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Hypoxia can contribute to the induction of the Epstein-Barr virus (EBV) lytic cycle
Journal of Clinical Virology 37 (2006) 98–103
Hypoxia can contribute to the induction of the Epstein-Barr virus (EBV) lytic cycle Ju-Hong Jiang a , Na Wang b , Ang Li b , Wen-Ting Liao b , Zhi-Gang Pan b , Shi-Juan Mai b , Da-Jiang Li b , Mu-Sheng Zeng b , Jian-ming Wen a , Yi-Xin Zeng b,∗ a
Department of Pathology, The First Affiliated Hospital, Sun Yat-sen University, 651 Dong-Feng Road East, Guangzhou 510080, China b State Key Laboratory of Oncology in South China and The Department of Experimental Research, Cancer Center, Sun Yat-sen University, 651 Dong-Feng Road East, Guangzhou 510060, China Received 28 November 2005; received in revised form 20 June 2006; accepted 24 June 2006
Abstract Background: Like other herpes viruses, latent Epstein-Barr virus (EBV) infection can be reactivated to lytic replication. Reactivation can be achieved by treatment with various reagents, including tetradecanoyl phorbol acetate (TPA) and Ca2+ ionophores. Relatively little is known about the physiological factors related to reactivation of EBV. Previous studies have demonstrated that G0 /G1 cell cycle arrest is associated with EBV activation, and that hypoxic conditions can induce cell cycle arrest. In the present study we investigated the effect of hypoxia on reactivation of EBV. Objective and methods: Hypoxic culture conditions were established and the expression of Zta protein and the number of EBV DNA copies were measured in B95-8 cells maintained under these conditions. Results: Hypoxia treatment not only increased the expression of the EBV immediate-early protein Zta (which mediates the switch between the latent and lytic form of infection), but also increased the number of EBV DNA copies in B95-8 cells. Conclusions: EBV in latent infection can be activated to lytic infection by hypoxia treatment. © 2006 Elsevier B.V. All rights reserved. Keywords: Hypoxia; Epstein-Barr virus; Reactivation; Nasopharyngeal carcinoma
1. Introduction Epstein-Barr virus (EBV) is a ubiquitous human herpes virus infecting over 90% of adults worldwide (Henle et al., 1969). In vivo, EBV primarily targets lymphoid and epithelial cells. Primary infection with EBV is followed by a lifelong presence of the virus within the host (Golden et al., 1973). Like other herpes viruses, EBV infection can remain latent and be reactivated to lytic replication. In vitro, latently infected cells can be reactivated by treatment with tetradecanoyl phorbol acetate (TPA), Ca2+ ionophore, anti-IgM, human herpes virus 6 infection, and transforming growth factor beta 1 (TGF-1) (di Renzo et al., 1994; Faggioni et al., 1986; Flamand et al., 1993; Gao et al., 2001; Liang et al., ∗
Corresponding author. Fax: +86 20 8734 3295. E-mail address: [email protected] (Y.-X. Zeng).
1386-6532/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2006.06.013
2002; Tovey et al., 1978). These treatments can induce the expression of the BZLF1 gene product ZEBRA, also known as Zta, which exhibits homology to the cellular transcription factor c-fos. Expression of Zta in latently infected cells is sufficient to trigger the viral lytic cycle (Lieberman et al., 1990). Zta expression is regulated by activation of the promoter Zp, which is located between the coding sequence of BRLF1 and BZLF1 (ranging from −221 to +13 of the BZLF1 gene) (Liang et al., 2002). With the exception of epithelial differentiation signals, there is relatively little information on the mechanism underlying EBV reactivation. An investigation of the EBV-related lesion, oral hairy leukoplakia (OHL), suggested that the trigger of the viral lytic cycle is associated with epithelial differentiation, as Zta immunoreactivity within nuclei was confined to the upper spinous layer and generally appeared as a single cell layer immediately preceding Viral
J.-H. Jiang et al. / Journal of Clinical Virology 37 (2006) 98–103
Capsid Antigen (VCA) expression. Zta staining was not observed in the lower epithelial layers (Young et al., 1991). This phenomenon was further supported by in vitro studies in epithelial cell lines (Karimi et al., 1995; MacCallum et al., 1999). In addition, studies by Rodriguez et al. (2001) demonstrated that treatment of latently infected cells with lytic cycle-inducing agents induced G0/G1 cell cycle arrest prior to detectable Zta expression. The finding that EBV replication is associated with cellular differentiation is consistent with EBV reactivation in growth-arrested cells and suggested that other physiological factors that induce cell cycle arrest may be associated with EBV reactivation. Hypoxic conditions can manifest during tumor development when the unregulated proliferation of tumour cells exceeds the available blood supply and subsequent oxygen supply. Hypoxic conditions are present in most solid tumours (Hockel and Vaupel, 2001; Vaupel et al., 1989). Exposing cultured cells to hypoxic conditions can induce cell cycle arrest (Krtolica and Ludlow, 1996). However, whether hypoxia is associated with lytic reactivation of EBV in nasopharyngeal carcinoma (NPC) remains unknown. The goal of the present study was to identify the role of hypoxia in EBV reactivation.
