Epstein–Barr Virus (EBV) Rta-Mediated EBV and Kaposi's Sarcoma-Associated Herpesvirus Lytic Reactivations in 293 Cells

Yen-Ju Chen,Yu-Lian Chen,Sheng-Ping Chou,Ying-Chieh Ko,Wan-Hua Tsai,Jen-Yang Chen,Su-Fang Lin,Micah Luftig

doi:10.1371/journal.pone.0017809

Abstract

Epstein–Barr virus (EBV) Rta belongs to a lytic switch gene family that is evolutionarily conserved in all gamma-herpesviruses. Emerging evidence indicates that cell cycle arrest is a common means by which herpesviral immediate-early protein hijacks the host cell to advance the virus's lytic cycle progression. To examine the role of Rta in cell cycle regulation, we recently established a doxycycline (Dox)-inducible Rta system in 293 cells. In this cell background, inducible Rta modulated the levels of signature G1 arrest proteins, followed by induction of the cellular senescence marker, SA-β-Gal. To delineate the relationship between Rta-induced cell growth arrest and EBV reactivation, recombinant viral genomes were transferred into Rta-inducible 293 cells. Somewhat unexpectedly, we found that Dox-inducible Rta reactivated both EBV and Kaposi's sarcoma-associated herpesvirus (KSHV), to similar efficacy. As a consequence, the Rta-mediated EBV and KSHV lytic replication systems, designated as EREV8 and ERKV, respectively, were homogenous, robust, and concurrent with cell death likely due to permissive lytic replication. In addition, the expression kinetics of EBV lytic genes in Dox-treated EREV8 cells was similar to that of their KSHV counterparts in Dox-induced ERKV cells, suggesting that a common pathway is used to disrupt viral latency in both cell systems. When the time course was compared, cell cycle arrest was achieved between 6 and 48 h, EBV or KSHV reactivation was initiated abruptly at 48 h, and the cellular senescence marker was not detected until 120 h after Dox treatment. These results lead us to hypothesize that in 293 cells, Rta-induced G1 cell cycle arrest could provide (1) an ideal environment for virus reactivation if EBV or KSHV coexists and (2) a preparatory milieu for cell senescence if no viral genome is available. The latter is hypothetical in a transient-lytic situation.

Highlights

Human oncogenic herpesviruses such as Epstein–Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV) are closely linked to a variety of malignancies including nonkeratinizing nasopharyngeal carcinoma, gastric adenocarcinoma, Burkitt’s lymphoma, Kaposi’s sarcoma, primary effusion lymphoma, multicentric Castleman’s disease, and various forms of lymphoproliferative disorders
To confirm whether the expression of EBV Rta in 293TetER cells is sufficient to promote EBV lytic replication from the latent stage, rAkata-G418 EBV genome was transferred into 293TetER cells, yielding an EREV8 derivative line [28]
The quantity of EBV genome equivalents encapsidated in the viral particles released from Dox-treated EREV8 cells at each time points were determined by comparative quantitative PCR (q-PCR)

Summary

Introduction

Human oncogenic herpesviruses such as Epstein–Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV) are closely linked to a variety of malignancies including nonkeratinizing nasopharyngeal carcinoma, gastric adenocarcinoma, Burkitt’s lymphoma, Kaposi’s sarcoma, primary effusion lymphoma, multicentric Castleman’s disease, and various forms of lymphoproliferative disorders. Both EBV and KSHV are latent residents in B lymphocytes and show sporadic reactivation in lymphoepithelial tissues such as tonsils [1,2,3]. Ablation of c-Myc in transgenic mouse models induced rapid tumor regression associated with senescence [16]; systemic expression of a dominant-interfering Myc mutant in a preclinical mouse model with Ras-initiated lung adenocarcinoma triggered rapid tumor regression accompanied by senescence [17]

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Results

Conclusion