Length of Epstein-Barr Virus Termini as a Determinant of Epithelial Cell Clonal Emergence

Abstract

Reiterated terminal sequences of Epstein-Barr virus (EBV) DNA are numerically heterogeneous among infectious virions, providing a viral measure of clonality in infected cells. After in vitro infection, carcinoma cells bearing EBV episomes with fewer terminal repeats (TRs) proliferated faster. In single-cell clones, TR number varied inversely to the quantity of latent membrane protein 2A (LMP2A) transcripts whose unspliced precursors cross joined TRs. Thus, EBV clonality may reflect selection for a TR number that optimizes LMP2A-enhanced tumor progression, with infection occurring after epithelial cell transformation.Epstein-Barr virus (EBV) has been linked to numerous human tumors of diverse tissue origin: nasopharyngeal carcinoma, Burkitt's lymphoma, Hodgkin's lymphoma, gastric carcinoma, T-cell lymphomas, leiomyosarcoma, and breast cancer (24). Although the association of EBV with cancers such as gastric carcinoma (ϳ15%), Hodgkin's lymphoma (40 to 60%), and sporadic Burkitt's lymphoma (ϳ30%) is less than complete, assignment of a causal role to EBV has been based in part on the perception that these diseases are clonal expansions of a single EBV-infected cell (2, 23). Upon entry, linear EBV DNA circularizes by homologous recombination of its terminal repeats (TRs), producing fused termini of unique length for each circularization event (13). Unlike the length heterogeneity found with a mixed-cell population at primary infection, fused TRs with an identical number of repeat units denote expansions of a single infected progenitor (Fig. 1A). Detection of a single form of EBV in all tumor cells indicates that cellular proliferation occurred after EBV infection and argues for an etiologic role for virus in these tumors (20,23).What is less evident from clonality data is whether EBV infection facilitates tumor initiation or subsequent malignant progression (22,32), an issue central to understanding viral pathogenesis. The fact that EBV is present prior to clonal expansion is intuitive evidence for a viral role in an early, initiating event. Indeed, it has been argued that if EBV were to infect preexisting neoplastic lesions, one would expect either a polyclonal pattern of TRs or the lack of EBV altogether in some tumor cells. Here, we present evidence from carcinoma cell lines infected in vitro that suggests that this need not be the case. Instead, EBV clonality postinfection may be a consequence of a selective growth advantage conferred by viral episomes with a minimal number of TRs.Our experiments stemmed from the unexpected observation, also made by others, that in vitro infection of established epithelial cell lines frequently gives rise to a clonal pattern by EBV TR analysis (3,15 ; American Type Culture Collection, Rockville, Md.), which were determined not to express the B-lymphocyte EBV receptor CD21 (data not shown), were incubated for 24 h with 0.5 ml of Akata culture supernatant containing EBV Neo r and then resuspended in Dulbecco's modified Eagle medium containing 350 g of G418/ml. When G418-resistant ...

Latent Membrane Protein 2A Inhibits Transforming Growth Factor-β1-Induced Apoptosis through the Phosphatidylinositol 3-Kinase/Akt Pathway

“…Previous studies were unable to discern any growth advantage in EBV LMP2A-expressing Akata cell clones compared to vector control cells (44,49). Previous studies already had indicated an important role for LMP2A in the regulation of the growth of epithelial cells in cultures (35,44). Members of the caspase family of proteases have been shown to be key executioners in the apoptotic process in many cell types in response to diverse apoptotic stimuli (8,14).…”

“…LMP2A not only alters cell growth in epithelial cells (35,44) but also inhibits TGF-␤1-induced apoptosis through activation of the PI3-K/Akt pathway in the gastric carcinoma cell line HSC-39. In particular, EBV-associated gastric carcinoma cells express a limited number of EBV genomes, including EBV nuclear antigen 1, EBV-encoded small RNAs, BARF0, and LMP2A (19,48).…”

“…LMP2A appears to be a key determinant in the alteration of epithelial cell growth by activating Akt and c-Jun in epithelial cells and leading to an enhancement of cell growth (7,44). In addition, LMP2A expression is important in epithelial cell clone outgrowth following infection of epithelial cells (35). Thus, the emerging importance of LMP2A in both B-cell and epithelial cell growth regulation led us to investigate the effect of LMP2A on transforming growth factor ␤1 (TGF-␤1) signaling.…”

Augmented Latent Membrane Protein 1 Expression from Epstein-Barr Virus Episomes with Minimal Terminal Repeats

“…Relative rates of DNA synthesis in cell clones were quantified by 5-bromo-2Ј-deoxyuridine (BrdU) incorporation assays using the in situ cell proliferation kit Competitive clonal outgrowth assays were performed as previously described (42). Briefly, cellular clones distinguishable by the molecular size of EBV-fused termini were combined in mixed culture, passaged every 48 h, and assessed after each passage for the emergence of a predominant cell clone as determined by changes in relative intensity of TR bands by Southern blotting.…”

Comparative analysis of oncogenic properties and nuclear factor- B activity of latent membrane protein 1 natural variants from Hodgkin's lymphoma's Reed-Sternberg cells and normal B-lymphocytes

“…The first source of diversity comes from the circularization of the EBV DNA double-stranded molecule via the terminal repeats during virion production in the saliva. 11 As a second source of diversity, allelic polymorphisms of EBNAs genes define type 1 and type 2 EBV isolates. 12 In addition, LMP1-BNLF1 gene polymorphisms, the most discriminating genetic locus, reflect intrastrain diversity.…”