2. Methods 2.1. Cell lines and hypoxia treatment B95-8 is a marmoset B-cell line transformed with EBV; CNE2 (EBV-negative) is a human undifferentiated NPC cell line. Cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 mg of streptomycin per milliliter, and 100 U of penicillin per milliliter in a humidified atmosphere at 37 ◦ C with 5% CO2 . Cells were cultured inside an air-tight chamber maintained under hypoxic conditions (1% O2 ) by pumping (via a vacuum pump) air out and pumping in a mixture of 1% O2 , 5% CO2 , and 94% N2 . 2.2. Plasmid construction The promoter sequence of the BZLF1 gene (Zp-236) was amplified by PCR using B95-8 genomic DNA as the template and primers Zp-F (5 -catctcgagaatgtctgctgcatgcca-3 ,) and Zp-R (5 -gtcagatctggcaaggtgcaatgttta-3 ) (Liang et al., 2002). The Zp-F and Zp-R primers were flanked by XhoI and BglII restriction sites (underlined), respectively. The PCR product of the Zp-236 fragment was first cloned into the pGEM-T vector (Promega, Madison, WI, USA), then cloned into the pGL3-Basic vector through XhoI and BglII double digestion. The constructed plasmid was named “pGL3-Zp-236.” 2.3. Cell cycle analysis CNE2 and B95-8 cells treated with normoxia, hypoxia, or TPA (20 ng/ml), were harvested at the indicated times, washed twice with phosphate-buffered saline (PBS), sus-
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pended in 0.5 ml cold PBS (4 ◦ C), fixed with 5 ml 70% cold (−20 ◦ C) ethanol, incubated at 4 ◦ C overnight, washed twice with PBS, and treated with RNase A (0.1 mg/ml) in 69 mM propidium iodide at 37 ◦ C for 45 min. Cell cycle analysis was carried out using a FACScan fluorescence-activated cell sorter and software of Phoenix Flow Systems. 2.4. RNA extraction and semi-quantitative reverse transcription-PCR (RT-PCR) B95-8 cells were treated with normoxia, hypoxia, or TPA (20 ng/ml), and harvested at the indicated times. Total RNA was extracted from cells by Trizol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription and amplification were carried out in a single step in 25 l of reaction mixture containing 0.2 M BZLF1 5 primer (5-agaggtgtgtcagccaaaga-3) and 3 primer (5 -cttcctccttctggggaata-3 ), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) 5 primer (5-caggggggagccaaaagg-3) and 3 primer (5-ggcagtggggacacggaa-3), dNTP mix (200 M for each), 1 l of one step RT-PCR enzyme mix (Qiagen, Hilden, Germany), and 1 g of total RNA. The thermal conditions of RT-PCR were established according to the recommendation of manufacturer and the annealing temperature of the primers. Five microliters of RTPCR product was analyzed on a 1.5% agarose gel. The size of PCR products of BZLF1 and GAPDH were 278 and 378 bp, respectively. 2.5. Western blot analysis B95-8 cells were treated with normoxia, hypoxia, or TPA (20 ng/ml) and harvested at the indicated times. Cells were collected by centrifugation and washed once in PBS, then lysed in sodium dodecyl sulfate (SDS) loading buffer containing -mercaptoethanol. Samples were heated to 100 ◦ C for 5 min and centrifuged at 10,000 × g for 10 min prior to a protein concentration assay and loading on an SDS–10% polyacrylamide gel. Following electrophoresis, the proteins were electro-transferred to a polyvinylidene fluoride (PVDF) transfer membrane. The blotting process was performed according to the protocol of the Phototope® -HRP Western Blot Detection System (Cell Signaling Technology, Inc., MA, USA) using a Zta monoclonal antibody (Santa Cruz Biotechnology Inc., CA, USA). 2.6. Transient DNA transfection and reporter gene assay The reporter plasmid pGL3-Zp-236 was introduced into the NPC cell line CNE2 using LipofectAMINETM 2000 (LF2000) reagent (Invitrogen). The day before transfection adherent CNE2 cells were trypsinized, counted, and plated at 5 × 105 cells per 25-ml flask to achieve 90% confluence on the day of transfection. 1.5 g of DNA and 2 l of LF2000 in 2 ml DMEM for transfection were added to each flask of cells. After incubation at 37 ◦ C for 4 h cells were divided into three groups. The positive control was treated with TPA
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(20 ng/ml), the negative-control was placed in a normoxic incubator, and the third group was placed in a hypoxic incubator. Cells were harvested at the indicated times with 1000 l of Glo lysis buffer (Promega) per flask. An equal volume of Bright-GloTM reagent (Promega) was mixed with cell lysate in each Eppendorf tube before luciferase activity was measured using a scintillation counter. 2.7. Quantitative analysis of EBV DNA copies in B95-8 cells B95-8 cells were treated as in the Western blot analysis and harvested at the indicated times. DNA was extracted using a QIAamp Blood Kit (Qiagen) using the “blood and body fluid protocol” as recommended by the manufacturer. Fifty nanograms of DNA was subjected to real-time quantitative PCR. The fluorogenic real-time quantitative PCR system developed for detecting the BamHI-W region of EBV DNA was described previously (Shao et al., 2004). Thermal cycling was initiated with a 2-min denaturation step at 93 ◦ C, followed by 40 cycles at 93 ◦ C for 30 s and a final extension at 55 ◦ C for 45 s.
Fig. 2. Induction of BZLF1 gene expression by hypoxia in B95-8 cell lines. (A) RT-PCR analysis of BZLF1 gene expression in response to hypoxia treatment. (B) Western blot analyses of Zta expression in response to hypoxia treatment. Cell extracts from TPA-treated B95–8 cells was used as the positive control for Zta expression. -Actin was blotted as the loading control.
3. Results 3.1. Effect of hypoxia on cell cycle arrest Previous studies have indicated that G0 /G1 cell growth arrest signaling occurs prior to detection of the EBV immediate-early gene product in EBV lytic induction systems (Rodriguez et al., 2001). In the present study, hypoxic culture conditions were established and HIF-1␣ protein expression was induced in CNE2 and B95-8 cells (data not shown). An examination of cell cycle changes in CNE2 cells and B95-8 cells after treatment with a lytic infection inducer, TPA, or with hypoxia, revealed that in both cell lines, the percentage of cells in G0 /G1 phase increased after either TPA treatment or hypoxia treatment (Fig. 1). These results suggest that both hypoxia and lytic infection inducers can induce alterations in the cell cycle. It is worth noting that a substantial percentage of the starting populations are in S phase. Because the cells were passed and then treated 12 h after passage with hypoxia
and TPA, at this time point, a significant amount of cultured cells were entering into S phase of the cell cycle. 3.2. Effect of hypoxia on EBV immediate-early gene expression We further examined whether Zta expression was induced following cellular entry into G0 /G1 cell growth arrest. As shown in Fig. 2A, BZLF1 transcripts were significantly elevated after hypoxia treatment. In addition, Zta protein was easily detected with an anti-Zta monoclonal antibody after hypoxia treatment (Fig. 2B). Hypoxia induced a timedependent increase in BZLF1 gene expression, which began at 36 h and reached a peak at 48 h. We noted that both Zta message and protein are detectable even at time zero. This may be partially due to the previous passage cells which were still in G0/G1 phase, as the consumption of serum in the medium,
Fig. 1. Analysis of CNE2 and B95-8 cell cycle after hypoxia or TPA treatment. CNE2 (A) and B95-8 (B) cells were cultured in the presence of hypoxic O2 conditions or TPA and harvested at the indicated times. Cell cycle analysis was carried out with FACScan fluorescence-activated cell sorter (FACS) and software from Phoenix Flow Systems. The bar graphs were derived from the means of three independent experiments.
