Download Inhibition of the Epstein–Barr virus lytic cycle by

Journal of General Virology (2004), 85, 1371–1379
DOI 10.1099/vir.0.79886-0
Inhibition of the Epstein–Barr virus lytic cycle
by Zta-targeted RNA interference
Yao Chang,1 Shih-Shin Chang,1 Heng-Huan Lee,1 Shin-Lian Doong,1
Kenzo Takada2 and Ching-Hwa Tsai1
1
Graduate Institute of Microbiology, College of Medicine, National Taiwan University, Room 714,
Number 1, Section 1, Jen-Ai Road, Taipei, Taiwan
Correspondence
Ching-Hwa Tsai
2
[email protected]
Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan
Received 9 December 2003
Accepted 13 February 2004
Epstein–Barr virus (EBV) reactivation into the lytic cycle plays certain roles in the development
of EBV-associated diseases, so an effective strategy to block the viral lytic cycle may be of
value to reduce the disease risk or to improve the clinical outcome. This study examined whether
the EBV lytic cycle could be inhibited using RNA interference (RNAi) directed against the
essential viral gene Zta. In cases of EBV reactivation triggered by chemicals or by exogenous
Rta, Zta-targeted RNAi prevented the induction of Zta and its downstream genes and further
blocked the lytic replication of viral genomes. This antiviral effect of RNAi was not likely to be
mediated by activation of the interferon pathway, as phosphorylation of STAT1 was not induced.
In addition, novel EBV-infected epithelial cells showing constitutive activation of the lytic cycle
were cloned; such established lytic infection was also suppressed by Zta-targeted RNAi.
These results indicate that RNAi can be used to inhibit the EBV lytic cycle effectively
in vitro and could also be of potential use to develop anti-EBV treatments.
INTRODUCTION
Epstein–Barr virus (EBV), a gammaherpesvirus associated
with several human diseases, has two phases in its life cycle:
latent and lytic stages (Kieff & Rickinson, 2001). During
EBV latency, only a few so-called latent genes are expressed,
whereas an expression cascade of numerous lytic genes occurs
following reactivation of the lytic cycle. In latency, the copy
number of viral DNA is maintained at a relatively low level
and no virion is produced but, in the EBV lytic cycle, there is
amplification of viral genomes and production of virus
particles (Kieff & Rickinson, 2001; Metzenberg, 1990).
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
0007-9886 G 2004 SGM
Printed in Great Britain
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
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Y. Chang and others
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 sequencespecific 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.
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 59-CAACAGCTAGCAGACATTG-39 (nucleotides 415–433 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 59-GCTGACCCTGAAGTTCATCTG-39. 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).
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.
4F10 (anti-Zta; Tsai et al., 1997), 467 (anti-Rta), 88A9 (anti-BMRF1;
Tsai et al., 1991), 3E8 (anti-BHRF1), 343D12 [anti-125 kDa viral
capsid antigen (VCA); Tsai & Glaser, 1991] and 201D6 [antimembrane antigen (MA); Tsai et al., 1991]. An NPC patient’s serum
was used to detect EBV nuclear antigen 1 (EBNA1) proteins (Chang
et al., 1999). The anti-GFP antibody was purchased from BD
Antibodies. mAbs used for detection of EBV lytic proteins included
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.
METHODS
Cells. NA is an EBV-positive NPC cell line previously generated by
in vitro infection of NPC-TW01 cells with recombinant Akata EBV
(Chang et al., 1999). To establish EBV-infected 293A cells, parental
293 cells were infected with recombinant Akata EBV and selected with
G418 as described previously (Chang et al., 1999). The 293A cell
clones were further isolated by limiting dilution and single-cell cloning.
EBV-negative 293-pZip cells were generated by transfection of 293
cells with the pZip-NeoSV(X)1 plasmid expressing a G418-resistance
gene (Chang et al., 1998). All the cells were maintained in Dulbecco’s
modified Eagle’s medium supplemented with 10 % fetal bovine serum.
Plasmids. The pSUPER plasmid was described by Brummelkamp
et al. (2002). siRNA-expressing plasmids were constructed by cloning
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Fig. 1. Specific knockdown of target genes by siRNAs.
