1 Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Room 714, Number 1, Section 1, Jen-Ai Road, Taipei, Taiwan
2 Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan
Correspondence
Ching-Hwa Tsai
chtsai{at}ha.mc.ntu.edu.tw
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ABSTRACT |
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INTRODUCTION |
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The majority of EBV infection in vivo is latent and it is this type of infection that is observed in peripheral B lymphocytes of healthy carriers and in most tumour cells of EBV-associated cancers such as nasopharyngeal carcinoma (NPC), Hodgkin's disease (HD) and endemic Burkitt's lymphoma (Rickinson & Kieff, 2001). Intriguingly, several clues indicate that EBV reactivation into the lytic cycle may play a role in the pathogenesis of these malignancies. Elevated antibody titres against EBV lytic antigens and increased viral DNA load in serum/plasma, two parameters that represent EBV reactivation in vivo, correlate with advanced cancer stages, poor prognosis or tumour recurrence after therapy (de-Vathaire et al., 1988
; Henle et al., 1969
, 1977
; Henle & Henle, 1976
; Lei et al., 2000
; Levine et al., 1971
; Lo, 2001
). Serological studies further suggest that EBV reactivation may occur months or years before the clinical diagnosis of NPC, HD and endemic Burkitt's lymphoma, serving as a risk factor of cancer development (Chien et al., 2001
; Geser et al., 1982
; Mueller et al., 1989
; Zeng et al., 1985
). Another notable clue comes from in vitro studies, in which the EBV lytic cycle was activated by extracts of some foodstuffs or plants that were identified as dietary or environmental risk factors of NPC or endemic Burkitt's lymphoma (Bouvier et al., 1995
; MacNeil et al., 2003
; Shao et al., 1988
).
The aetiological role of the EBV lytic cycle is further identified in the disease oral hairy leukoplakia (OHL), a tongue lesion with epithelial hyperplasia in immunodeficient patients (Rickinson & Kieff, 2001; Triantos et al., 1997
). OHL is characterized by an unusual activation of the EBV lytic cycle, so that abundant EBV lytic proteins, viral genomes and virus particles are detected in the squamous epithelial cells (Becker et al., 1991
; Greenspan et al., 1985
; Young et al., 1991
). Inhibition of the productive EBV replication by antiviral agents, such as acyclovir, results in effective resolution of OHL, indicating that EBV lytic replication is necessary for the pathogenesis of the lesion (Resnick et al., 1988
; Triantos et al., 1997
). However, such treatment is not always satisfactory, because the EBV lytic cycle and OHL lesions frequently recur after withdrawal of the drugs (Resnick et al., 1988
; Triantos et al., 1997
; Walling et al., 2003
).
As EBV reactivation and productive replication are involved in EBV-related diseases, development of an effective strategy to inhibit the EBV lytic cycle may be of value in reducing the disease risk or improving clinical outcome. One attractive approach is RNA interference (RNAi), in which sequence-specific RNA degradation leading to gene silencing is directed by a short RNA duplex, small interfering RNA (siRNA) (Dykxhoorn et al., 2003; Gitlin & Andino, 2003
). The siRNA-mediated inhibition of virus replication is successful in some viral systems that exhibit productive infection (Ge et al., 2003
; Gitlin et al., 2002
; Jia & Sun, 2003
; Novina et al., 2002
). However, such a permissive system for EBV productive replication has not been available, in that EBV infection in vitro is predominantly restricted to latency (Rickinson & Kieff, 2001
). The EBV lytic cycle can be conditionally activated in vitro, but it has still not been tested whether RNAi can prevent the viral switch from latency to lytic replication.
Based on the following reasons, our first RNAi target gene for inhibiting EBV reactivation is Zta, an immediate-early gene of the lytic cycle. (i) Exogenous stimuli triggering EBV reactivation induce Zta expression (Mellinghoff et al., 1991). Being a key transcriptional activator, Zta protein is sufficient to disrupt EBV latency (Grogan et al., 1987
). (ii) A study of a Zta-deleted EBV mutant showed that Zta is essential for full expression of lytic genes and for viral DNA replication (Feederle et al., 2000
). (iii) Theoretically, Zta-targeted RNAi will knock down not only Zta but also endogenous Rta, another essential transcriptional activator of the lytic cycle, because endogenous Rta is expressed from the Rta/Zta bicistronic mRNAs that should be degraded by Zta-specific RNAi (Feederle et al., 2000
; Manet et al., 1989
). (iv) In the initial step of EBV reactivation, Zta and Rta autostimulate their own expression, reciprocally activate each other and cooperatively induce the downstream lytic cascade (Adamson et al., 2000
; Holley-Guthrie et al., 1990
; Liu & Speck, 2003
; Ragoczy et al., 1998
). Therefore, Zta-targeted RNAi may block the positive feedback loop at the beginning of the lytic cycle.
In this study, we examined the potential of Zta-targeted RNAi to inhibit the EBV lytic cycle in two situations: one was EBV reactivation induced by exogenous stimuli and the other was a novel infection state showing constitutive activation of the EBV lytic cycle in epithelial cells.