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4. Discussion
Fig. 3. Zp activation in response to hypoxia treatment. CNE2 cells were transfected with the reporter plasmid pGL3 inserted with the BZLF1 gene promoter (Zp-236), and were treated with hypoxic O2 conditions or TPA. Subsequent luciferase activity (fold increase) was measured as described in Section 2. The relative luciferase activity was the ratio of Zp-236 luciferase activity measured in the presence of hypoxia or TPA to that measured without treatment. The values presented are means of three independent experiments.
and the activation of EBV could occur in these residual cells in G0/G1 phase. 3.3. Activation of BZLF1 gene promoter by hypoxia CNE2 (EBV-negative) cells were used to investigate the activation of the BZLF1 gene promoter. A luciferase-reporter plasmid with the BZLF1 gene promoter (ZP-236) was constructed and introduced into CNE2 cells. We calculated the relative luciferase activity as the ratio of Zp-236 luciferase activity of cells cultured under hypoxic conditions or in the presence of TPA to that cultured under normoxic conditions. As illustrated in Fig. 3, ZP-236 was activated two- to threefold and four- to five-fold by a 24-h and 48-h hypoxia treatment, respectively. The increment was relatively small when compared to the positive control of TPA treatment, which induced a more than 10-fold increase in luciferase reporter gene activity over the basal levels at both time points. 3.4. Hypoxia increased the copy number of viral DNA in B95-8 cells To examine whether the full cycle of viral replication was induced by hypoxia treatment, real-time PCR was used to quantify the increase in EBV DNA copy number in B95-8 cells. In this system, data are initially expressed as a Ct value. The Ct value corresponds to the cycle number at which the amplification plot for a given sample crosses the threshold, which is normally set at the point where the fluorescent signal equals 10 times the standard deviation of background fluorescence (Heid et al., 1996). As shown in Fig. 4, the Ct values were 20.5, 17.2, 16, 14.6, and 11.7 for B95-8 cells treated with hypoxia for 0, 12, 24, 36, and 48 h, respectively, and 9.3 after TPA treatment for 48 h.
Due to inadequate vasculature, primary tumors and related metastases in humans are exposed to significant hypoxia (Brizel et al., 1995). Hypoxic cells express the transcription factor HIF-1, which is a heterodimer composed of HIF-1␣ and HIF-1 subunits (Iyer et al., 1998; Wang et al., 1995). Under normoxic conditions, the HIF-1␣ gene is continuously transcribed and translated, but the HIF-1␣ protein is expressed at very low levels owing to rapid destruction via the ubiquitin-proteasome pathway. Under hypoxic conditions, HIF-1␣ is protected from ubiquitination and proteasomal degradation. However, either in hypoxic or normoxic conditions, HIF-1, also known as aryl hydrocarbon receptor nuclear translocator, is constitutively expressed (Huang et al., 1996; Salceda and Caro, 1997). Previous work has demonstrated that hypoxia can induce lytic replication of Kaposi sarcoma-associated herpes virus (KSHV) (Davis et al., 2001). Correspondingly, in the present study we described a relationship between hypoxia and the reactivation of EBV, another ubiquitous human herpes virus, and hypoxic culture conditions for EBV latently infected cells were established. Protein expression of HIF-1␣ (data not shown) was detectable after cells were treated under these conditions, indicating that the established hypoxia system was effective. Notably, hypoxia treatment induced G0 /G1 cell growth arrest in B95-8 and CNE2 cells, a cell cycle alteration similar to that caused by other lytic infection inducers. We further examined the in vitro reactivation of EBV by hypoxia. Hypoxia induced a time-dependent increase in BZLF1 gene transcription and protein expression. In addition, a luciferase-reporter plasmid with the BZLF1 gene promoter (Zp) was transfected into the NPC cell line, CNE2. Promoter activity was increased two- to three-fold and four- to fivefold by a 24-h and 48-h hypoxia treatment, respectively. The effect of hypoxia on Zp activation is similar to that of TGF1, which induced 3.5-fold activation of Zp (Liang et al., 2002). The protein product of the BZLF1 gene is Zta, which triggers the transition from latent infection to virus replication in EBV infected cells. Furthermore, we evaluated whether the full cycle of viral replication was induced by hypoxia treatment, using realtime polymerase chain reaction. We observed an increase in the number of EBV DNA copies in B95-8 cells after hypoxia treatment. Importantly, TPA was used as a positive control in our investigation of EBV reactivation. It is worth noting that there was a marked difference in Zp induction between hypoxia and TPA treatment (Fig. 3). This difference also occurred in the amount of viral DNA induced by treatment with hypoxia and TPA, but was not as significant as for Zp induction (Fig. 4). These results suggest that TPA is potentially stronger than hypoxia in inducing EBV reactivation. Other stressors, such as chemotherapy drugs and radiation, are also able to convert latent EBV infection into the lytic form (Feng et al., 2002; Westphal et al., 2000). Unlike small DNA tumour viruses, which use the host cell DNA
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Fig. 4. Detection of EBV DNA in the BamHI-W region by real-time quantitative PCR. B95-8 cells were exposed to hypoxic O2 conditions or treated with TPA and harvested at the indicated times. DNA extracted from cells was subjected to real-time quantitative PCR. The results were scored as the cycle number (Ct ) at which the amplification plot of fluorescence intensity crosses the threshold. The X-axis denotes the cycle number of a quantitative PCR reaction. The Y-axis denotes the Delta Rn, which is the fluorescence intensity over the background. Three independent experiments were carried out and similar results were obtained. 1, hypoxia 0 h; 2, hypoxia 12 h; 3, hypoxia 24 h; 4, hypoxia 36 h; 5, hypoxia 48 h; 6, TPA48.
