Induction Of Epstein-Barr Virus Lytic Cycle Research Articles

Abstract 986: Induction of lytic cycle of Epstein-Barr virus by a novel organic compound through activation of the c-Jun N-terminal kinase pathway

Abstract The reactivation of lytic cycle of Epstein-Barr Virus (EBV) is an important component of the emerging lytic induction therapy to specifically target the virus in EBV-associated malignancies. The potency of the lytic induction is central to the effectiveness of the lytic induction therapy. It is hampered by cancer cells that are refractory to lytic induction. This study aims to elucidate novel pathways involved in inducing EBV lytic cycle in epithelial cancer cells using a small organic compound, E11 intermediate (E11.1), and develop new drug combinations to overcome the refractoriness of the cancer cells to lytic induction. E11.1 was found to be the active component in the original E11 compound which has a superior lytic induction capability in both gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC) cells. Timepoint and dose dependent studies were performed to characterize E11.1's lytic reactivation kinetics of EBV in AGS-BDneo cells. Reactive oxygen species (ROS) scavengers and chemical pathway inhibitors were used to investigate the involvement of ROS and major cellular pathways in inducing lytic cycle of EBV by E11.1. Concomitant drug treatment of E11.1 and histone deacetylase (HDAC) inhibitor (suberoylanilide hydroxamic acid or romidepsin) was tested for synergistic action in lytic induction and cell death. E11.1 elicited a rapid kinetics of lytic induction through the activation of c-Jun N-terminal kinase (JNK) pathway in GC and NPC cells and induced the production of ROS and cell death at later timepoints. The combination of E11.1 and HDAC inhibitor reactivated the lytic cycle of EBV synergistically in both GC and NPC lines at 48 hours of treatment. In conclusion, we provided the evidence of activation of JNK pathway in the induction of EBV lytic cycle by E11.1 in EBV-positive epithelial malignancies. E11.1 and HDAC inhibitor induced the lytic cycle of EBV in EBV-positive epithelial cancer cells synergistically resulting in enhanced cell death. Citation Format: Mike Dorothea, Stephanie PT Yiu, Kwai Fung Hui, Alan K. S. Chiang. Induction of lytic cycle of Epstein-Barr virus by a novel organic compound through activation of the c-Jun N-terminal kinase pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 986.

Identification of Novel Small Organic Compounds with Diverse Structures for the Induction of Epstein-Barr Virus (EBV) Lytic Cycle in EBV-Positive Epithelial Malignancies.

Phorbol esters, which are protein kinase C (PKC) activators, and histone deacetylase (HDAC) inhibitors, which cause enhanced acetylation of cellular proteins, are the main classes of chemical inducers of Epstein-Barr virus (EBV) lytic cycle in latently EBV-infected cells acting through the PKC pathway. Chemical inducers which induce EBV lytic cycle through alternative cellular pathways may aid in defining the mechanisms leading to lytic cycle reactivation and improve cells’ responsiveness towards lytic induction. We performed a phenotypic screening on a chemical library of 50,240 novel small organic compounds to identify novel class(es) of strong inducer(s) of EBV lytic cycle in gastric carcinoma (GC) and nasopharyngeal carcinoma (NPC) cells. Five hit compounds were selected after three successive rounds of increasingly stringent screening. All five compounds are structurally diverse from each other and distinct from phorbol esters or HDAC inhibitors. They neither cause hyperacetylation of histone proteins nor significant PKC activation at their working concentrations, suggesting that their biological mode of action are distinct from that of the known chemical inducers. Two of the five compounds with rapid lytic-inducing action were further studied for their mechanisms of induction of EBV lytic cycle. Unlike HDAC inhibitors, lytic induction by both compounds was not inhibited by rottlerin, a specific inhibitor of PKCδ. Interestingly, both compounds could cooperate with HDAC inhibitors to enhance EBV lytic cycle induction in EBV-positive epithelial cancer cells, paving way for the development of strategies to increase cells’ responsiveness towards lytic reactivation. One of the two compounds bears structural resemblance to iron chelators and the other strongly activates the MAPK pathways. These structurally diverse novel organic compounds may represent potential new classes of chemicals that can be used to investigate any alternative mechanism(s) leading to EBV lytic cycle reactivation from latency.