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METHODS |
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Plasmids.
The pSUPER plasmid was described by Brummelkamp et al. (2002). siRNA-expressing plasmids were constructed by cloning siRNA sequences into pSUPER via BglII and HindIII sites. siRNAs targeted against Zta and green fluorescence protein (GFP) were designed using a program available online (https://www.genscript.com/ssl-bin/app/rnai) and the top-ranked sequence for each gene was chosen in this study. The siRNA sequences were further subjected to a BLAST search against human genome and EST databases to ensure that no human gene was targeted. The Zta-targeted siRNA, siZ1, is directed against the Zta sequence 5'-CAACAGCTAGCAGACATTG-3' (nucleotides 415433 downstream of the start codon). The targeted Zta sequence is conserved among Akata, B95-8 and P3HR-1 strains of EBV. The predicted structure of siZ1 after transcription and processing in cells is shown in Fig. 1
(a). The sequence of GFP-targeted siRNA, siGFP, is 5'-GCTGACCCTGAAGTTCATCTG-3'. The plasmids expressing Zta and GFP were derived from the pRc/CMV plasmid (Lu et al., 2000
). The Rta-expressing plasmid RTS15 was described by Ragoczy et al. (1998)
.
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Expression of siRNA and induction of the EBV lytic cycle.
In the experiment shown in Fig. 1(b), 3 µg pSUPER-derived siRNA-producing plasmids were co-transfected with 3 µg plasmids expressing GFP, Zta or Rta into 293 cells using Lipofectamine 2000 transfection reagent (Invitrogen). For siRNA-mediated inhibition of EBV reactivation, NA cells were pre-transfected with 5 µg siRNA-producing plasmids for 48 h and then either treated with 12-O-tetradecanoylphorbol-13-acetate (TPA; 40 ng ml1) plus sodium n-butyrate (3 mM) for 24 h or post-transfected with 4 µg Rta-expressing plasmids for 24 h. For siRNA-mediated suppression of the constitutive lytic cycle, 293A-2 and 293A98 cell clones were subcultured every 4 days and each passage was followed by transfection once with siRNA-expressing plasmids.
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Detection of viral and cellular proteins.
The immunoblotting assay was performed as described previously (Chang et al., 1999). The positive control for expression of EBV lytic proteins was the protein lysate of NA cells treated with TPA and n-butyrate for 48 h (Chang et al., 1999
). The positive control for activation of the interferon pathway was the protein lysate of NA cells treated with interferon
(1000 U ml1) for 10 min. An immunofluorescence assay was used to examine the percentage of cells expressing lytic proteins, as described previously (Chang et al., 1999
).
Detection and quantification of EBV DNA.
Cells were lysed and digested by proteinase K as described previously and then subjected to the following PCR analysis (Chang et al., 2002). Conventional PCR detection of BamHI W fragments of the EBV genome was performed following our previous protocol (Chang et al., 2002
). For quantification of EBV DNA, real-time PCR was used according to the manufacturer's instructions (Applied Biosystems). The detection target of real-time PCR was the EBNA1 region of the EBV genome; details of the primers and probes were provided in a previous study (Lo et al., 1999
). We used H2B4 cells harbouring one EBV genome per cell to generate a standard curve for quantification (Chang et al., 2002
) and EBV copy number was calculated by comparison with the standard. All samples were tested in duplicate.
Titration of infectious EBV particles.
Filtered culture supernatants of tested cells were subjected to serial twofold dilution and used to infect human peripheral blood mononuclear cells as described previously (Miller & Lipman, 1973). The titre of EBV was determined by its ability to transform primary B lymphocytes into lymphoblastoid cell lines in 4 weeks.
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RESULTS |
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Zta-targeted RNAi inhibited EBV reactivation induced by chemicals
Next, we tested whether siZ1 could prevent EBV reactivation induced by TPA and n-butyrate in an EBV-positive NPC cell line, NA (Chang et al., 1999). The cells were transiently transfected with siRNA-expressing plasmids and treated with the chemicals. After chemical induction in the absence of siRNA, three major species of viral transcripts encoding Zta were detected: 1 kb Zta monocistronic mRNA and 4 kb and 3 kb Rta/Zta bicistronic mRNAs (Fig. 2
a) (Manet et al., 1989
; Mellinghoff et al., 1991
). In agreement with expectations, all three transcripts were significantly diminished in the presence of siZ1 (Fig. 2a
). Consistently, the protein levels of Zta and Rta induced by the chemicals were apparently decreased by siZ1 (Fig. 2b
). We also observed siZ1-mediated reduction of expression of the viral BMRF1 and BHRF1 genes, both of which are downstream lytic genes regulated by Zta and Rta (Fig. 2b
) (Cox et al., 1990
; Holley-Guthrie et al., 1990
). To examine whether siZ1 further suppressed lytic replication of the viral genomes, EBV DNA copies in NA cells were quantified using a real-time PCR method (Lo et al., 1999
). Untreated NA cells harboured about 15 copies of EBV DNA per cell on average, while viral genomes increased more than tenfold after 1 day of chemical induction in the absence of siRNA (Fig. 2c
). Such viral DNA amplification in the lytic cycle was almost completely blocked by siZ1 (Fig. 2c
). EBV reactivation triggered by TPA and n-butyrate was inhibited specifically by siZ1-directed RNAi, since the inhibition could not be achieved by either siGFP or the control pSUPER vector (Fig. 2
). Meanwhile, siZ1 had no effect on the expression of a latent gene, EBNA1 (Fig. 2b
).