(a) Predicted structure of siZ1 after transcription from a
pSUPER-based
plasmid
and
intracellular
processing
(Brummelkamp et al., 2002). It consists of two 21-nt singlestranded RNAs forming a 19 bp duplex with 2-nt 39 overhangs
(Dykxhoorn et al., 2003). (b) Specific inhibition of gene expression by siRNA. Transfection of plasmids expressing Zta, GFP
or Rta was in combination with transfection of the control
pSUPER vector or pSUPER-derived plasmids expressing siZ1
or siGFP in 293 cells. Gene expression was detected by the
immunoblotting assay using mAbs against Zta, Rta, GFP or
b-actin.
Journal of General Virology 85
Inhibition of EBV lytic cycle by RNAi
Biosciences Clontech and the anti-b-actin antibody was from Sigma.
Specific rabbit antibodies were used to detect total STAT1 proteins
or phosphorylated STAT1 (at tyrosine 701) (Cell Signalling).
Expression of siRNA and induction of the EBV lytic cycle.
In the experiment shown in Fig. 1(b), 3 mg pSUPER-derived
siRNA-producing plasmids were co-transfected with 3 mg 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 mg siRNAproducing plasmids for 48 h and then either treated with 12-Otetradecanoylphorbol-13-acetate (TPA; 40 ng ml21) plus sodium
n-butyrate (3 mM) for 24 h or post-transfected with 4 mg Rtaexpressing 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.
Detection of viral and cellular RNAs. The Northern blotting
assay was carried out as described previously (Lu et al., 2000). In
each lane, 15 mg total RNA was electrophoresed and hybridized with
32
P-labelled DNA probes recognizing Zta or the internal control
GAPDH.
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 a (1000 U ml21) 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 super-
natants 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.
RESULTS
Specific inhibition of gene expression by RNAi
We used a plasmid-based pSUPER system to drive siRNA
synthesis in transfected cells (Brummelkamp et al., 2002).
The specificity of siRNA-mediated gene silencing was tested
in EBV-negative 293 cells that were co-transfected with
plasmids expressing siRNA and target genes. Expression of
Zta was inhibited only by Zta-targeted siRNA (siZ1) and
GFP expression was inhibited only by its specific siRNA
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(siGFP) (Fig. 1b). The control vector pSUPER did not affect
the expression of Zta and GFP, and the level of exogenous
Rta was not influenced by either siZ1 or siGFP (Fig. 1b).
Therefore, our siRNAs function to knock down specifically
their own target genes.
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. 2a) (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; HolleyGuthrie 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).
Zta-targeted RNAi inhibited Rta-induced EBV
reactivation
Previous reports have shown that expression of Rta is
sufficient to disrupt EBV latency in several cell lines (Ragoczy
et al., 1998; Zalani et al., 1996). Here, we further examined
whether siZ1 could block the EBV reactivation induced by
ectopic expression of Rta. Exogenous Rta induced the
production of Zta-encoded monocistronic and bicistronic
mRNAs, the expression of Zta, BMRF1 and BHRF1 proteins
and the amplification of EBV DNA copies, indicating that
Rta efficiently disrupted EBV latency in NA cells (Fig. 3). The
Rta-induced expression of Zta and BMRF1 was significantly
diminished by siZ1 but not by siGFP, while siZ1 did not
affect exogenous Rta (Fig. 3a, b). Furthermore, siZ1 also prevented Rta-induced amplification of viral genomes (Fig. 3c),
indicating that siZ1-mediated inhibition of EBV lytic replication is sustained even in the presence of Rta proteins. On
the other hand, Rta-mediated induction of BHRF1 was not
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Y. Chang and others
Fig. 2. siZ1-mediated inhibition of chemically induced EBV reactivation. NA cells
were untransfected (control) or transfected
with empty vectors (pSUPER) or plasmids
expressing siZ1 or siGFP for 48 h and then
treated with (+) or without (”) TPA plus
sodium n-butyrate (T/S) for 24 h. (a)
Detection of viral transcripts by the Northern
blotting assay. Total RNAs were electrophoresed and hybridized with 32P-labelled DNA
probes recognizing Zta or the internal
control GAPDH. The predicted mRNAs are
indicated. Using the Zta probe, both 1 kb
monocistronic Zta mRNAs (Z) and 4 kb and
3 kb Rta/Zta bicistronic mRNAs (R+Z)
were detected in the EBV lytic cycle (Manet
et al., 1989; Mellinghoff et al., 1991).
