Courtney M. Shirley,1Jianmeng Chen,1Meir Shamay,1Huili Li,1CynthiaA. Zahnow,1S. Diane Hayward,1and
1Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD
Epstein-Barr virus (EBV) is associated
with a variety of lymphoid malignancies.
Bortezomib activates EBV lytic gene ex-
pression. Bortezomib, a proteasome inhibi-
tor, leads to increased levels of CCAAT/
enhancer-binding protein? (C/EBP?) in a
variety of tumor cell lines. C/EBP? acti-
vates the promoter of the EBV lytic switch
gene ZTA. Bortezomib treatment leads to
increased binding of C/EBP to previously
recognized binding sites in the ZTA pro-
moter. Knockdown of C/EBP? inhibits
bortezomib activation of EBV lytic gene
expression. Bortezomib also induces the
unfolded protein response (UPR), as evi-
denced by increases in ATF4, CHOP10,
and XBP1s and cleavage ofATF6. Thapsi-
gargin, an inducer of the UPR that does
not interfere with proteasome function,
also induces EBV lytic gene expression.
The effects of thapsigargin on EBV lytic
gene expression are also inhibited by
C/EBP? knock-down. Therefore, C/EBP?
mediates the activation of EBV lytic gene
expression associated with bortezomib
and another UPR inducer. (Blood. 2011;
Epstein-Barr virus (EBV)–associated lymphoid malignancies in-
clude a subset of Burkitt lymphoma, AIDS lymphoma, Hodgkin
lymphoma, posttransplant lymphoma, age-associated B-cell lym-
phoma, and peripheral T- and NK–cell lymphoma.1-3The EBV life
cycle includes distinct lytic and latent genetic programs.4-6In the
lytic program, linear, double-stranded genomes are produced and
packaged as virions that spread infection from cell to cell. In the
latent program, only a few viral genes are transcribed, no viral
progeny are produced, and infected cells are protected from
apoptotic stimuli and in some circumstances driven to proliferate.
The latent program predominates in tumors. There is increasing
interest in pharmacologic activation of lytic viral gene expression
in tumors.7,8Several therapeutic strategies requiring activation of
EBV lytic genes for tumor cell killing have been described,9,10but
concerns have been raised about the possible adverse effects of
viral gene activation in patients treated with pharmacologic
To better understand the drugs that induce EBV lytic gene
expression, we screened a library of drugs approved by the Food
and Drug Administration for human use and identified bortezomib
as among the most potent.10,13Bortezomib is a proteasome inhibitor
approved for use in the treatment of myeloma and mantle cell
lymphoma. The cellular pathways that mediate the apoptotic
effects of bortezomib in malignancy remain the focus of active
investigation, but activation of the unfolded protein response
(UPR) may play a critical role.14-16In the investigations described
herein, we sought to better understand the pathways by which
bortezomib may activate EBV lytic program gene expression. The
screening assay that identified bortezomib as a lytic inducer
focused on transcriptional activation of the promoter of the EBV
ZTA gene (also referred to as BZLF1, ZEBRA, Z, or EB1).17-21
ZTA is an immediate early transcriptional transactivator that
induces other lytic genes and transcriptional trans-activators,
resulting in virion production in permissive cell types. We were
particularly interested in the possibility that CCAAT/enhancer-
binding protein (C/EBP) family members might be important in
ZTA promoter activation, because there is evidence that some
C/EBP family members are polyubiquitinylated and degraded by
the proteasome,22,23and also that C/EBP? and C/EBP? bind the
C/EBP family members share highly conserved C-terminal
basic DNA-binding domains and basic leucine zipper domains
(bZIPs).26,27As homo- or heterodimers, they modulate various
tion, proliferation, and malignancy. C/EBP? and C/EBP? each
give rise to several different isoforms. The full-length isoforms of
each protein include trans-activating and regulatory domains that
can induce differentiation and inhibit proliferation. Truncated
isoforms of these proteins lacking the trans-activating domains but
retaining the DNA-binding and dimerization domains function as
trans-dominant repressors. C/EBP? has been identified as a critical
transcription factor in multiple myeloma, regulating growth, prolif-
eration, and antiapoptotic responses by regulating the expression of
other key transcription factors.28In addition, a role for C/EBP? in
regulating the transition from the protective to the death-promoting
phase of the UPR has been proposed.29We present evidence that
bortezomib activation of the ZTA promoter is mediated by
increased C/EBP? in the context of the UPR.
