Abstract
Infection of Epstein–Barr virus (EBV), a ubiquitous human gamma herpesvirus, is closely linked to various lymphoid and epithelial malignancies. Previous studies demonstrated that the efficiency of EBV infection in epithelial cells is significantly enhanced by coculturing them with latently infected B cells relative to cell-free infection, suggesting that cell-to-cell contact-mediated viral transmission is the dominant mode of infection by EBV in epithelial cells. However, a detailed mechanism underlying this process has not been fully understood. In the present study, we assessed the role of transforming growth factor β (TGF-β), which is known to induce EBV's lytic cycle by upregulation of EBV's latent-lytic switch BZLF1 gene. We have found that 5 days of cocultivation facilitated cell-to-cell contact-mediated EBV transmission. Replication of EBV was induced in cocultured B cells both with and without a direct cell contact in a time-dependent manner. Treatment of a blocking antibody for TGF-β suppressed both induction of the lytic cycle in cocultured B cells and subsequent viral transmission. Cocultivation with epithelial cells facilitated expression of TGF-β receptors in B cells and increased their susceptibility to TGF-β. Finally, we confirmed the spontaneous secretion of TGF-β from epithelial cells, which was not affected by cell-contact. In contrast, the extracellular microvesicles, exosomes derived from cocultured cells partly contributed to cell-to-cell contact-mediated viral transmission. Taken together, our findings support a role for TGF-β derived from epithelial cells in efficient viral transmission, which fosters induction of the viral lytic cycle in the donor B cells.
Highlights
We have found that cocultivation increased the lytic cycle in B cells and subsequent viral transmission into epithelial cell lines derived from gastric carcinoma (GC) in a time-dependent manner
We observed that the transmission of eGFP-positive AGS and NU-GC-3 cells increased in a time-dependent manner and approximately 20% of epithelial cells were infected with Epstein–Barr virus (EBV)-eGFP after 5 days of cocultivation (Figures 1A,B)
Our observations indicate that direct cocultivation of latently EBV-infected B cells and epithelial cells increases viral transmission into epithelial cells in a timedependent manner (Figure 1), which is likely associated with the induction of the lytic cycle in cocultured B cells (Figure 2)
Summary
Epstein–Barr virus (EBV), a ubiquitous human gamma herpesvirus, infects approximately 95% of the population worldwide and establishes persistent lifelong, mostly asymptomatic infection. We established an assay to assess the efficiency of EBV transmission mediated by cell-to-cell contact by coculturing EBV-infected BL cells and EBV-negative epithelial cells including human GC and NPC cell lines. By use of this assay, we demonstrated that direct cell contact induces bi-directional cell signaling pathways in cocultured cells, which leads to induction of the viral lytic cycle in BL cells and the subsequent enhancement of viral transmission into epithelial cells (Nanbo et al, 2012). These studies indicate that EBV-infected B cells that migrate in the epithelial stroma or intraepithelial space contribute to efficient viral transmission into epithelial cells via cell contact
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