(b) Immunoblotting analysis of EBV protein
expression. Expression of Zta, Rta, BMRF1,
BHRF1, EBNA1 and cellular b-actin was
examined in the analysis. (c) Quantification
of EBV DNA using real-time PCR. Shown
are the copy numbers of EBV DNA per cell.
Each sample was tested in duplicate and
error bars are given.
Fig. 3. siZ1-mediated inhibition of Rtainduced EBV reactivation. NA cells were
untransfected (control) or transfected with
plasmids expressing siZ1 or siGFP for 48 h
and then post-transfected with Rtaexpressing plasmids (R) or control vectors
(V) for 24 h. The detection procedures were
the same as outlined in Fig. 2. (a) Detection
of viral transcripts by the Northern blotting
assay. The predicted mRNAs are indicated.
(b) Immunoblotting analysis to detect expression of Zta, Rta, BMRF1, BHRF1, EBNA1
and cellular b-actin. (c) Quantification of
EBV DNA by real-time PCR.
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Journal of General Virology 85
Inhibition of EBV lytic cycle by RNAi
affected by siZ1 (Fig. 3b), consistent with a previous study
that suggested that the BHRF1 promoter can be activated by
Rta alone in the absence of Zta (Cox et al., 1990).
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 siZ1mediated 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 a induced significant phosphorylation
of STAT1 in NA cells, expression of siZ1 caused little
enhancement of STAT1 phosphorylation (Fig. 4a). 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).
Zta-targeted RNAi suppressed constitutive
activation of the EBV lytic cycle
As induction of EBV reactivation was effectively inhibited by
siZ1, we next asked whether siZ1 could also suppress a lytic
cycle that was already in a constitutively activated state, a
state like EBV infection in OHL (Greenspan et al., 1985).
Although spontaneous activation of the EBV lytic cycle is
rare, previous observations indicated that a small proportion of EBV-infected epithelial cells in vitro were permissive
for the lytic cycle without exogenous stimuli (Chang et al.,
1999; Mauser et al., 2002). In this study, we sought to isolate
such permissive cells from EBV-infected 293 cells, 293A,
through limiting dilution and single-cell cloning (Fig. 5a).
Within the 258 cell clones we examined, EBV infection was
restricted to latency in most clones, designated ‘latent
clones’, as no lytic protein was detected (Fig. 5b, c). Only
five cell clones, designated ‘lytic clones’, constitutively
expressed lytic proteins such as Zta, Rta, BMRF1, BHRF1
and 125 kDa VCA (Fig. 5b). In 293A-2 and 293A-98 cells,
lytic proteins were detected most prominently and about
30 % of the cells expressed Zta and BMRF1 (Fig. 5c). The
Zta-positive rates in these lytic clones were stable for more
than 1 year and cell viability of the lytic clones was
indistinguishable from that of latent clones (data not
shown). The copy numbers of EBV genomes in latent clones
ranged from 1 to 30 copies per cell (Fig. 5d). In contrast, the
lytic clones had a higher EBV DNA copy number (ranging
from 400 to more than 1000 copies per cell), reflecting the
amplification of viral genomes in the lytic cycle (Fig. 5d).
We further examined what level the lytic cycle had reached
in the 293A lytic clones in comparison with the maximal
level that was induced by ectopic expression of Zta and Rta.
Such induction increased by two- to fourfold the positive
rates of cells expressing lytic proteins in 293A-2 clones
(Fig. 6a, b). Production of infectious EBV was detected in
the culture supernatants of untreated lytic clones and virus
titres were further raised about 16-fold by exogenous Zta
and Rta (Fig. 6c). These results suggest that the EBV lytic
cycle in 293A lytic clones is maintained at a moderate level
below the maximum that can be potentially activated.
Fig. 4. Lack of effect of Zta-targeted RNAi on STAT1 phosphorylation and cell viability. (a) Examination of STAT1. NA cells
were treated the same way as for Fig. 2 (left panel) or Fig. 3
(right panel) and phosphorylated STAT1 (at tyrosine 701) and
total STAT1 proteins were detected by immunoblotting analysis.
NA cells treated with interferon a were used as the positive
control. (b) Examination of cell viability. NA and 293 cells were
transfected with indicated plasmids for 48 h and their viability
was determined using a trypan blue exclusion method.
