Telomerase activity impacts on Epstein-Barr virus infection of AGS cells.

Jürgen Rac,David Nadal,Andrina Schumacher,Henri-Jacques Delecluse,Florian Haas,Michele Bernasconi,Jaap M Middeldorp,Roberto F Speck

doi:10.1371/journal.pone.0123645

Abstract

The Epstein-Barr virus (EBV) is transmitted from host-to-host via saliva and is associated with epithelial malignancies including nasopharyngeal carcinoma (NPC) and some forms of gastric carcinoma (GC). Nevertheless, EBV does not transform epithelial cells in vitro where it is rapidly lost from infected primary epithelial cells or epithelial tumor cells. Long-term infection by EBV, however, can be established in hTERT-immortalized nasopharyngeal epithelial cells. Here, we hypothesized that increased telomerase activity in epithelial cells enhances their susceptibility to infection by EBV. Using HONE-1, AGS and HEK293 cells we generated epithelial model cell lines with increased or suppressed telomerase activity by stable ectopic expression of hTERT or of a catalytically inactive, dominant negative hTERT mutant. Infection experiments with recombinant prototypic EBV (rB95.8), recombinant NPC EBV (rM81) with increased epithelial cell tropism compared to B95.8, or recombinant B95.8 EBV with BZLF1-knockout that is not able to undergo lytic replication, revealed that infection frequencies positively correlate with telomerase activity in AGS cells but also partly depend on the cellular background. AGS cells with increased telomerase activity showed increased expression mainly of latent EBV genes, suggesting that increased telomerase activity directly acts on the EBV infection of epithelial cells by facilitating latent EBV gene expression early upon virus inoculation. Thus, our results indicate that infection of epithelial cells by EBV is a very selective process involving, among others, telomerase activity and cellular background to allow for optimized host-to-host transmission via saliva.

Highlights

Epstein-Barr virus (EBV) is transmitted via saliva and has to pass the oral mucosal epithelium after exiting from B cells, the site where the virus establishes latency
To address the question whether human telomerase reverse transcriptase (hTERT) expression level influences EBV infection in epithelial cells, we set out to establish an in vitro model. For this we chose three EBV-negative epithelial cell lines: HONE-1, which originates from an EBV-positive nasopharyngeal carcinoma (NPC) but lost EBV in vitro [20,21], suggesting that they do no longer support EBV infection; the gastric carcinoma cell line AGS, which is often used to study epithelial cell infection with EBV [5,11,19,56]; and HEK293, deriving from human epithelial kidney and anatomically remote from the oropharynx [43]
In this study we investigate the impact of hTERT expression and telomerase activity on the infection frequency of epithelial cells by EBV in vitro and how infection develops over time

Summary

Introduction

Epstein-Barr virus (EBV) is transmitted via saliva and has to pass the oral mucosal epithelium after exiting from B cells, the site where the virus establishes latency. It has been demonstrated that differentiation of memory B cells into plasma cells results in reactivation of latent EBV and virus replication [4]. EBV is believed to reside and replicate in oropharyngeal epithelium [5,6]. Cell-free EBV predominantly infects epithelial cells from the basolateral membranes [7], and cell-associated virus efficiently infects cells from the apical surface [8] especially after cell-to-cell contact [9]. Recent work has shown that cell-associated EBV infects in vitro reconstituted stratified epithelium from its mucosal surface [10]. Since EBV egressing from epithelial cells is more lymphotropic than EBV egressing from B cells [11], lytic replication in oropharyngeal epithelial cells might be important for efficient host-to-host transmission

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Conclusion