Epstein-Barr Virus (EBV) replication and transcription activator (Rta/BRLF1) is an immediate-early transcription factor that controls the conversion of the latent viral genome into one undergoing lytic replication. By using a doxycycline-inducible expression system, the present study demonstrates that EBV Rta efficiently elicits growth arrest in the human epithelial cell line HEK293. In cells arrested by EBV Rta, the expression of p21 (CDKN1A), p27 (CDKN1B) and cyclin E were increased. In contrast, the levels of cyclin D1, CDK4 and CDK6 were sharply decreased. Activation of the host cell DNA damage response (DDR), indicated by the increasing phosphorylation of H2AX and p53 Ser15, was observed on day 3 and day 5 after EBV Rta expression, respectively. Finally, EBV Rta arrested cells exhibited strong senescence-associated β-galactosidase staining on day 10 after doxycycline induction. Together, these results indicate that, in addition to triggering viral lytic replication in epithelial cells, EBV Rta concurrently initiates a cellular senescence program that was previously undocumented. This finding, showing Rta may be centrally involved in inducing a host cell state amenable to efficient viral reproduction, in addition to its previously characterized regulation of viral transcription, provides new perspectives in understanding EBV pathogenesis.

Interferon (IFN)-alpha upregulates serotonin (5-HT) uptake and serotonin transporter (5-HTT) messenger ribonucleic acid (mRNA) expression in immune cells, which implies the mechanism underlying IFN-alpha-induced depression. However, the signal transduction of this effect remains unclear. We investigated whether the effects of IFN-alpha on the functions of 5-HTT were related to mitogen-activated protein kinase (MAPK). By performing Western blotting, real-time reverse transcriptase-polymerase chain reaction and [3H]5-HT labelling, we examined MAPK phosphorylation, 5-HTT mRNA expression and 5-HT uptake in Jurkat T cells. The cells had been cultured for different time periods (1) with IFN-alpha alone and (2) preincubated with either MAPK inhibitors or with the selective serotonin reuptake inhibitor, fluoxetine, and subsequently cultured along with IFN-alpha. The levels of MAPK phosphorylation, 5-HTT mRNA expression and 5-HT uptake all increased in the IFN-alpha-treated cells but were blocked in those that were pretreated with MAPK inhibitors and fluoxetine. These results appear to clarify the association of depression with IFN-alpha-induced 5-HT uptake that reduces the 5-HT levels and IFN-alpha-regulated transcription of 5-HTT; further, the results suggest the involvement of MAPK in this process.

Epstein-Barr virus (EBV) has been associated with several human malignancies including nasopharyngeal carcinoma (NPC). Reactivation of latent EBV has been considered to contribute to the carcinogenesis of NPC. Blocking the EBV lytic cycle has been shown effective in the treatment of EBV-associated diseases. We have searched for natural dietary compounds inhibiting EBV reactivation in NPC cells. Among them, sulforaphane (SFN) was found to be effective in the inhibition of EBV reactivation in latent EBV-positive NPC cells, NA and HA. SFN is a histone deacetylase (HDAC) inhibitor and has been recognized as an antioxidant and antitumor compound for chemoprevention. However, its antiviral effect is less well elucidated. In this study, after determination of the cytotoxicity of SFN on various epithelial cells, we showed that SFN treatment inhibits EBV reactivation, rather than induction, by detection of EBV lytic gene expression in EBV-positive NPC cells. We also determined that the number of cells supporting the EBV lytic cycle is decreased using immunofluorescence and flow cytometric analysis. Moreover, we have found that this inhibitory effect decreases virus production. To elucidate the inhibitory mechanism of SFN on the EBV lytic cycle, luciferase reporter assays were carried out on the Zta and Rta promoters. The results show that SFN inhibits transactivation activity of the EBV immediate-early gene Rta but not Zta. Together, our results suggest that SFN has the capability to inhibit EBV lytic cycle and the potential to be taken as a dietary compound for prevention of EBV reactivation.

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.

BackgroundThe replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV) is a molecular switch that initiates a productive replication of latent KSHV genomes. KSHV RTA (K-RTA) is composed of 691 amino acids with high Ser and Thr content (17.7%), but to what extent these Ser and Thr are modified in vivo has not been explored.MethodsBy using tandem mass spectrometric analysis of affinity-purified FLAG tagged K-RTA, we sought to identify Ser and Thr residues that are post-translationally modified in K-RTA.ResultsWe found that K-RTA is an O-GlcNAcylated protein and Thr-366/Thr-367 is the primary motif with O-GlcNAcylation in vivo. The biological significance of O-GlcNAc modified Thr-366 and Thr-367 was assessed by site-specific amino acid substitution. Replacement of Thr with Ala at amino acid 366 or 367 caused a modest enhancement of K-RTA transactivation activity in a luciferase reporter assay and a cell model for KSHV reactivation. By using co-immunoprecipitation coupled with western blot analysis, we showed that the capacity of K-RTA in associating with endogenous PARP1 was significantly reduced in the Thr-366/Thr-367 O-GlcNAc mutants. PARP1 is a documented negative regulator of K-RTA that can be ascribed by the attachment of large negatively charged polymer onto K-RTA via PARP1's poly (ADP-ribose) polymerase activity. In agreement, shRNA-mediated depletion of O-GlcNAc transferase (OGT) in KSHV infected cells augmented viral reactivation and virus production that was accompanied by diminished K-RTA and PARP1 complexes.ConclusionsKSHV latent-lytic switch K-RTA is modified by cellular O-GlcNAcylation, which imposes a negative effect on K-RTA transactivation activity. This inhibitory effect involves OGT and PARP1, two nutritional sensors recently emerging as chromatin modifiers. Thus, we speculate that the activity of K-RTA on its target genes is continuously checked and modulated by OGT and PARP1 in response to cellular metabolic state.