Akata and Rael are EBV-associated Burkitt lymphoma cell lines.Asubclone of
the Akata cell line that lacks EBV was a gift from L. Hutt-Fletcher (Louisiana
State University, Shreveport, LA).30SNU-719 is an EBV-associated gastric
Submitted January 24, 2011; accepted March 18, 2011. Prepublished online as
Blood First Edition paper, March 29, 2011; DOI 10.1182/blood-2011-01-332379.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
© 2011 by TheAmerican Society of Hematology
6297BLOOD, 9 JUNE 2011?VOLUME 117, NUMBER 23
carcinoma cell line (a gift from J. M. Lee, Yonsei University, Seoul, Korea).31
These cell lines were maintained in RPMI 1640 medium containing 10% fetal
bovine serum, 100 units/mLpenicillin, 100 ?g/mLstreptomycin, and 100mM
L-glutamine. AGS, a gastric carcinoma cell line lacking EBV, was
maintained in Ham F12 medium containing 10% FBS, 100 units/mL
penicillin, 100 ?g/mL streptomycin, and 100mM L-glutamine. Each of
these cell lines tested negative for mycoplasma.
Cells were treated for 24-48 hours with 20nM bortezomib (Millennium
Pharmaceuticals), 1?M thapsigargin (Enzo Life Sciences), or 2 ?g/mL
tunicamycin (Enzo Life Sciences).
Zp-CAT wt (pHC41) is a chloramphenicol acetyltransferase (CAT) reporter
driven by the ZTA promoter (position ?221 to ?39).26Zp-CAT 2M,
Zp-CAT 3M, and Zp-CAT 2/3M are derivatives with mutations of the
C/EBP–binding sites. Zp-Luc (pHC133) is a luciferase reporter containing
the ZTApromoter from position ?221 to ?39.27
SNU-719 cells were transfected using Lipofectamine 2000 (Invitrogen)
according to the manufacturer’s protocol. One day before transfection,
1 ? 106cells were added to each well in a 6-well plate. All cells were
transfected with 4 ?g of DNA. The medium was changed and cells were
treated with 20nM bortezomib 24 hours after transfection. Cells were
harvested at 48 hours after transfection. The CAT assay was performed
using the CAT ELISAsystem (RocheApplied Science).
Preparation of nuclear protein extracts and immunoblotting
To prepare nuclear protein extracts, 2 ? 107cells were pelleted by
centrifugation and washed in PBS. The pellet was resuspended in a low-salt
buffer (10mM HEPES, pH 7.9, 10mM KCl, 100?M EDTA, 1mM DTT,
0.5mM PMSF, 10 ?g/mL pepstatin A, 10 ?g/mL leupeptin, and 10 ?g/mL
aprotinin) and incubated on ice for 15 minutes. NP-40 was added to a final
concentration of 0.6%, and the cells were lysed by brief vortexing. The
nuclear pellet was isolated by centrifugation at 9000g for 30 seconds at 4°C,
followed by resuspension in a high-salt buffer (20mM HEPES, pH 7.9,
400mM NaCl, 1mM EDTA, 1mM DTT, 0.5mM PMSF, 10 ?g/mL pepstatin
A, 10 ?g/mL leupeptin, and 10 ?g/mL aprotinin), and rotation for 15 minutes.
Nuclear debris was pelleted by centrifugation at 15 000g for 5 minutes at
4°C and the nuclear extract (supernatant) was stored at ?80°C. The nuclear
extract was subjected to SDS-PAGE and Western blotting. Proteins were
detected with ECLdetection reagents (GE Healthcare) and exposed to film.
