Novel mechanisms of EBV-induced oncogenesis

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

Viral effects on the content and function of extracellular vesicles

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

Rhesus lymphocryptovirus latent membrane protein 2A activates β-catenin signaling and inhibits differentiation in epithelial cells

AbstractRhesus lymphocryptovirus (LCV) is a γ-herpesvirus closely related to Epstein–Barr virus (EBV). The rhesus latent membrane protein 2A (LMP2A) is highly homologous to EBV LMP2A. EBV LMP2A activates the phosphatidylinositol 3-kinase (PI3K) and β-catenin signaling pathways in epithelial cells and affects differentiation. In the present study, the biochemical and biological properties of rhesus LMP2A in epithelial cells were investigated. The expression of rhesus LMP2A in epithelial cells induced Akt activation, GSK3β inactivation and accumulation of β-catenin in the cytoplasm and nucleus. The nuclear translocation, but not accumulation of β-catenin was dependent on Akt activation. Rhesus LMP2A also impaired epithelial cell differentiation; however, this process was not dependent upon Akt activation. A mutant rhesus LMP2A lacking six transmembrane domains functioned similarly to wild-type rhesus LMP2A indicating that the full number of transmembrane domains is not required for effects on β-catenin or cell differentiation. These results underscore the similarity of LCV to EBV and the suitability of the macaque as an animal model for studying EBV pathogenesis

Analysis of the Phosphorylation Status of Epstein–Barr Virus LMP2A in Epithelial Cells

LMP2A deletion and point mutants, with mutations in phosphotyrosine-containing protein-protein interaction motifs, were transiently expressed in 293 cells and their phosphorylation was examined in immune complex kinase assays as well as in vivo. In vitro LMP2A phosphorylation depended on tyrosine 112. In vivo, mutations of single tyrosines did not eliminate LMP2 phosphorylation, although mutation of the LMP2A ITAM decreased LMP2A phosphorylation. The relationship between LMP2A in vitro phosphorylation and that induced by cell-extracellular matrix (ECM) interactions was also investigated. While LMP2A was phosphorylated to higher levels in whole-cell extracts of stimulated cells, a difference in in vitro kinase assays with extracts from stimulated and unstimulated cells was not detected, indicating that the ECM-mediated regulation of LMP2A phosphorylation is lost in vitro. In the presence of LMP2A, several cellular proteins with molecular weights between 70 and 80 kDa were phosphorylated on tyrosine. This increase in cellular protein phosphorylation depended on the LMP2A ITAM motif and suggests that the ITAM may participate in signal-transduction events in epithelial cells

LMP1 Promotes Expression of Insulin-Like Growth Factor 1 (IGF1) To Selectively Activate IGF1 Receptor and Drive Cell Proliferation

ABSTRACT 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

Epstein Barr Virus: Potential Immune Selection in Associated Cancers

The EBV latent membrane protein 1 (LMP1) is expressed in most of the EBV-associated malignancies including nasopharyngeal carcinoma (NPC), Hodgkin's Lymphoma, and immunosuppression-associated lymphoma. have been identified by distinguishing amino acids. We have identified seven sequence variants of LMP1 that can be distinguished using a heteroduplex tracking assay and have determined that most healthy individuals are infected with multiple strains of EBV. Striking differences were found between NPC and matching blood samples with one specific variant, China 1, prevalent in NPC samples and multiple other variants of LMP1 prevalent in the blood samples. The possible selection against some strains appearing in the tumor was highly significant with a p < 0.0001. Many of the LMP1 variants had changes in predicted HLA epitopes of various restrictions. The potential negative selection of the immune system on strains detected in the blood would be reflected in the striking predominance of the China 1 strain in the tumors. In lymphoma samples, changes were also frequently detected in known HLA-restricted epitopes of EBNA3A, 3B, or 3C, in addition to LMP1. Variation in potential immune recognition could contribute to the development of EBV-associated diseases in distinct populations and individuals

Signature Amino Acid Changes in Latent Membrane Protein 1 Distinguish Epstein–Barr Virus Strains

Sequence variations in the Epstein-Barr virus (EBV) latent membrane protein 1 gene have been described in numerous EBV-associated tumors with some of these variations, most notably a 30-base pair deletion in the cytoplasmic carboxyl-terminal domain, suggested as associated with an increase in tumorigenicity. In this study, EBV DNA sequence was determined from 92 tissue specimens or cell lines, including nasopharyngeal carcinoma, oral hairy leukoplakia, post-transplant lymphoma, post-transplant without pathology, mononucleosis, Burkitt's lymphoma, parotid tumor, and normal from distinct geographical regions. The amino- and carboxyl-terminal sequences and, in some cases, the full-length sequences of latent membrane protein 1 were determined. Characteristic sequence patterns distinguished strains, with the carboxyl-terminal sequence being the most informative in distinguishing among the strains. Phylogenetic relationships between strains were determined, as were signature amino acid changes that discriminate between them. A correlation between strain and disease or strain and geographic location was not detected. The sequence variation and signature sequences identified at least seven distinct strains, as well as hybrid strains that apparently result from recombination

The A20 Protein Interacts with the Epstein–Barr Virus Latent Membrane Protein 1 (LMP1) and Alters the LMP1/TRAF1/TRADD Complex

The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) interacts with the tumor necrosis factor receptor (TNFR)-associated factor (TRAF) molecules, which are important for LMP1-mediated signaling. Two domains of LMP1 can independently activate NF-kB, carboxyl-terminal activating region 1 (CTAR1) and CTAR2. The activation of NF-kB by CTAR1 occurs through direct interaction of LMP1 with the TRAF molecules, whereas CTAR2 interacts with the TNFR-associated death domain protein (TRADD) to activate NF-kB and the c-Jun N-terminal kinase (JNK). A20, which is induced by LMP1 through NF-kB, can block NF-kB activation from both domains of LMP1 and inhibit JNK activation from CTAR2. A20 also has been shown to associate with TRAF1 and TRAF2. In this study, an interaction between LMP1 and A20 was detected that was increased by TRAF2 overexpression. A20 did not affect the association of TRAF1 with TRAF2 but did displace TRAF1 from the LMP1 complex. The interaction of LMP1 and TRADD was decreased in the presence of A20, and the LMP1-A20 association was decreased by TRADD, suggesting that A20 and TRADD both interact with LMP1 and may compete for binding. These data indicate that A20 alters the interactions between LMP1 and the TRAF molecules and TRADD, affecting the activation of NF-kB, JNK, and perhaps other TRAF-mediated signaling events

Epstein-Barr Virus Latent Membrane Protein 2 Induces Autophagy To Promote Abnormal Acinus Formation

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

The role of miRNAs and EBV BARTs in NPC

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