Inhibition of class I histone deacetylases by romidepsin potently induces Epstein-Barr virus lytic cycle and mediates enhanced cell death with ganciclovir.

Pan-histone deacetylase (HDAC) inhibitors, which inhibit 11 HDAC isoforms, are widely used to induce Epstein-Barr virus (EBV) lytic cycle in EBV-associated cancers in vitro and in clinical trials. Here, we hypothesized that inhibition of one or several specific HDAC isoforms by selective HDAC inhibitors could potently induce EBV lytic cycle in EBV-associated malignancies such as nasopharyngeal carcinoma (NPC) and gastric carcinoma (GC). We found that inhibition of class I HDACs, particularly HDAC-1, -2 and -3, was sufficient to induce EBV lytic cycle in NPC and GC cells in vitro and in vivo. Among a panel of selective HDAC inhibitors, the FDA-approved HDAC inhibitor romidepsin was found to be the most potent lytic inducer, which could activate EBV lytic cycle at ∼0.5 to 5 nM (versus ∼800 nM achievable concentration in patients' plasma) in more than 75% of cells. Upregulation of p21(WAF1) , which is negatively regulated by class I HDACs, was observed before the induction of EBV lytic cycle. The upregulation of p21(WAF1) and induction of lytic cycle were abrogated by a specific inhibitor of PKC-δ but not the inhibitors of PI3K, MEK, p38 MAPK, JNK or ATM pathways. Interestingly, inhibition of HDAC-1, -2 and -3 by romidepsin or shRNA knockdown could confer susceptibility of EBV-positive epithelial cells to the treatment with ganciclovir (GCV). In conclusion, we demonstrated that inhibition of class I HDACs by romidepsin could potently induce EBV lytic cycle and mediate enhanced cell death with GCV, suggesting potential application of romidepsin for the treatment of EBV-associated cancers.

P0127 Curcuminoids as putative ebv lytic induction agents for adjuvant treatment in EBV-positive carcinoma

Background Epstein-Barr virus (EBV) is causally involved in various lymphoid and non-lymphoid cancers, including nasopharyngeal (NPC) and stomach carcinomas (EBVaGC). NPC is endemic in south east Asia, including Indonesia. EBVaGC accounts for about 10% of GC worldwide. We recently initiated efforts to target EBV inside tumour cells by cytolytic virus activation therapy (CLVA), currently being validated in a phase 1/2 trial. We aim to develop oral drugs to improve CLVA treatment availability for low-income countries. Curcumin (diferuloylmethane) from Curcuma longa is used as spice in Asian countries without any toxic effects and has anti-cancer activity. Several curcumin analogues (curcuminoids), developed to increase efficacy and bioavailability, are currently being tested in clinical studies. Curcuminoids affect multiple pathways, including inhibition of COX2 and NF-kB and have potential as oral CLVA regimens for EBV-positive carcinoma. Methods We investigated EBV-reactivating effects of curcuminoids on EBV-positive NPC (C666.1) and EBVaGC cell lines (SNU179 and AGS-BX1). EBV-negative GC (AGS) line was used as a negative control. Standard CLVA, SAHA, and bortezomib were used as positive controls. EBV lytic (Zebra, EA-D) activity was measured by semi-quantitative immunoblot, FACS and indirect immunofluorescence. Effects on cell viability were measured by MTT assay. Findings Curcumin induced EBV lytic activity in AGS-BX1 cells but had marginal effect on natural EBV lines (C666.1,SNU179). However, several curcumin analogues induced EBV reactivation in all three EBV-positive cell lines in a dose- and time-dependent manner. MTT assay showed curcuminoids to be more toxic to EBVaGC cells than to C666.1 cells. Noteworthy curcuminoids showed synergistic effects with current lytic induction agents. Interpretation Curcumin analogues serve as non-toxic drugs to reactivate EBV from latently infected NPC and GC cells, but seem to reactivate EBV differently in NPC versus GC. Curcuminoids at pharmacologically relevant concentrations may promote cytolysis of EBV-positive carcinoma when combined with CLVA drugs. The molecular mechanisms of EBV lytic cycle induction by curcuminoids will be studied to gain further insights in EBV reactivation from latency in different carcinoma models.