Primary antibodies used included anti–C/EBP? (14AA; Santa Cruz Biotech-
nology), anti–C/EBP? (H-7; Santa Cruz Biotechnology), anti-EBV Zebra
(Argene), anti–XBP-1s (BioLegend), anti-ATF6 (Abcam), anti-ATF4 (C-20;
A total of 4 ? 107EBV?Akata cells were treated with 20nM bortezomib
for 24 hours. ChIP was performed using the SimpleChIP Enzymatic
Chromatin IPKit (Cell SignalingTechnology) according to the manufactur-
er’s protocol. For immunoprecipitation, 2 ?Lof anti–C/EBP? (C-19; Santa
Cruz Biotechnology) was used. To detect the immunoprecipitated EBV
ZTA promoter region, 2 primers, CMO-005 (5? GAGGAGGAG-
GCAGTTTTCAG 3?) and CMO-006 (5? CTGACCCCCGAACTTAATGA
3?), were used. DNA was analyzed using both standard PCR and real-time
quantitative PCR (qPCR). For standard PCR, each 50-?L reaction con-
tained 1? Platinum PCR SuperMix (Invitrogen), 200nM primers, and
2 ?L of DNA. Thermocycling conditions were 95°C for 5 minutes for
1 cycle; 95°C for 30 seconds, 54°C for 30 seconds, and 72°C for 30 seconds
for 34 cycles; and 72°C for 5 minutes for 1 cycle. The PCR products were
then analyzed on a 2% agarose gel. For real-time qPCR, each 20-?L
reaction contained 1? SsoFast EvaGreen Supermix (Bio-Rad), primer
concentrations of 500nM, and 2 ?L of DNA. Thermocycling conditions
were 95°C for 30 seconds for 1 cycle and 95°C for 5 seconds and 60°C for
10 seconds for 40 cycles.
DNA transfection and luciferase assay
SNU-719 cells were transfected using Lipofectamine 2000 (Invitrogen)
according to the manufacturer’s protocol. One day before transfection,
5 ? 105cells were added to each well in a 6-well plate. All cells were
transfected with 2 ?g of DNA and harvested at 48 hours after transfection.
The luciferase assay was performed using a luciferase assay system kit
(Promega) according to the manufacturer’s protocol. Light intensity was
measured after a 2-second delay for 10 seconds.
C/EBP? knock-down was performed using Expression Arrest TRIPZ
lentiviral shRNAmir constructs (Open Biosystems). The TRIPZ lentiviral
vector is an inducible vector in which the shRNAmir is only expressed in
the presence of doxycycline. In addition, miRNA is a polycistronic RNA
with red fluorescent protein, making it easy to visualize shRNAmir
expression. First, the shRNAmir was packaged using lentivirus. Then,
21 ?g of gag/pol, 7 ?g of vesicular stomatitis virus glycoprotein, and 7 ?g
of shRNAmir were transfected into HEK293T cells using calcium/
phosphate. Packaged lentivirus was concentrated 48 hours after transfec-
tion. Briefly, the supernatant was filtered through a 0.45-?m syringe-driven
filter unit into a filtering conical tube, then the supernatant was centrifuged
at 1750g for 30 minutes at 4°C, 2 mL of cold 1? PBS was added, and the
centrifugation step was repeated. The concentrated virus was stored at
?80°C. Next, lentiviral particles containing shRNAmir were transduced
into EBV?Akata cells. 2 ? 106cells were added to each well in a 6-well
plate. A final concentration of 8 ?g/mL Polybrene and 100 ?L of
concentrated packaged lentivirus were added to the cells, allowed to
incubate for 8 hours, and then the medium was changed. After 48 hours,
2 ?g/mL puromycin was added to select for stably transduced cells (Akata
C/EBP?si). To induce shRNAmir expression, 1 ?g/mL doxycycline was
added to the cells for 72 hours. Because the half-life of doxycycline is
24 hours in culture, it was refreshed every 48 hours during the duration of
Extraction of RNA, cDNA synthesis, and real-time qPCR
RNA was isolated using the RNeasy Plus Kit (QIAGEN) according to the
manufacturer’s protocol. cDNA was synthesized from 1 ?g of RNA using
the iScript cDNA Synthesis Kit (Bio-Rad) according to the manufacturer’s
protocol. The synthesized cDNAwas used as a template for C/EBP? PCRs
with primers CMO-021 (5?TTTCGAAGTTGATGCAATCG 3?) and CMO-
022 (5? CAACAAGCCCGTAGGAACAT 3?), and ZTAPCRs with primers
CMO-013 (5? ACATCTGCTTCAACAGGAGG 3?) and CMO-014
(5? AGCAGACATTGGTGTTCCAC 3?). Each PCR reaction contained
1? SsoFast EvaGreen Supermix (Bio-Rad), 500nM primer concentrations,
cDNA corresponding to 25 ng of total RNA, and nuclease-free water to a
final volume of 20 ?L. Themocycler conditions were 95°C for 30 seconds
for 1 cycle and 95°C for 5 seconds and 60°C for 10 seconds for 40 cycles.