polymerase, depend completely upon the host cell for replication, and drive host cells into S phase through the expression of viral proteins, large DNA viruses (e.g. herpes) have a large genome and prefer to replicate in a growth-arrested environment. This preference of EBV may be attributed to two factors. First, the large genome encodes its own DNA polymerase as well as a number of DNA-modifying enzymes. Thus, the replication of herpes viruses is much less dependent on the host cell than is the replication of smaller DNA viruses (Swanton and Jones, 2001). Second, if the replications of the virus and host cell occur at the same time, cellular resources required for replication may be limited. Thus, when latently infected cells are hypoxic, their DNA replication is arrested, permitting viral DNA replication to proceed. Seemingly, therefore, EBV has developed a clever and sensitive system for detecting stress and impending death in the host cell, allowing the virus to convert to the lytic form of infection and subsequently re-infect a healthy cell. Moreover, previous work suggests that LMP1, the major oncoprotein of EBV, can induce HIF-1␣ as well as other invasive and angiogenic factors (Wakisaka et al., 2004). Taken with the findings presented here, we hypothesize that hypoxia, HIF and EBV may collectively contribute to the development and progression of EBV-associated tumors. There are at least two promoters within the KSHV genome that are activated by hypoxia. One is within the promoter region of the Rta gene, the primary lytic switch gene, and the other is within the promoter region of ORF34, a lytic gene of unknown function. The ORF34 promoter contains three
putative consensus HREs oriented in the direction of the gene. These promoters could be strongly up regulated by HIF-1␣ and HIF-2␣ through activation of functional viral hypoxia response elements (HREs) (Haque et al., 2003). However, the signaling pathway involved in the reactivation of EBV by hypoxia was not defined in this study. Therefore, further experiments will be required to determine if this is a direct effect, and if so, which transcription factor is involved in the regulation of Zta expression and the characterization of the hypoxia responsive sites within the Zp promoter. The activation of the switch from latency to lytic infection of EBV by hypoxia that was demonstrated may play an important role in the pathogenesis of EBV associated tumours. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (2002BA711A03) and Guangdong Provincial Science and Technology Foundation (A1080202). References Brizel DM, Rosner GL, Prosnitz LR, Dewhirst MW. Patterns and variability of tumor oxygenation in human soft tissue sarcomas, cervical carcinomas, and lymph node metastases. Int J Rad Oncol Biol Phys 1995;32:1121–5. Davis DA, Rinderknecht AS, Zoeteweij JP, Aoki Y, Read-Connole EL, Tosato G, Blauvelt A, Yarchoan R. Hypoxia induces lytic replica-
J.-H. Jiang et al. / Journal of Clinical Virology 37 (2006) 98–103 tion of Kaposi sarcoma-associated herpesvirus. Blood 2001;97:3244– 50. di Renzo L, Altiok A, Klein G, Klein E. Endogenous TGF-beta contributes to the induction of the EBV lytic cycle in two Burkitt lymphoma cell lines. Int J Cancer 1994;57:914–9. Faggioni A, Zompetta C, Grimaldi S, Barile G, Frati L, Lazdins J. Calcium modulation activates Epstein-Barr virus genome in latently infected cells. Science 1986;232:1554–6. Feng WH, Israel B, Raab-Traub N, Busson P, Kenney SC. Chemotherapy induces lytic EBV replication and confers ganciclovir susceptibility to EBV-positive epithelial cell tumors. Cancer Res 2002;62:1920–6. Flamand L, Stefanescu I, Ablashi DV, Menezes J. Activation of the Epstein-Barr virus replicative cycle by human herpesvirus 6. J Virol 1993;67:6768–77. Gao X, Ikuta K, Tajima M, Sairenji T. 12-O-tetradecanoylphorbol-13-acetate induces Epstein-Barr virus reactivation via NF-kappaB and AP-1 as regulated by protein kinase C and mitogen-activated protein kinase. Virology 2001;286:91–9. Golden HD, Chang RS, Prescott W, Simpson E, Cooper TY. Leukocytetransforming agent: prolonged excretion by patients with mononucleosis and excretion by normal individuals. J Infect Dis 1973;127:471–3. Haque M, Davis DA, Wang V, Widmer I, Yarchoan R. Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8) contains hypoxia response elements: relevance to lytic induction by hypoxia. J Virol 2003;77:6761–8. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996;6:986–94. Henle G, Henle W, Clifford P, Diehl V, Kafuko GW, Kirya BG, Klein G, Morrow RH, Munube GM, Pike P, Tukei PM, Ziegler JL. Antibodies to Epstein-Barr virus in Burkitt’s lymphoma and control groups. J Natl Cancer Inst 1969;43:1147–57. Hockel M, Vaupel P. Tumor hypoxia: definitions and current clinical, biologic, and molecular aspects. J Natl Cancer Inst 2001;93:266–76. Huang LE, Arany Z, Livingston DM, Bunn HF. Activation of hypoxiainducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit. J Biol Chem 1996;271:32253–9. Iyer NV, Kotch LE, Agani F, Leung SW, Laughner E, Wenger RH, Gassmann M, Gearhart JD, Lawler AM, Yu AY, Semenza GL. Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1 alpha. Genes Dev 1998;12:149–62. Karimi L, Crawford DH, Speck S, Nicholson LJ. Identification of an epithelial cell differentiation responsive region within the BZLF1 promoter of the Epstein-Barr virus. J Gen Virol 1995;76:759–65. Krtolica A, Ludlow JW. Hypoxia arrests ovarian carcinoma cell cycle progression, but invasion is unaffected. Cancer Res 1996;56:1168–73.