Signal transducer and activator of transcription 3 (STAT3) controls susceptibility to Epstein-Barr virus reactivation in B cells

Epstein-Barr Virus (EBV) is known to cause malignancies in immunocompromised individuals, including lymphomas and lymphoproliferative disorders. Reactivation of EBV from latency is important in the pathogenesis of such malignancies. Therapeutically, oncolysis of tumors harboring lytic EBV holds promise. However, exposure of latently infected cultured B cells to lytic cycle inducing stimuli results in virus reactivation within only 20% to 50% of cells. Host cell determinants, that govern this susceptibility to lytic reactivat ion within latently infected B cells, are not well understood. Our research has previously identified that higher levels of signal transducer and activator of transcription 3 (STAT3) within cells correlate with resistance to EBV lytic reactivation. We now investigate whether inhibiting function of STAT3 promotes susceptibility of latently infected B cells to known lytic cycle inducing stimuli or results in induction of EBV lytic cycle. We found that pharmacological inhibition of STAT3 phosphorylation by Janus Kinase 2 (JAK2) inhibitors (AG490 or WP1066) in latently infected cells (HH514-16 Burkitt lymphoma and B95-8 lymphoblastoid cells) increased lytic reactivation by chemical stimuli that function by distinct mechanisms. Moreover, functional inhibition of STAT3 alone resulted in lytic reactivation in these cells. EBV-Lymphoblastoid cell lines (LCL) newly generated from healthy individuals also demonstrated increased susceptibility to lytic cycle inducing stimuli in the presence of AG490 but variable susceptibility to lytic reactivation when exposed to AG490 alone. Since this lack of uniform response to AG490 alone could be due to inadequate functional suppression of STAT3, we examined EBV-LCLs derived from patients with Autosomal Dominant Hyperimmunoglobulin-E syndrome (AD-HIES or Job syndrome). Patients with AD-HIES carry a dominant negative mutation in their Stat3 gene resulting in lower basal levels of functional STAT3. When LCLs from AD-HIES patients were exposed to a JAK2 inhibitor alone, we observed a strong increase in lytic reactivation by expression of early and late lytic antigens over LCLs derived from healthy individuals. Lytic reactivation in the presence of AG490 occurred in these cells despite lack of discernable increase from basal levels of expression of ZEBRA, the viral lytic switch protein, when compared to cells not exposed to AG490. Thus, STAT3 is important in determining susceptibility to EBV reactivation. Fully understanding how STAT3 governs such susceptibility can lead to novel therapeutic strategies for EBV-related diseases.

Abstract 4715: Activation of lytic cycle of Epstein-Barr virus by suberoylanilide hydroxamic acid affects apoptosis and tumor growth suppression of nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV). We examined the in vitro and in vivo effects of suberoylanilide hydroxamic acid (SAHA), a FDA-approved histone deacetylase inhibitor, on EBV lytic cycle induction in NPC and investigated the cellular consequences. Micromolar concentrations of SAHA potently induced EBV lytic cycle with replication of EBV DNA, expression of immediate early (Zta and Rta), early (BMRF1) and late (gp350) lytic proteins and production of infectious viral particles in culture supernatants in three of four EBV-positive NPC cell lines. One NPC cell line had abortive EBV lytic cycle (no EBV DNA replication or late lytic protein expression) induction. Same concentrations of SAHA effected enhanced killing of EBV-positive NPC cells in comparison with EBV-negative counterparts, as measured by MTT assay. Increased percentages of annexin V-positive and TUNEL-positive cells and proteolytic cleavage of poly (ADP-ribose) polymerase (PARP), caspase-3, -7 and -9 in EBV-positive versus EBV-negative NPC were also observed. Immunofluorescence staining and flow cytometry showed that the majority of NPC cells (>85%) expressing Zta, BMRF1 or gp350 lytic protein co-expressed cleaved caspase-3. Tracking of expression of EBV lytic protein and cleaved caspase-3 showed that proportion of NPC cells co-expressing EBV lytic protein and cleaved caspase-3 increased over time. Furthermore, knockdown of Zta expression by short-hairpin RNA significantly decreased proteolytic cleavage of caspase-3 in SAHA-treated EBV-positive NPC cells whilst overexpression of Zta induced apoptosis of EBV-positive NPC cells, confirming that lytic cycle induction programmed NPC cells to apoptosis. Interestingly, inhibition of EBV DNA replication and late lytic protein expression by phosphonoformic acid did not impact on SAHA's induced cell death in NPC, suggesting that early rather than late phase of EBV lytic cycle contributed to the apoptotic effect. In vivo effects of SAHA on EBV lytic cycle induction and tumor growth suppression were observed in NPC xenografts established in nude mice. Taken together, our data indicated that activation of lytic cycle from latent cycle of EBV by SAHA effects apoptosis and tumor growth suppression of NPC thereby providing experimental evidence for virus-targeted therapy against EBV-positive cancer. This project is funded by NPC Area of Excellence (AoE/M 06/08 Center for Nasopharyngeal Carcinoma Research) and Epstein-Barr virus research (# 20004525) grants of AKSC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4715. doi:1538-7445.AM2012-4715