?-Actin was used as an internal control with primers CMO-023
(5? ACAGAGCCTCGCCTTTGCCG 3?) and CMO-024 (5? ACATGCCG-
Quantification of EBV viral load by real-time qPCR
Akata C/EBP?si cells were treated with 20nM bortezomib for 48 hours.
Genomic DNA was isolated used the QIAamp DNA mini kit (QIAGEN)
according to the manufacturer’s protocol. To detect the EBV, 2 primers,
BamH-W 5? (5? CCCAACACTCCACCACACC 3?) and BamH-W
3? (5? TCTTAGGAGCTGTCCGAGGG 3?), and the BamH-W fluorescent
probe (5? (FAM) CACACACTACACACACCCACCCGTCTC (BH1) 3?)
were used. Each 50 ?L of real-time qPCR reaction contained 1? IQ
SuperMix (Bio-Rad), a 100nM concentration of each primer, a 100nM
concentration of the probe, and 20 ?L of DNA. Thermocycling conditions
6298SHIRLEY et al BLOOD, 9 JUNE 2011?VOLUME 117, NUMBER 23
were 95°C for 10 minutes for 1 cycle and 95°C for 15 seconds and 60°C for
1 minute for 40 cycles. ?-actin was used as an internal control (TaqMan
?-actin detection reagents;Applied Biosystems).
Bortezomib leads to accumulation of C/EBP? and increased
association with Zp
C/EBP? and C/EBP? activate ZTA promoter reporter plasmids in
transient transfection experiments.24,26,27Evidence to support a role
for the ubiquitin-proteasome system in the degradation of some
C/EBP family members has been reported previously.22,23There-
fore, we investigated the impact of bortezomib on C/EBP? and
C/EBP? by immunoblot analysis. In a variety of cancer cell lines,
we found that bortezomib had no apparent effect on C/EBP?, but
did lead to increased C/EBP? (Figure 1A). C/EBP–binding sites
had previously been identified in the ZTA promoter. Using
promoter reporter plasmids with 2 binding sites, we found that
mutation of either binding site diminished reporter activation,
whereas mutation of both binding sites nearly abolished activation
(Figure 1B). The ChIP assay showed that after bortezomib
treatment there was a 3-fold increase in the binding of the
endogenous C/EBP? to ZTA promoter sites of viral genomes
naturally present in an EBV-associated lymphoma cell line (Figure
1C). C/EBP? is expressed as 3 isoforms generated by alternative
Figure 1. Bortezomib modulates C/EBP? but not C/EBP?. (A) Western blot showing C/EBP? and C/EBP? protein levels after treatment with bortezomib in the EBV(?)
gastric carcinoma cell lineAGS, the EBV?gastric carcinoma cell line SNU-719, and the Burkitt lymphoma cell linesAkata and Rael. Western blots were also probed for ?-actin
as a loading control. All cells were treated with 20nM bortezomib. After 24 hours of bortezomib treatment, cells were harvested and nuclear protein extracts were prepared.