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Liang CL, Chen JL, Hsu YP, Ou JT, Chang YS. Epstein-Barr virus BZLF1 gene is activated by transforming growth factor-beta through cooperativity of Smads and c-Jun/c-Fos proteins. J Biol Chem 2002;277:23345–57. Lieberman PM, Hardwick JM, Sample J, Hayward GS, Hayward SD. The zta transactivator involved in induction of lytic cycle gene expression in Epstein-Barr virus-infected lymphocytes binds to both AP-1 and ZRE sites in target promoter and enhancer regions. J Virol 1990;64: 1143–55. MacCallum P, Karimi L, Nicholson LJ. Definition of the transcription factors which bind the differentiation responsive element of the EpsteinBarr virus BZLF1 Z promoter in human epithelial cells. J Gen Virol 1999;80:1501–12. Rodriguez A, Jung EJ, Flemington EK. Cell cycle analysis of Epstein-Barr virus-infected cells following treatment with lytic cycle-inducing agents. J Virol 2001;75:4482–9. Salceda S, Caro J. Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J Biol Chem 1997;272:22642–7. Shao JY, Li YH, Gao HY, Wu QL, Cui NJ, Zhang L, Cheng G, Hu LF, Ernberg I, Zeng YX. Comparison of plasma Epstein-Barr virus (EBV) DNA levels and serum EBV immunoglobulin A/virus capsid antigen antibody titers in patients with nasopharyngeal carcinoma. Cancer 2004;100:1162–70. Swanton C, Jones N. Strategies in subversion: de-regulation of the mammalian cell cycle by viral gene products. Int J Exp Pathol 2001;82:3–13. Tovey MG, Lenoir G, Begon-Lours J. Activation of latent Epstein-Barr virus by antibody to human IgM. Nature 1978;276:270–2. Vaupel P, Kallinowski F, Okunieff P. Blood flow, oxygen and nutrient supply, and metabolic microenvironment of human tumors: a review. Cancer Res 1989;49:6449–65. Wakisaka N, Kondo S, Yoshizaki T, Murono S, Furukawa M, Pagano JS. Epstein-Barr virus latent membrane protein 1 induces synthesis of hypoxia-inducible factor 1 alpha. Mol Cell Biol 2004;24:5223– 34. Wang GL, Jiang BH, Rue EA, Semenza GL. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA 1995;92:5510–4. Westphal EM, Blackstock W, Feng W, Israel B, Kenney SC. Activation of lytic Epstein-Barr virus (EBV) infection by radiation and sodium butyrate in vitro and in vivo: a potential method for treating EBV-positive malignancies. Cancer Res 2000;60:5781–8. Young LS, Lau R, Rowe M, Niedobitek G, Packham G, Shanahan F, Rowe DT, Greenspan D, Greenspan JS, Rickinson AB. Differentiationassociated expression of the Epstein-Barr virus BZLF1 transactivator protein in oral hairy leukoplakia. J Virol 1991.
Hypoxia can contribute to the induction of the Epstein-Barr virus (EBV) lytic cycle Ju-Hong Jiang a , Na Wang b , Ang Li b , Wen-Ting Liao b , Zhi-Gang Pan b , Shi-Juan Mai b , Da-Jiang Li b , Mu-Sheng Zeng b , Jian-ming Wen a , Yi-Xin Zeng b,∗ a
Department of Pathology, The First Affiliated Hospital, Sun Yat-sen University, 651 Dong-Feng Road East, Guangzhou 510080, China b State Key Laboratory of Oncology in South China and The Department of Experimental Research, Cancer Center, Sun Yat-sen University, 651 Dong-Feng Road East, Guangzhou 510060, China Received 28 November 2005; received in revised form 20 June 2006; accepted 24 June 2006
Abstract Background: Like other herpes viruses, latent Epstein-Barr virus (EBV) infection can be reactivated to lytic replication. Reactivation can be achieved by treatment with various reagents, including tetradecanoyl phorbol acetate (TPA) and Ca2+ ionophores. Relatively little is known about the physiological factors related to reactivation of EBV. Previous studies have demonstrated that G0 /G1 cell cycle arrest is associated with EBV activation, and that hypoxic conditions can induce cell cycle arrest. In the present study we investigated the effect of hypoxia on reactivation of EBV. Objective and methods: Hypoxic culture conditions were established and the expression of Zta protein and the number of EBV DNA copies were measured in B95-8 cells maintained under these conditions. Results: Hypoxia treatment not only increased the expression of the EBV immediate-early protein Zta (which mediates the switch between the latent and lytic form of infection), but also increased the number of EBV DNA copies in B95-8 cells. Conclusions: EBV in latent infection can be activated to lytic infection by hypoxia treatment. © 2006 Elsevier B.V. All rights reserved. Keywords: Hypoxia; Epstein-Barr virus; Reactivation; Nasopharyngeal carcinoma
1. Introduction Epstein-Barr virus (EBV) is a ubiquitous human herpes virus infecting over 90% of adults worldwide (Henle et al., 1969). In vivo, EBV primarily targets lymphoid and epithelial cells. Primary infection with EBV is followed by a lifelong presence of the virus within the host (Golden et al., 1973). Like other herpes viruses, EBV infection can remain latent and be reactivated to lytic replication. In vitro, latently infected cells can be reactivated by treatment with tetradecanoyl phorbol acetate (TPA), Ca2+ ionophore, anti-IgM, human herpes virus 6 infection, and transforming growth factor beta 1 (TGF-1) (di Renzo et al., 1994; Faggioni et al., 1986; Flamand et al., 1993; Gao et al., 2001; Liang et al., ∗
Corresponding author. Fax: +86 20 8734 3295. E-mail address: [email protected] (Y.-X. Zeng).