Activation of lytic cycle of Epstein‐Barr virus by suberoylanilide hydroxamic acid leads to apoptosis and tumor growth suppression of nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is strongly associated with Epstein-Barr virus (EBV). We reported that suberoylanilide hydroxamic acid (SAHA) induced EBV lytic cycle in EBV-positive gastric carcinoma cells and mediated enhanced cell death. However, expression of EBV lytic proteins was thought to exert antiapoptotic effect in EBV-infected cells. Here, we examined the in vitro and in vivo effects of SAHA on EBV lytic cycle induction in NPC cells and investigated the cellular consequences. Micromolar concentrations of SAHA significantly induced EBV lytic cycle in EBV-positive NPC cells. Increased apoptosis and proteolytic cleavage of poly(ADP-ribose) polymerase and caspase-3, -7 and -9 in EBV-positive versus EBV-negative NPC cells were observed. More than 85% of NPC cells expressing immediate-early (Zta), early (BMRF1) or late (gp350/220) lytic proteins coexpressed cleaved caspase-3. Tracking of expression of EBV lytic proteins and cleaved caspase-3 over time demonstrated that NPC cells proceeded to apoptosis following EBV lytic cycle induction. Inhibition of EBV DNA replication and late lytic protein expression by phosphonoformic acid did not impact on SAHA's induced cell death in NPC, indicating that early rather than late phase of EBV lytic cycle contributed to the apoptotic effect. In vivo effects of SAHA on EBV lytic cycle induction and tumor growth suppression were also observed in NPC xenografts in nude mice. Taken together, our data indicated that activation of lytic cycle from latent cycle of EBV by SAHA leads to apoptosis and tumor growth suppression of NPC thereby providing experimental evidence for virus-targeted therapy against EBV-positive cancer.

Reactive Oxygen Species Production and Antioxidant Enzyme Expression after Epstein–Barr Virus Lytic Cycle Induction in Raji Cell Line

In a previous study, we have described oxidative stress during Epstein-Barr virus lytic cycle induction. Oxidative stress was evidenced by the observed high MDA levels and the decreased activities of antioxidant enzymes. We hypothesised that the lower activities of the antioxidant enzymes decrease were the result of either the excessive production of reactive oxygen radical species (ROS) or a negative regulation of the antioxidant enzyme gene expressions. In an attempt to clarify this situation, EBV lytic cycle was induced in Raji cell line by a non-stressing dose of 12-0-tetradecanoylphorbol-13-acetate. BZLF-1, superoxide dismutase, and catalase gene expressions were then analysed using semi-quantitative RT-PCR, simultaneously at a kinetic of 6, 12, 24, 36, and 48h. ROS production was evaluated by chemiluminescence. A study was conducted to establish whether ROS production, BZLF-1, and the expression of antioxidant genes were inter-correlated. Induction of the lytic cycle resulted in increased expressions of the genes of superoxide dismutase and catalase, which began at 24h (p < 0.05) and reached a peak at 48h (p < 0.05). Significant increases of the ROS levels were observed in TPA-treated Raji cell line at 12h, as compared with untreated cells, reaching a peak at 48h after EBV lytic cycle induction. ROS production correlates positively with BZLF-1, SOD, and CAT gene expressions (p < 0.05; r = 0.913, r = 0.978, and r = 0.955, respectively). A positive correlation was also observed between BZLF-1 and antioxidant gene expressions (p < 0.05; r = 0.961 and r = 0.987, respectively). In conclusion, the observed increases of the SOD and CAT gene expressions eliminate the hypothesis of a repression of the respective genes during the induction of the lytic cycle. On the other hand, the observed direct correlation between the BZLF-1 gene expression and the ROS production is indicative of a role of this gene in oxidative stress.