(B) CAT assay performed in EBV?gastric carcinoma SNU-719 cells to assess effects of C/EBP–binding sites on activation of Zp by bortezomib. Cells were transfected with a
plasmid expressing Zp-CAT with wild-type C/EBP–binding sites, one C/EBP–binding site mutated, or both sites mutated. Cells were treated with 20nM bortezomib (BZ) or
DMSO for 24 hours. Assays were repeated 3 times. Error bars indicate SEM. (C) ChIP assay performed in Akata cells after treatment with 20nM bortezomib for 24 hours to
show ZTApromoter (Zp) DNAbound to C/EBP?. Immunoprecipitated Zp DNAwas measured by standard PCR (top panel) and analyzed on a 2% agarose gel and by real-time
qPCR (bottom panel). (D) Luciferase assay performed in SNU-719 cells to assess the effects of C/EBP? isoforms on the Zp-Luc reporter. Cells were transfected with a plasmid
expressing all 3 C/EBP? isoforms (C/EBP?) or the indicated C/EBP? isoforms (LAPand LIP).Transfection with an empty parent vector (vector) was used as an internal control.
Assays were repeated 3 times. Error bars indicate SEM.
BORTEZOMIBAND EPSTEIN-BARR VIRUS6299 BLOOD, 9 JUNE 2011?VOLUME 117, NUMBER 23
translation initiation of the same mRNA transcript: LAP1, LAP2,
and LIP.32The antibody used binds to the C terminus of C/EBP?
and thus detects all 3 isoforms; it does not cross react with C/EBP?.
Suppression of C/EBP? expression diminishes EBV lytic
induction by bortezomib
We studied individual C/EBP? isoforms in short-term transfection
experiments. A plasmid expressing only the LAP2 isoform led to
Zp-luciferase activation, whereas a plasmid expressing only the
LIP isoform led to Zp-luciferase inhibition, which is consistent
with the presence of the N-terminal transcriptional activating
domain in the LAP2 but not the LIP isoform (Figure 1D). A
wild-type expression vector expressing all 3 isoforms was also
endogenous C/EBP? isoforms, we also evaluated the effects of
mouse C/EBP? isoforms in a mouse C/EBP?–knockout cell line,
and again found that LAP2 was associated with activation of the
ZTApromoter whereas LIP was associated with its inhibition (data
These observations are consistent with the possibility that
C/EBP? mediates the effects of bortezomib on the ZTA promoter.
To determine whether suppression of C/EBP? would interfere with
bortezomib-induced lytic activation, we created a stable regulated
C/EBP? knockdown in an EBV-associated Burkitt lymphoma cell
line (Akata). The cell line was engineered to express C/EBP?
shRNAfrom a doxycycline-activated promoter so that doxycycline
treatment would reduce C/EBP? expression. As seen in Figure
2, the addition of doxycycline was associated with a reduction in
C/EBP? RNA, and the effects of bortezomib on ZTA RNA were
diminished by C/EBP? knockdown. Immunoblot analysis showed
that C/EBP? protein expression was also inhibited by doxycycline
and that bortezomib failed to increase ZTAprotein in doxycycline-
treated cells. In this EBV-associated Burkitt cell line, bortezomib
led to the induction of lytic cycle gene expression and replication of
viral DNA, but, as with ZTA RNA and ZTA protein expression,
replication of viral DNAin response to bortezomib was diminished
(Figure 2D). This indicates that C/EBP? mediates EBV lytic
induction by bortezomib.
Bortezomib alters C/EBP? transcription
The increase in C/EBP? protein after bortezomib treatment might
be explained as a direct consequence of bortezomib-induced
inhibition of the proteasomal degradation of C/EBP?, or as an
tion.22,23To distinguish between these possibilities, we investigated
with bortezomib led to an increase in C/EBP? RNA, followed by
an increase in ZTARNA(Figure 3A-B).
