Smaller mitochondria are found along the sarcoplasmic reticulum among the columns of myofilaments at the level of z-bands

Virus Rta-Mediated EBV and Kaposi’s Sarcoma-Associated Herpesvirus Lytic Reactivations in 293 Cells Yen-Ju Chen1,2, Wan-Hua Tsai2, Yu-Lian Chen2, Ying-Chieh Ko2, Sheng-Ping Chou2, Jen-Yang Chen1,2, Su-Fang Lin2 1 College of Medicine, Graduate Institute of Microbiology, National Taiwan University, Taipei, Taiwan, 2 National Institute of Cancer Research, National Health Research Institutes, Miaoli County, Taiwan Abstract Epsteinarr virus Rta belongs to a lytic switch gene family that is evolutionarily conserved in all gammaherpesviruses. 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 -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-b-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, 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 an ideal environment for virus reactivation if EBV or KSHV coexists and a preparatory milieu for cell senescence if no viral genome is available. The latter is hypothetical in a transient-lytic situation. Citation: Chen Y-J, Tsai W-H, Chen Y-L, Ko Y-C, Chou S-P, et al. Epsteinarr Virus Rta-Mediated EBV and Kaposi’s Sarcoma-Associated Herpesvirus Lytic Reactivations in 293 Cells. PLoS ONE 6: e17809. doi:10.1371/journal.pone.0017809 Editor: Micah Luftig, Duke University Medical Center, United States of America Received November 10, 2010; Accepted February 11, 2011; Published March 10, 2011 Copyright: 2011 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was Paritaprevir supported by Taiwan NHRI CA-099-PP-17 and Department of Health DOH99-TD-C-111-004 to S.-F. Lin; NHRI CA-099-PP-13, National Science Council NSC98-3112-B-400-002, and NSC99-3112-B-400-009 to J.-Y. Chen. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. E-mail: [email protected]; [email protected] Introduction Human oncogenic herpesviruses such asVirus Rta-Mediated EBV and Kaposi’s Sarcoma-Associated Herpesvirus Lytic Reactivations in 293 Cells Yen-Ju Chen1,2, Wan-Hua Tsai2, Yu-Lian Chen2, Ying-Chieh Ko2, Sheng-Ping Chou2, Jen-Yang Chen1,2, Su-Fang Lin2 1 College of Medicine, Graduate Institute of Microbiology, National Taiwan University, Taipei, Taiwan, 2 National Institute of Cancer Research, National Health Research Institutes, Miaoli County, Taiwan Abstract Epsteinarr virus Rta belongs to a lytic switch gene family that is evolutionarily conserved in all gammaherpesviruses. 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 -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-b-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, 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 an ideal environment for virus reactivation if EBV or KSHV coexists and a preparatory milieu for cell senescence if no viral genome is available. The latter is hypothetical in a transient-lytic situation. Citation: Chen Y-J, Tsai W-H, Chen Y-L, Ko Y-C, Chou S-P, et al. Epsteinarr Virus Rta-Mediated EBV and Kaposi’s Sarcoma-Associated Herpesvirus Lytic Reactivations in 293 Cells. PLoS ONE 6: e17809. doi:10.1371/journal.pone.0017809 Editor: Micah Luftig, Duke University Medical Center, United States of America Received November 10, 2010; Accepted February 11, 2011; Published March 10, 2011 Copyright: 2011 Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by Taiwan NHRI CA-099-PP-17 and Department of Health DOH99-TD-C-111-004 to S.-F. Lin; NHRI CA-099-PP-13, National Science Council NSC98-3112-B-400-002, and NSC99-3112-B-400-009 to J.-Y. Chen. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. E-mail: [email protected]; [email protected] Introduction Human oncogenic herpesviruses such as