Induction of epstein-barr virus (EBV) lytic cycle in vitro causes lipid peroxidation, protein oxidation and DNA damage in lymphoblastoid B cell lines

BackgroundWe investigated the oxidative modifications of lipids, proteins and DNA, potential molecular targets of oxidative stress, in two lymphoblastoid cell lines: B95-8 and Raji, after EBV lytic cycle induction. Conjugated dienes level was measured as biomarker of lipid peroxidation. Malondialdehyde adduct and protein carbonyl levels, as well as protein thiol levels were measured as biomarkers of protein oxidation. DNA fragmentation was evaluated as biomarker of DNA oxidation.ResultsAfter 48 h (peak of lytic cycle), a significant increase in conjugated dienes level was observed in B95-8 and Raji cell lines (p = 0.0001 and p = 0.019 respectively). Malondialdehyde adduct, protein carbonyl levels were increased in B95-8 and Raji cell lines after EBV lytic cycle induction as compared to controls (MDA-adduct: p = 0.008 and p = 0.006 respectively; Carbonyl: p = 0.003 and p = 0.0039 respectively). Proteins thiol levels were decreased by induction in B95-8 and Raji cell lines (p = 0.046; p = 0.002 respectively). DNA fragmentation was also detected in B95-8 and Raji cell lines after EBV lytic cycle induction as compared to controls.ConclusionThe results of this study demonstrate the presence of increased combined oxidative modifications in lipids, proteins in B95-8 and Raji cells lines after EBV lytic cycle induction. These results suggest that lipid peroxidation, protein oxidation and DNA fragmentation are generally induced during EBV lytic cycle induction and probably contribute to the cytopathic effect of EBV.

Abstract 5445: Suberoylanilide hydroxamic acid induces Epstein-Barr virus lytic cycle and enhanced apoptosis in nasopharyngeal carcinoma

Abstract Nasopharyngeal carcinoma (NPC) has uniquely high prevalence in Southern Chinese populations and a predilection to affect young adult males. Epstein-Barr virus (EBV) is harbored in every tumour cell of NPC but it persists in tightly latent form expressing few viral proteins and evading the immune system. Induction of EBV lytic cycle will lead to expression of a much larger number of viral proteins which may serve as potential therapeutic targets against the cancer. We reported that suberoylanilide hydroxamic acid (SAHA), a FDA-approved histone deacetylase inhibitor, could strongly induce viral lytic cycle in EBV-positive gastric carcinoma cells. Here, we demonstrated that SAHA (at 5-10 μM) could potently induce EBV lytic cycle in a panel of EBV-positive NPC cell lines, as evidenced by increased viral DNA replication, expression of immediate early (Zta and Rta), early (BMRF1) and late (VCA-p40 and gp350/220) viral lytic proteins and production of infectious viral particles. Immunofluorescent staining demonstrated expression of viral lytic proteins in more than 40% of the NPC cells. Enhanced killing of EBV-positive NPC cells when compared with that of EBV-negative counterparts was observed and cleaved form of PARP was predominantly expressed in EBV-positive than EBV-negative NPC cells, following treatment with SAHA for 24, 48 and 72 hours. The kinetics of expression of cleaved PARP correlated with that of Zta viral protein in EBV-positive NPC cells. In conclusion, SAHA is found to be a potent EBV lytic cycle inducing agent and mediates enhanced apoptosis of NPC cells. Further investigation into SAHA's development as a novel virus-targeted therapeutic drug in NPC is warranted. This work is funded by EBV research and NPC AoE grants of AKSC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5445.