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Although the mechanism of constitutive EBV activation in
293A lytic clones remains unclear, these cells were useful for
testing the efficacy of siZ1. Two lytic clones, 293A-2 and
293A-98, were subcultured every 4 days and each passage was
followed by transfection once with plasmids producing
siRNA. Fig. 7(a) shows that repeated transfection with
plasmids expressing siZ1 but not siGFP gradually suppressed
the levels of lytic proteins. Consistently, the copy numbers of
EBV DNA in the lytic clones were also significantly reduced
by siZ1 (Fig. 7b). Meanwhile, expression of siZ1 did not affect
the level of latent EBNA1 proteins (Fig. 7a).
DISCUSSION
In summary, siZ1-directed RNAi can inhibit not only
EBV reactivation induced by chemicals or by exogenous
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Y. Chang and others
Fig. 5. Constitutive activation of the lytic
cycle in some clones of EBV-infected 293
cells, 293A. For comparison, five cell clones
showing restricted EBV latency (latent
clones: 293A-19, -10, -24, -52 and -12)
and five clones exhibiting lytic infection (lytic
clones: 293A-55, -85, -14, -98 and -2)
were included in the analysis. (a) Detection
of EBV DNA by PCR. The target was the
BamHI W fragment of the EBV genome.
Negative controls were parental 293 cells
and two vector transfectants, 293-pZip-1
and -2. H2B4 cells harbouring one EBV
genome per cell were used as the positive
control (Chang et al., 2002). (b) Immunoblotting analysis of EBV protein expression.
NA cells treated with TPA and sodium
n-butyrate (NA+T/S) were used as a positive control of the EBV lytic cycle.
(c) Detection of EBV lytic proteins by the
immunofluorescence assay. Nuclear fluorescence staining in 293A-2 and 293A-98
cells represents the expression of Zta or
BMRF1. (d) Quantification of EBV DNA by
real-time PCR. Viral copy numbers per cell
are presented on a log scale.
Rta but also the lytic cycle exhibiting a constitutively active
state. This is the first report that the EBV lytic cycle can
be effectively blocked by RNAi. One advantage of this
study is the use of NA and 293A cells, both of which can
be transfected with high efficiency. The efficiency was
higher than 90 %, as monitored by transfection with GFPexpressing plasmids (data not shown), so a near-complete
gene knockdown by RNAi could be achieved in these cells.
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
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(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
Journal of General Virology 85
Inhibition of EBV lytic cycle by RNAi
Fig. 6. Further induction of the EBV lytic cycle in 293A lytic
clones. Cells were mock-transfected (uninduced) or cotransfected with plasmids expressing Zta and Rta (induced) for
48 h and then subjected to the following studies. (a) Immunofluorescence staining to detect Zta, BHRF1 and MA proteins
in uninduced and induced 293A-2 cells. (b) Expression rate
of lytic proteins in uninduced and induced 293A-2 cells.
Expression of Zta, BMRF1, BHRF1 and MA was detected
using an immunofluorescence assay and positive rates were
determined under a fluorescence microscope. A summary of
three repeated experiments is shown. (c) Titration of infectious
EBV. A transformation assay was used to determine the titres
of infectious EBV in the culture supernatants of 293A-2 and
293A-98 cells that were either uninduced or further induced.
analyse how the viral expression cascade is regulated during
the lytic cycle.
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
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
Fig. 7. siZ1-mediated suppression of the
EBV lytic cycle in 293A lytic clones. Two
lytic clones, 293A-2 and 293A-98, were
subcultured every 4 days and each passage
was followed by transfection once with
siRNA-producing plasmids or the control
vector pSUPER. Detection procedures were
the same as for Fig. 5. Cells at passage 1,
2 or 3 were subjected to the following studies. (a) Immunoblotting analysis to detect
Zta, Rta, BMRF1, BHRF1, VCA, EBNA1 and
b-actin. (b) Quantification of EBV DNA by
real-time PCR.
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Y. Chang and others
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.
ACKNOWLEDGEMENTS
We thank Dr Reuven Agami for kindly providing the pSUPER plasmid,
Dr S. Diane Hayward for kindly providing the Rta-expressing plasmid
RTS15, Dr Tsuey-Ying Hsu for providing the anti-Rta monoclonal
antibody, Dr Jen-Yang Chen for providing the anti-BHRF1 monoclonal antibody and Dr Mei-Ru Chen of National Taiwan University
for critically reading the manuscript. The skilful technical assistance of
Chin-Min Ho is appreciated. This work was supported by the National
Science Council (grants NSC 92-3112-B-002-014, NSC 91-2320-B002-177 and NSC 92-2320-B-002-167).
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