C/EBP? and ER stress
Increased transcription of C/EBP? in the setting of endoplasmic
reticulum (ER) stress has been described previously,33and bor-
tezomib has been shown to induce ER stress.14,34,35ER stress
activates a homeostatic response called the UPR. To better
understand the context of changes in C/EBP? and ZTA expression
with regard to the UPR, we analyzed the impact of bortezomib on
C/EBP? isoforms, key UPR response indicator proteins, and ZTA
by immunoblot analysis. Three ER sensors led to activation of
different branches of the UPR: (1) PERK activation led to
attenuation of global translation initiation but to increased transla-
tion of ATF4, a transcription factor that leads to transcription of
stress response genes including CHOP10; (2) IRE1 activation led
to splicing of XBP1 mRNA; and (3) ATF6 activation led to
translocation from the ER to the Golgi, where it was cleaved to
produce an active transcription factor. As shown in Figure
4, bortezomib led to a transient increase in ATF4, followed by a
transient increase in CHOP10, a transient increase in the spliced
form of XBP1 (XBP1s), and cleavage of ATF6. C/EBP? isoforms
increased in parallel withATF4 and XBP1s and before ZTA. These
inducible vector in which the shRNAmir is only expressed in the presence of doxycycline (DOX). To induce shRNAmir expression, 1 ?g/mL doxycycline was added to cells for
72 hours. Cells were treated with 20nM bortezomib (BZ) and harvested after 24 hours. RNAwas isolated and subjected to real-time qPCR with primers for C/EBP? (A) and ZTA
(B). Primers to ?-actin were used as an internal control. (C) Nuclear protein extracts were prepared and subjected to Western blot analysis. Blots were probed for C/EBP? and
ZTA. Blots were also probed for ?-actin as a loading control. (D) Viral DNA was isolated and EBV viral load was measured using real-time PCR. Primers to the EBV BamW
region were used to quantify EBV copy number. Primers to ?-actin were used as an internal control. Error bars indicate SEM.
Figure 3. Time course of C/EBP? and ZTA RNA induction by bortezomib. EBV?
Akata cells were treated with 20nM bortezomib (BZ) and harvested at the indicated
time points. RNA was isolated and subjected to real-time qPCR using primer for
C/EBP? (A) and ZTA (B). Primers for ?-actin were used as an internal control. Error
bars indicate SEM.
6300 SHIRLEY et al BLOOD, 9 JUNE 2011?VOLUME 117, NUMBER 23
results are consistent with the hypothesis that the effects of
bortezomib on ZTA transcription reflect activation of the UPR. If
this is the case, then ZTA transcription might also be induced by
very different ER stressors. We investigated thapsigargin, which
disrupts the Ca2?gradient in the ER, and tunicamycin, which
inhibits the N-linked glycosylation required for proper protein
folding. Both agents led to ZTA RNA expression and EBV DNA
replication (Figure 5A-B). Thapsigargin elevated C/EBP? (Figure
5C) and doxycycline-regulated knockdown of C/EBP? expression
inhibited EBV lytic induction by thapsigargin (Figure 5D).
We have presented evidence that a proteasome inhibitor activates
EBV lytic gene expression in the context of ER stress, and that
C/EBP? plays a role in mediating EBV lytic gene expression in
these circumstances. Proteasome inhibitors alter transcription di-
rectly by inhibiting the degradation of transcription factors, but also
indirectly by activating the UPR. The observation that C/EBP?
isoforms increased after bortezomib treatment (Figure 1A) in
tumor cell lines that do not harbor EBV (AGS) and in those that do
harbor EBV (SNU-719, Akata, and Rael), together with previous
reports that C/EBP? activates the ZTA promoter, suggested that
C/EBP? might mediate ZTA activation.26,27The observation that
bortezomib increased C/EBP? RNA suggested that the increase in
protein detected on immunoblot analysis did not merely reflect
inhibition of degradation of the protein, but also transcriptional
activation of C/EBP?.
We found that other activators of the UPR also activated the
ZTA promoter and were also associated with increased expression
of C/EBP? isoforms, and that inhibition of C/EBP? expression
attenuated activation of ZTA in response to these agents. Markers
of the UPR—ATF4, CHOP10, and XBP1(s)—appeared 6 hours
after bortezomib treatment and had returned to baseline or near
baseline by 24 hours after treatment, which may reflect cell
recovery, cell death, or a combination of both.
We have provided evidence here and in previous reports that
bortezomib leads to increased Zta RNA expression, ZTA protein
expression, and viral DNA replication.10,13However, not all
investigators have reported EBV lytic activation with bortezomib
treatment. Zou et al studied cell lines in vivo and in vitro and saw
no induction of ZTA.36However, our previous studies with tumor
xenografts in mice10,13and those presented here involved assay at
24 hours or fewer, whereas in the report by Zou et al, mice bearing
xenografts were treated with bortezomib for 6 weeks and then
observed off-treatment for 5 weeks before tumor tissues were
assayed.36We suspect that the long time interval between bort-
ezomib treatment and assays explains why these investigators did
not observe lytic activation. Zou et al also reported no evidence of
lytic activation in vitro in EBV-immortalized lymphoblastoid cell
lines.36We suspect that the different observations reflect the fact
that lymphoblastoid cell lines express EBNA-3C, which modulates
the UPR by inhibiting splicing of XBP-1 and activation cleavage of
ATF6.37The cell lines studied in the present study and in our
previous studies do not express EBNA-3C.