Suberoylanilide hydroxamic acid induces viral lytic cycle in Epstein‐Barr virus‐positive epithelial malignancies and mediates enhanced cell death

In Epstein-Barr virus (EBV)-associated malignancies, the virus is harbored in every tumor cell and persists in tightly latent forms expressing a very limited number of viral latent proteins. Induction of EBV lytic cycle leads to expression of a much larger number of viral proteins, which may serve as potential therapeutic targets. We found that 4 histone deacetylase inhibitors, trichostatin A (TSA), sodium butyrate (SB), valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA), all significantly induced EBV lytic cycle in EBV-positive gastric carcinoma cells (AGS/BX1, latency II) but only weakly induced in Burkitt lymphoma cells (AK2003, latency I) and did not induce in lymphoblastoid cells (LCLs, latency III). Interestingly, SAHA potently induced viral lytic cycle in AGS/BX1 cells at micromolar concentrations (evidenced by 8-fold increase in viral DNA replication, strong expression of viral lytic proteins and production of infectious virus particles) and mediated enhanced cell death of EBV-positive AGS/BX1 cells when compared with that of EBV-negative AGS cells, possibly related to cell cycle arrest at G2/M phase. Furthermore, SAHA effected strong induction of EBV lytic cycle in nasopharyngeal carcinoma but not in NK lymphoma cells (both expressing EBV latency II pattern), indicating preferential viral lytic induction in epithelial rather than lymphoid malignancies. In conclusion, SAHA is found to be a potent EBV lytic cycle inducing agent, which warrants further investigation into its potential application as a novel virus-targeted drug for treatment of EBV-associated epithelial malignancies.

Upregulation of STAT3 Marks Burkitt Lymphoma Cells Refractory to Epstein-Barr Virus Lytic Cycle Induction by HDAC Inhibitors

A fundamental problem in studying the latent-to-lytic switch of Epstein-Barr virus (EBV) and the viral lytic cycle itself is the lack of a culture system fully permissive to lytic cycle induction. Strategies to target EBV-positive tumors by inducing the viral lytic cycle with chemical agents are hindered by inefficient responses to stimuli. In vitro, even in the most susceptible cell lines, more than 50% of cells latently infected with EBV are refractory to induction of the lytic cycle. The mechanisms underlying the refractory state are not understood. We separated lytic from refractory Burkitt lymphoma-derived HH514-16 cells after treatment with an HDAC inhibitor, sodium butyrate. Both refractory- and lytic-cell populations responded to the inducing stimulus by hyperacetylation of histone H3. However, analysis of host cell gene expression showed that specific cellular transcripts Stat3, Fos, and interleukin-8 (IL-8) were preferentially upregulated in the refractory-cell population, while IL-6 was upregulated in the lytic population. STAT3 protein levels were also substantially increased in refractory cells relative to untreated or lytic cells. This increase in de novo expression resulted primarily in unphosphorylated STAT3. Examination of single cells revealed that high levels of STAT3 were strongly associated with the refractory state. The refractory state is manifest in a unique subpopulation of cells that exhibits different cellular responses than do lytic cells exposed to the same stimulus. Identifying characteristics of cells refractory to lytic induction relative to cells that undergo lytic activation will be an important step in developing a better understanding of the regulation of the EBV latent to lytic switch.

Induction of Epstein-Barr virus (EBV) lytic cycle in vitro causes oxidative stress in lymphoblastoid B cell lines

Here, we investigated the effect of induction of the Epstein-Barr virus (EBV) viral lytic cycle on the oxidant/antioxidant balance in three lymphoblastoid cell lines: B95-8, Raji, and LCL C1. The induction of the EBV lytic cycle was done by a non-stressing dose of 12-0-tetradecanoylphorbol-13-acetate (8 nM). Oxidative stress was assessed by measuring malondialdehyde as a parameter of lipid peroxidation, the levels of glutathione, and the activities of three antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase). After 48 h (peak of lytic cycle), a significant decrease in superoxide dismutase activity was observed in B95-8, Raji, and LCL C1 cells (P < 0.05). In addition, in B95-8 cells also a significant decrease of catalase activity was detected (P < 0.05). The glutathione peroxidase activity and the glutathione level were not significantly modified by the induction in any of the cell lines. We found a significant rise in malondialdehyde levels in B95-8, Raji, and LCL C1 cells after the induction of the lytic cycle compared to controls (P < 0.05). In conclusion, induction of EBV lytic cycle in lymphoblastoid cells causes increased oxidative stress in the host cells within 48 h, a process that could be involved in malignant transformations.