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Zta-targeted RNAi did not activate the interferon pathway
Although the action of RNAi was thought to be independent of the interferon pathway, a recent study showed that some siRNAs activate the interferon response (Gitlin & Andino, 2003; Sledz et al., 2003
). To examine whether the siZ1-mediated inhibition of EBV reactivation involved the antiviral effect of interferon, we measured the phosphorylation of STAT1, a common signalling event triggered by interferon (Sledz et al., 2003
; Stark et al., 1998
). While treatment with interferon
induced significant phosphorylation of STAT1 in NA cells, expression of siZ1 caused little enhancement of STAT1 phosphorylation (Fig. 4
a). Therefore, the prevention of EBV reactivation by siZ1 was not likely to be mediated by activation of the interferon pathway. In addition, the inhibitory effect of RNAi was not likely to be caused by toxicity to the cells, as expression of siZ1 did not affect the viability of 293 or NA cells (Fig. 4b
).
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DISCUSSION |
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Compared with conventional anti-EBV agents such as acyclovir, Zta-targeted RNAi has some advantages. Acyclovir inhibits EBV DNA synthesis but does not cause suppression of immediate-early or early lytic genes such as Zta, Rta and BMRF1, whose expression is independent of viral DNA replication (Takase et al., 1996). These unaffected lytic proteins may facilitate recurrence of the lytic cycle, perhaps explaining the frequent relapse of OHL and EBV reactivation after withdrawal of acyclovir treatment (Resnick et al., 1988
; Triantos et al., 1997
). Zta-targeted RNAi may prevent the lytic cycle more completely because it blocks the initial and essential step of EBV reactivation, the expression of endogenous Zta and Rta. In addition, RNAi may bring much less unfavourable side-effects than the antiviral drugs as it knocks down gene expression in a sequence-specific manner and may cause little cytotoxicity (Figs 1b and 4b
). Our results indicate that RNAi is of potential use in developing a new anti-EBV approach. Before its application to clinical therapy, however, an effective strategy to deliver siRNA into target cells of patients is required. We are testing the feasibility using viral vectors for the delivery of siRNA-expressing genes.
Zta-targeted RNAi is also useful to clarify the roles of Zta and Rta in regulation of downstream lytic genes. A good example provided here is the regulation of BMRF1 and BHRF1. Although the promoters of both genes contain responsive elements for Zta and Rta, they are regulated differentially by the two transactivators (Cox et al., 1990; Holley-Guthrie et al., 1990
). In lymphoid cells, the induction of BMRF1 requires cooperation of Zta and Rta, while, in epithelial cells, Zta alone seems sufficient to stimulate BMRF1 expression (Feederle et al., 2000
; Ragoczy & Miller, 1999
). In this study, siZ1 blocked BMRF1 expression even in the presence of exogenous Rta (Fig. 3b
), indicating that Zta is the sole and essential transactivator for the BMRF1 gene in epithelial cells. On the other hand, it has been reported that the BHRF1 promoter could be activated by Rta alone and synergistically enhanced by Zta and Rta (Cox et al., 1990
; Ragoczy & Miller, 1999
). Our results showed that Rta-mediated induction of BHRF1 was not affected by siZ1 (Fig. 3b
), suggesting that expression of BHRF1 is regulated mainly by Rta and independent of Zta in NA cells. This study indicates that use of siRNAs targeted against lytic genes can be a feasible approach to analyse how the viral expression cascade is regulated during the lytic cycle.
It is notable that we have established EBV-positive 293A cell clones with constitutive activation of the lytic cycle, a permissive state resembling EBV infection in OHL (Greenspan et al., 1985). According to our observations, the EBV lytic cycle in 293A lytic clones was maintained at a moderate level below the maximum that could be fully activated (Fig. 6
). The moderate level of the lytic cycle in these lytic clones may represent a balanced state of EBV productive infection with minimized cytotoxicity, as the cell viability of lytic clones was similar to that of the latent clones (data not shown). Two possible mechanisms may account for the permissive EBV infection: a rearranged viral genome causing constitutive expression of Zta or Rta or a unique cellular factor supporting spontaneous activation of the Zta or Rta promoter (Grogan et al., 1987
; Young et al., 1991
). No matter which mechanism is the case, the expression of Zta is essentially required as siZ1 suppressed the constitutive EBV activation in 293A lytic clones (Fig. 7
). These cell clones are useful for screening and evaluation of agents for inhibiting the EBV lytic cycle. In addition, they also provide good opportunities to explore the mechanisms of spontaneous EBV reactivation and the effects of viral productive infection on epithelial cells.
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ACKNOWLEDGEMENTS |
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Received 9 December 2003;
accepted 13 February 2004.