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. =20

The BamHI A rightward transcripts are a set of alternatively splicing transcripts produced by Epstein-Barr Virus that are highly expressed in nasopharyngeal carcinoma. These transcripts contain several open reading frames as well as precursors for twenty-two miRNAs. Although the putative proteins corresponding to these open reading frames have not been detected, several studies have identified properties that are interesting and potentially significant with respect to cellular transformation. The miRNAs, however, are very abundant in all nasopharyngeal carcinomas and several potentially significant functions have been identified for some of the miRNAs. This article will focus on the nature of this complicated set of transcripts and the evidence that they contribute to the development of nasopharyngeal carcinoma. Copyright A 2011 Elsevier Ltd. All rights reserved.

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. Copyright =C2=A9 2012 Elsevier B.V. All rights reserved.

;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.

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.=20

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) is expressed in multiple human malignancies and has potent effects on cell growth. It has been detected in exosomes and shown to inhibit immune function. Exosomes are small secreted cellular vesicles that contain proteins, mRNAs, and microRNAs (miRNAs). When produced by malignant cells, they can promote angiogenesis, cell proliferation, tumor-cell invasion, and immune evasion. In this study, exosomes released from nasopharyngeal carcinoma (NPC) cells harboring latent EBV were shown to contain LMP1, signal transduction molecules, and virus-encoded miRNAs. Exposure to these NPC exosomes activated the ERK and AKT signaling pathways in the recipient cells. Interestingly, NPC exosomes also contained viral miRNAs, several of which were enriched in comparison with their intracellular levels. LMP1 induces expression of the EGF receptor in an EBV-negative epithelial cell line, and exosomes produced by these cells also contain high levels of EGF receptor in exosomes. These findings suggest that the effects of EBV and LMP1 on cellular expression also modulate exosome content and properties. The exosomes may manipulate the tumor microenvironment to influence the growth of neighboring cells through the intercellular transfer of LMP1, signaling molecules, and viral miRNAs.

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. =20

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-kappa B (NF-kappa B) signaling. Inhibitors of PI3K, Akt, and NF-kappa B signaling dramatically reduced these enhanced properties. All I kappa B alpha super-repressor also blocked these effects. However, constitutive activation of Akt alone did not alter cell growth, suggesting that both PI3K/Akt and NF-kappa B 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-kappa B-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-kappa B and shows that the loss of plakoglobin expression by LMP1 is a significant factor in the enhanced migration.

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-kappa B, phosphatidylinositol 3-kinase (PI2K)-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). CTARI and CTAR2 induce signal transduction pathways through their direct (CTARI) or indirect (CTAR2) recruitment of tumor necrosis factor receptor-associated factors (TRAFs). CTARI is necessary for LMP1-mediated transformation as well as activation of PI3K signaling and induction of cell cycle markers associated with G,(I)/S transition. In this study, activation of PI3K-Akt signaling and deregulation of cell cycle markers were mapped to the TRAF-binding domain within CTARI and to the residues between CTAR1 and CTAR2. LMP1 CTARI 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.

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that infects more than 90% of the world's adult population and is linked to multiple malignancies, including Burkitt lymphoma, Hodgkin disease, and nasopharyngeal carcinoma (NPC). The EBV oncoprotein LMP1 induces transcription of the epidermal growth factor receptor (EGFR), which is expressed at high levels in NPC. EGFR transcription is induced by LMP1 through a p50 NF kappa B1-Bcl-3 complex, and Bcl-3 is induced by LMP1-mediated activation of STAT3. This study reveals that LMP1, through its carboxyl-terminal activation domain 1 (LMP1-CTAR1), activates both STAT3 and EGFR in a serum-independent manner with constitutive serine phosphorylation of STAT3. Upon treatment with EGF, the LMP1-CTAR1-induced EGFR was additionally phosphorylated and STAT3 became phosphorylated on tyrosine, concomitant with upregulation of a subset of STAT3 target genes. The kinase responsible for LMP1-CTAR1-mediated serine phosphorylation of STAT3 was identified to be PKC delta using specific RNAi, a dominant negative PKC delta, and the PKC delta inhibitor rottlerin. Interestingly, inhibition of PKC delta also inhibited constitutive phosphorylation of EGFR and LMP1-CTAR1-induced phosphorylation of ERK. Inhibition of PKC delta blocked LMP1-CTAR1-mediated transformation of Rat-1 cells, likely through the inhibition of ERK activation. These findings indicate that LMP1 activates multiple distinct signaling pathways and suggest that PKC delta functions as a master regulator of EGFR, STAT3, and ERK activation by LMP1-CTAR1.

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.

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. Copyright =C2=A9 2015, American Society for Microbiology. All Rights Reserved.

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 TO 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. (C) 2008 Published by Elsevier Inc.

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.

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-kappaB, phosphatidylinositol 3'-OH kinase (PI3K) signaling, and notch signaling. Activation of PI3K can affect the activity of beta-catenin, the target of the wnt signaling pathway. Deregulation of beta-catenin is associated with a number of malignancies. To determine if beta-catenin is regulated by EBV infection, EBV-infected cells were examined for beta-catenin levels and localization. beta-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 beta-catenin in the cytoplasm but not the nucleus. The beta-catenin regulating kinase, glycogen synthase kinase 3beta (GSK3beta), was shown to be phosphorylated and inactivated in EBV-infected lymphocytes. Inactivated GSK3beta was localized to the nucleus of EBV-infected LCL. Neither the cytoplasmic accumulation of beta-catenin nor the nuclear inactivation of GSK3beta was affected by the inhibition of PI3K signaling. These data indicate that latent infection with EBV has unique effects on beta-catenin signaling that are distinct from activation of wnt and independent of its effects on PI3K.