EBV‐miR‐BHRF1‐1 contributes to Epstein‐Barr virus lytic cycle induction via inhibits p53

EBV‐miR‐BHRF1‐1 contributes to Epstein‐Barr virus lytic cycle induction via inhibits p53

De Novo Protein Synthesis Is Required for Lytic Cycle Reactivation of Epstein-Barr Virus, but Not Kaposi's Sarcoma-Associated Herpesvirus, in Response to Histone Deacetylase Inhibitors and Protein Kinase C Agonists

The oncogenic human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), are latent in cultured lymphoma cells. We asked whether reactivation from latency of either virus requires de novo protein synthesis. Using Northern blotting and quantitative reverse transcriptase PCR, we measured the kinetics of expression of the lytic cycle activator genes and determined whether abundance of mRNAs encoding these genes from either virus was reduced by treatment with cycloheximide (CHX), an inhibitor of protein synthesis. CHX blocked expression of mRNAs of EBV BZLF1 and BRLF1, the two EBV lytic cycle activator genes, when HH514-16 Burkitt lymphoma cells were treated with histone deacetylase (HDAC) inhibitors, sodium butyrate or trichostatin A, or a DNA methyltransferase inhibitor, 5-Aza-2'-deoxycytidine. CHX also inhibited EBV lytic cycle activation in B95-8 marmoset lymphoblastoid cells by phorbol ester phorbol-12-myristate-13-acetate (TPA). EBV lytic cycle induction became resistant to CHX between 4 and 6 h after application of the inducing stimulus. KSHV lytic cycle activation, as assessed by ORF50 mRNA expression, was rapidly induced by the HDAC inhibitors, sodium butyrate and trichostatin A, in HH-B2 primary effusion lymphoma cells. In HH-B2 cells, CHX did not inhibit, but enhanced, expression of the KSHV lytic cycle activator gene, ORF50. In BC-1, a primary effusion lymphoma cell line that is dually infected with EBV and KSHV, CHX blocked EBV BRLF1 lytic gene expression induced by TPA and sodium butyrate; KSHV ORF50 mRNA induced simultaneously in the same cells by the same inducing stimuli was resistant to CHX. The experiments show, for the cell lines and inducing agents studied, that the EBV BZLF1 and BRLF1 genes do not behave with "immediate-early" kinetics upon reactivation from latency. KSHV ORF50 is a true "immediate-early" gene. Our results indicate that the mechanism by which HDAC inhibitors and TPA induce lytic cycle gene expression of the two viruses differs and suggest that EBV but not KSHV requires one or more proteins to be newly synthesized between 4 and 6 h after application of an inducing stimulus.

Down-regulation of proteolytic complexes following EBV activation in BL cells

In Burkitt’s lymphoma cells, Epstein Barr virus (EBV) latency products interact with the ubiquitin–proteasome system to promote episomal maintenance and immunological evasion while the tripeptidylpeptidase II (TPPII) functions as an alternative protease. In the present study, we have examined the activities and levels of the proteasome and TPPII complex in Raji and in Akata cells after induction of EBV lytic cycle. The results show that the chymotrypsin-like and caspase-like activities of the proteasome were substantially reduced in Raji and Akata cells. Similarly, TPPII activity was diminished in both cell lines but was recovered in Akata cells at longer time after induction. Protein levels of the α/β subunits of the 20S proteasome and TPPII concentration decreased to different extents after EBV activation, whereas the ubiquitin binding S6’ subunit of the 19S regulatory complex increased three to fourfold along with the levels of ubiquitin-conjugates. Collectively, these observations demonstrate impairment of two major cellular proteolytic systems at the onset of EBV lytic infection.

Interaction of EBV latent origin of replication with the nuclear matrix: identification of S/MAR sequences and protein components

During latency, Epstein Barr virus (EBV) genome, as an episome, is attached to the nuclear matrix (NM) via the latent origin of replication ori P. Within this element, we have found that a region, 580 bp long, encompassing the replicator DS element, shows the strongest affinity for the NM. In addition, by cross-linking with cis-diamminedichloroplatinum, we have identified two NM proteins with an apparent molecular weight of 85 and 60 kDa that, with high affinity and specificity, bind ori P. These proteins are not induced by EBV infection, but their interaction with ori P is lost upon induction of EBV lytic cycle. These data strongly suggest that the binding of ori P to specific components of the NM is required for EBV latent replication.