we now compare signaling from the LMP1 CT with that from the CD40 CT fused to the LMP1 transmembrane domains. The LMPCD40 chimera was similar to LMP1 in nuclear factor-κB activation and in up-regulation of epidermal growth factor receptor expression. CD40 ligation was known to activate the stress-activated protein kinase, and both LMPCD40 and LMP1 are now shown to induce stress-activated protein kinase activity in the absence of ligand. Deletion of the first four transmembrane domains of LMP1 abrogated LMP1 aggregation in the plasma membrane and nearly abolished signaling from LMP1 or the LMPCD40 chimera. These results highlight the role of LMP1 as a constitutively active receptor similar to CD40 and provide a novel approach for the generation of ligand-independent receptors.