1386-6532/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2006.06.013
2002; Tovey et al., 1978). These treatments can induce the expression of the BZLF1 gene product ZEBRA, also known as Zta, which exhibits homology to the cellular transcription factor c-fos. Expression of Zta in latently infected cells is sufficient to trigger the viral lytic cycle (Lieberman et al., 1990). Zta expression is regulated by activation of the promoter Zp, which is located between the coding sequence of BRLF1 and BZLF1 (ranging from −221 to +13 of the BZLF1 gene) (Liang et al., 2002). With the exception of epithelial differentiation signals, there is relatively little information on the mechanism underlying EBV reactivation. An investigation of the EBV-related lesion, oral hairy leukoplakia (OHL), suggested that the trigger of the viral lytic cycle is associated with epithelial differentiation, as Zta immunoreactivity within nuclei was confined to the upper spinous layer and generally appeared as a single cell layer immediately preceding Viral
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Capsid Antigen (VCA) expression. Zta staining was not observed in the lower epithelial layers (Young et al., 1991). This phenomenon was further supported by in vitro studies in epithelial cell lines (Karimi et al., 1995; MacCallum et al., 1999). In addition, studies by Rodriguez et al. (2001) demonstrated that treatment of latently infected cells with lytic cycle-inducing agents induced G0/G1 cell cycle arrest prior to detectable Zta expression. The finding that EBV replication is associated with cellular differentiation is consistent with EBV reactivation in growth-arrested cells and suggested that other physiological factors that induce cell cycle arrest may be associated with EBV reactivation. Hypoxic conditions can manifest during tumor development when the unregulated proliferation of tumour cells exceeds the available blood supply and subsequent oxygen supply. Hypoxic conditions are present in most solid tumours (Hockel and Vaupel, 2001; Vaupel et al., 1989). Exposing cultured cells to hypoxic conditions can induce cell cycle arrest (Krtolica and Ludlow, 1996). However, whether hypoxia is associated with lytic reactivation of EBV in nasopharyngeal carcinoma (NPC) remains unknown. The goal of the present study was to identify the role of hypoxia in EBV reactivation.
2. Methods 2.1. Cell lines and hypoxia treatment B95-8 is a marmoset B-cell line transformed with EBV; CNE2 (EBV-negative) is a human undifferentiated NPC cell line. Cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 mg of streptomycin per milliliter, and 100 U of penicillin per milliliter in a humidified atmosphere at 37 ◦ C with 5% CO2 . Cells were cultured inside an air-tight chamber maintained under hypoxic conditions (1% O2 ) by pumping (via a vacuum pump) air out and pumping in a mixture of 1% O2 , 5% CO2 , and 94% N2 . 2.2. Plasmid construction The promoter sequence of the BZLF1 gene (Zp-236) was amplified by PCR using B95-8 genomic DNA as the template and primers Zp-F (5 -catctcgagaatgtctgctgcatgcca-3 ,) and Zp-R (5 -gtcagatctggcaaggtgcaatgttta-3 ) (Liang et al., 2002). The Zp-F and Zp-R primers were flanked by XhoI and BglII restriction sites (underlined), respectively. The PCR product of the Zp-236 fragment was first cloned into the pGEM-T vector (Promega, Madison, WI, USA), then cloned into the pGL3-Basic vector through XhoI and BglII double digestion. The constructed plasmid was named “pGL3-Zp-236.” 2.3. Cell cycle analysis CNE2 and B95-8 cells treated with normoxia, hypoxia, or TPA (20 ng/ml), were harvested at the indicated times, washed twice with phosphate-buffered saline (PBS), sus-
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pended in 0.5 ml cold PBS (4 ◦ C), fixed with 5 ml 70% cold (−20 ◦ C) ethanol, incubated at 4 ◦ C overnight, washed twice with PBS, and treated with RNase A (0.1 mg/ml) in 69 mM propidium iodide at 37 ◦ C for 45 min. Cell cycle analysis was carried out using a FACScan fluorescence-activated cell sorter and software of Phoenix Flow Systems. 2.4. RNA extraction and semi-quantitative reverse transcription-PCR (RT-PCR) B95-8 cells were treated with normoxia, hypoxia, or TPA (20 ng/ml), and harvested at the indicated times. Total RNA was extracted from cells by Trizol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription and amplification were carried out in a single step in 25 l of reaction mixture containing 0.2 M BZLF1 5 primer (5-agaggtgtgtcagccaaaga-3) and 3 primer (5 -cttcctccttctggggaata-3 ), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) 5 primer (5-caggggggagccaaaagg-3) and 3 primer (5-ggcagtggggacacggaa-3), dNTP mix (200 M for each), 1 l of one step RT-PCR enzyme mix (Qiagen, Hilden, Germany), and 1 g of total RNA. The thermal conditions of RT-PCR were established according to the recommendation of manufacturer and the annealing temperature of the primers. Five microliters of RTPCR product was analyzed on a 1.5% agarose gel. The size of PCR products of BZLF1 and GAPDH were 278 and 378 bp, respectively. 2.5. Western blot analysis B95-8 cells were treated with normoxia, hypoxia, or TPA (20 ng/ml) and harvested at the indicated times. Cells were collected by centrifugation and washed once in PBS, then lysed in sodium dodecyl sulfate (SDS) loading buffer containing -mercaptoethanol. Samples were heated to 100 ◦ C for 5 min and centrifuged at 10,000 × g for 10 min prior to a protein concentration assay and loading on an SDS–10% polyacrylamide gel. Following electrophoresis, the proteins were electro-transferred to a polyvinylidene fluoride (PVDF) transfer membrane. The blotting process was performed according to the protocol of the Phototope® -HRP Western Blot Detection System (Cell Signaling Technology, Inc., MA, USA) using a Zta monoclonal antibody (Santa Cruz Biotechnology Inc., CA, USA). 2.6. Transient DNA transfection and reporter gene assay The reporter plasmid pGL3-Zp-236 was introduced into the NPC cell line CNE2 using LipofectAMINETM 2000 (LF2000) reagent (Invitrogen). The day before transfection adherent CNE2 cells were trypsinized, counted, and plated at 5 × 105 cells per 25-ml flask to achieve 90% confluence on the day of transfection. 1.5 g of DNA and 2 l of LF2000 in 2 ml DMEM for transfection were added to each flask of cells. After incubation at 37 ◦ C for 4 h cells were divided into three groups. The positive control was treated with TPA
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(20 ng/ml), the negative-control was placed in a normoxic incubator, and the third group was placed in a hypoxic incubator. Cells were harvested at the indicated times with 1000 l of Glo lysis buffer (Promega) per flask. An equal volume of Bright-GloTM reagent (Promega) was mixed with cell lysate in each Eppendorf tube before luciferase activity was measured using a scintillation counter. 2.7. Quantitative analysis of EBV DNA copies in B95-8 cells B95-8 cells were treated as in the Western blot analysis and harvested at the indicated times. DNA was extracted using a QIAamp Blood Kit (Qiagen) using the “blood and body fluid protocol” as recommended by the manufacturer. Fifty nanograms of DNA was subjected to real-time quantitative PCR. The fluorogenic real-time quantitative PCR system developed for detecting the BamHI-W region of EBV DNA was described previously (Shao et al., 2004). Thermal cycling was initiated with a 2-min denaturation step at 93 ◦ C, followed by 40 cycles at 93 ◦ C for 30 s and a final extension at 55 ◦ C for 45 s.
Fig. 2. Induction of BZLF1 gene expression by hypoxia in B95-8 cell lines. (A) RT-PCR analysis of BZLF1 gene expression in response to hypoxia treatment. (B) Western blot analyses of Zta expression in response to hypoxia treatment. Cell extracts from TPA-treated B95–8 cells was used as the positive control for Zta expression. -Actin was blotted as the loading control.
3. Results 3.1. Effect of hypoxia on cell cycle arrest Previous studies have indicated that G0 /G1 cell growth arrest signaling occurs prior to detection of the EBV immediate-early gene product in EBV lytic induction systems (Rodriguez et al., 2001). In the present study, hypoxic culture conditions were established and HIF-1␣ protein expression was induced in CNE2 and B95-8 cells (data not shown). An examination of cell cycle changes in CNE2 cells and B95-8 cells after treatment with a lytic infection inducer, TPA, or with hypoxia, revealed that in both cell lines, the percentage of cells in G0 /G1 phase increased after either TPA treatment or hypoxia treatment (Fig. 1). These results suggest that both hypoxia and lytic infection inducers can induce alterations in the cell cycle. It is worth noting that a substantial percentage of the starting populations are in S phase. Because the cells were passed and then treated 12 h after passage with hypoxia
and TPA, at this time point, a significant amount of cultured cells were entering into S phase of the cell cycle. 3.2. Effect of hypoxia on EBV immediate-early gene expression We further examined whether Zta expression was induced following cellular entry into G0 /G1 cell growth arrest. As shown in Fig. 2A, BZLF1 transcripts were significantly elevated after hypoxia treatment. In addition, Zta protein was easily detected with an anti-Zta monoclonal antibody after hypoxia treatment (Fig. 2B). Hypoxia induced a timedependent increase in BZLF1 gene expression, which began at 36 h and reached a peak at 48 h. We noted that both Zta message and protein are detectable even at time zero. This may be partially due to the previous passage cells which were still in G0/G1 phase, as the consumption of serum in the medium,
Fig. 1. Analysis of CNE2 and B95-8 cell cycle after hypoxia or TPA treatment. CNE2 (A) and B95-8 (B) cells were cultured in the presence of hypoxic O2 conditions or TPA and harvested at the indicated times. Cell cycle analysis was carried out with FACScan fluorescence-activated cell sorter (FACS) and software from Phoenix Flow Systems. The bar graphs were derived from the means of three independent experiments.
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4. Discussion
Fig. 3. Zp activation in response to hypoxia treatment. CNE2 cells were transfected with the reporter plasmid pGL3 inserted with the BZLF1 gene promoter (Zp-236), and were treated with hypoxic O2 conditions or TPA. Subsequent luciferase activity (fold increase) was measured as described in Section 2. The relative luciferase activity was the ratio of Zp-236 luciferase activity measured in the presence of hypoxia or TPA to that measured without treatment. The values presented are means of three independent experiments.
and the activation of EBV could occur in these residual cells in G0/G1 phase. 3.3. Activation of BZLF1 gene promoter by hypoxia CNE2 (EBV-negative) cells were used to investigate the activation of the BZLF1 gene promoter. A luciferase-reporter plasmid with the BZLF1 gene promoter (ZP-236) was constructed and introduced into CNE2 cells. We calculated the relative luciferase activity as the ratio of Zp-236 luciferase activity of cells cultured under hypoxic conditions or in the presence of TPA to that cultured under normoxic conditions. As illustrated in Fig. 3, ZP-236 was activated two- to threefold and four- to five-fold by a 24-h and 48-h hypoxia treatment, respectively. The increment was relatively small when compared to the positive control of TPA treatment, which induced a more than 10-fold increase in luciferase reporter gene activity over the basal levels at both time points. 3.4. Hypoxia increased the copy number of viral DNA in B95-8 cells To examine whether the full cycle of viral replication was induced by hypoxia treatment, real-time PCR was used to quantify the increase in EBV DNA copy number in B95-8 cells. In this system, data are initially expressed as a Ct value. The Ct value corresponds to the cycle number at which the amplification plot for a given sample crosses the threshold, which is normally set at the point where the fluorescent signal equals 10 times the standard deviation of background fluorescence (Heid et al., 1996). As shown in Fig. 4, the Ct values were 20.5, 17.2, 16, 14.6, and 11.7 for B95-8 cells treated with hypoxia for 0, 12, 24, 36, and 48 h, respectively, and 9.3 after TPA treatment for 48 h.
Due to inadequate vasculature, primary tumors and related metastases in humans are exposed to significant hypoxia (Brizel et al., 1995). Hypoxic cells express the transcription factor HIF-1, which is a heterodimer composed of HIF-1␣ and HIF-1 subunits (Iyer et al., 1998; Wang et al., 1995). Under normoxic conditions, the HIF-1␣ gene is continuously transcribed and translated, but the HIF-1␣ protein is expressed at very low levels owing to rapid destruction via the ubiquitin-proteasome pathway. Under hypoxic conditions, HIF-1␣ is protected from ubiquitination and proteasomal degradation. However, either in hypoxic or normoxic conditions, HIF-1, also known as aryl hydrocarbon receptor nuclear translocator, is constitutively expressed (Huang et al., 1996; Salceda and Caro, 1997). Previous work has demonstrated that hypoxia can induce lytic replication of Kaposi sarcoma-associated herpes virus (KSHV) (Davis et al., 2001). Correspondingly, in the present study we described a relationship between hypoxia and the reactivation of EBV, another ubiquitous human herpes virus, and hypoxic culture conditions for EBV latently infected cells were established. Protein expression of HIF-1␣ (data not shown) was detectable after cells were treated under these conditions, indicating that the established hypoxia system was effective. Notably, hypoxia treatment induced G0 /G1 cell growth arrest in B95-8 and CNE2 cells, a cell cycle alteration similar to that caused by other lytic infection inducers. We further examined the in vitro reactivation of EBV by hypoxia. Hypoxia induced a time-dependent increase in BZLF1 gene transcription and protein expression. In addition, a luciferase-reporter plasmid with the BZLF1 gene promoter (Zp) was transfected into the NPC cell line, CNE2. Promoter activity was increased two- to three-fold and four- to fivefold by a 24-h and 48-h hypoxia treatment, respectively. The effect of hypoxia on Zp activation is similar to that of TGF1, which induced 3.5-fold activation of Zp (Liang et al., 2002). The protein product of the BZLF1 gene is Zta, which triggers the transition from latent infection to virus replication in EBV infected cells. Furthermore, we evaluated whether the full cycle of viral replication was induced by hypoxia treatment, using realtime polymerase chain reaction. We observed an increase in the number of EBV DNA copies in B95-8 cells after hypoxia treatment. Importantly, TPA was used as a positive control in our investigation of EBV reactivation. It is worth noting that there was a marked difference in Zp induction between hypoxia and TPA treatment (Fig. 3). This difference also occurred in the amount of viral DNA induced by treatment with hypoxia and TPA, but was not as significant as for Zp induction (Fig. 4). These results suggest that TPA is potentially stronger than hypoxia in inducing EBV reactivation. Other stressors, such as chemotherapy drugs and radiation, are also able to convert latent EBV infection into the lytic form (Feng et al., 2002; Westphal et al., 2000). Unlike small DNA tumour viruses, which use the host cell DNA
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Fig. 4. Detection of EBV DNA in the BamHI-W region by real-time quantitative PCR. B95-8 cells were exposed to hypoxic O2 conditions or treated with TPA and harvested at the indicated times. DNA extracted from cells was subjected to real-time quantitative PCR. The results were scored as the cycle number (Ct ) at which the amplification plot of fluorescence intensity crosses the threshold. The X-axis denotes the cycle number of a quantitative PCR reaction. The Y-axis denotes the Delta Rn, which is the fluorescence intensity over the background. Three independent experiments were carried out and similar results were obtained. 1, hypoxia 0 h; 2, hypoxia 12 h; 3, hypoxia 24 h; 4, hypoxia 36 h; 5, hypoxia 48 h; 6, TPA48.
polymerase, depend completely upon the host cell for replication, and drive host cells into S phase through the expression of viral proteins, large DNA viruses (e.g. herpes) have a large genome and prefer to replicate in a growth-arrested environment. This preference of EBV may be attributed to two factors. First, the large genome encodes its own DNA polymerase as well as a number of DNA-modifying enzymes. Thus, the replication of herpes viruses is much less dependent on the host cell than is the replication of smaller DNA viruses (Swanton and Jones, 2001). Second, if the replications of the virus and host cell occur at the same time, cellular resources required for replication may be limited. Thus, when latently infected cells are hypoxic, their DNA replication is arrested, permitting viral DNA replication to proceed. Seemingly, therefore, EBV has developed a clever and sensitive system for detecting stress and impending death in the host cell, allowing the virus to convert to the lytic form of infection and subsequently re-infect a healthy cell. Moreover, previous work suggests that LMP1, the major oncoprotein of EBV, can induce HIF-1␣ as well as other invasive and angiogenic factors (Wakisaka et al., 2004). Taken with the findings presented here, we hypothesize that hypoxia, HIF and EBV may collectively contribute to the development and progression of EBV-associated tumors. There are at least two promoters within the KSHV genome that are activated by hypoxia. One is within the promoter region of the Rta gene, the primary lytic switch gene, and the other is within the promoter region of ORF34, a lytic gene of unknown function. The ORF34 promoter contains three
putative consensus HREs oriented in the direction of the gene. These promoters could be strongly up regulated by HIF-1␣ and HIF-2␣ through activation of functional viral hypoxia response elements (HREs) (Haque et al., 2003). However, the signaling pathway involved in the reactivation of EBV by hypoxia was not defined in this study. Therefore, further experiments will be required to determine if this is a direct effect, and if so, which transcription factor is involved in the regulation of Zta expression and the characterization of the hypoxia responsive sites within the Zp promoter. The activation of the switch from latency to lytic infection of EBV by hypoxia that was demonstrated may play an important role in the pathogenesis of EBV associated tumours. Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (2002BA711A03) and Guangdong Provincial Science and Technology Foundation (A1080202). References Brizel DM, Rosner GL, Prosnitz LR, Dewhirst MW. Patterns and variability of tumor oxygenation in human soft tissue sarcomas, cervical carcinomas, and lymph node metastases. Int J Rad Oncol Biol Phys 1995;32:1121–5. Davis DA, Rinderknecht AS, Zoeteweij JP, Aoki Y, Read-Connole EL, Tosato G, Blauvelt A, Yarchoan R. Hypoxia induces lytic replica-
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