Viruses have long been recognized as important inducers of the
UPR. From the perspective of the virus, the UPR presents
formidable obstacles to be overcome. To assemble virions, viral
proteins must be synthesized. Investigations have identified several
different strategies for overcoming translational inhibition among
the human herpesviruses.37-39Several previous investigators have
reported that UPR-associated pathways affect EBV gene expres-
sion. The latency membrane protein 1 (LMP1) promoter is
Figure 4. Bortezomib induces the UPR. EBV?Akata cells were treated with 20nM
bortezomib (BZ) and harvested at the indicated time points. Nuclear protein extracts
were prepared and subjected to Western blot analysis. Blots were probed for
C/EBP?, ATF4, CHOP10, XBP1(s), ATF6, and ZTA, and for ?-actin as a loading
Figure 5. C/EBP? modulates EBV lytic gene expression and replication by ER stressors. EBV?Akata cells and Rael were treated with 20nM bortezomib (BZ), 1?M
thapsigargin (TG), or 2 ?g/mLtunicamycin (TU) for 24 hours. (A) RNAwas isolated and subjected to real-time qPCR using primers for ZTA. Primers for ?-actin were used as an
internal control. (B) Viral DNAwas isolated and EBV viral load was measured using real-time PCR. Primers to the EBV BamW region were used to quantify EBV copy number.
Primers to ?-actin were used as an internal control. (C) EBV?Akata and Rael cells were treated with 1?M thapsigargin (TG) and harvested after 24 hours. Western blots were
probed for C/EBP?, CHOP10, and ZTA. (D) C/EBP? knock-down was performed in EBV?Akata cells using an inducible vector in which the shRNAmir is only expressed in the
presence of doxycycline (DOX).To induce shRNAmir expression, 1 ?g/mLdoxycycline was added to the cells for 72 hours. Cells were treated with 1?M thapsigargin (TG), and
RNAwas isolated after 24 hours and subjected to real-time qPCR with primers for ZTA. Primers to ?-actin were used as an internal control. Error bars indicate SEM.
BORTEZOMIBAND EPSTEIN-BARR VIRUS 6301 BLOOD, 9 JUNE 2011?VOLUME 117, NUMBER 23
activated by ATF4 and by XBP1(s).40,41The ZTA promoter is
activated by XBP1(s), which functions both as an effector of the
UPR and as a plasma cell differentiation factor.42-44The role of
C/EBP? in the UPR has only recently been recognized. Early in the
UPR, cells are protected from further accumulation of unfolded or
misfolded proteins by inhibition of global translation, synthesis of
chaperone and other proteins important for protein folding, and by
stasis is not restored, then apoptotic pathways are later activated.
C/EBP? isoforms play a critical role in triggering the transition
from the early protective phase to the late apoptotic phase.33
C/EBP? is a member of the bZIPfamily of transcription factors and
may homo- or heterodimerize with other bZIP proteins. LAP and
LIP isoforms may dimerize with each other, but C/EBP? isoforms
may also dimerize with other partners, such as CHOP10. Recently,
evidence for direct interactions between ZTAand C/EBP? has been
reported.45The dimerization partner and the promoter-binding site
are both important determinants of transcriptional activity.
In closing, this study puts bortezomib activation of the EBV
lytic cycle into a broader context. C/EBP? induction by bortezomib
mediates ZTA induction. Similarly, C/EBP? induction by thapsi-
gargin mediates ZTA induction. Both agents lead to ER stress and
activation of the UPR. A better understanding of the interplay
between viral gene regulation and cellular stress responses, includ-
ing the UPR, promise to facilitate the development of new
ing the potential adverse effects of viral reactivation.
This work was supported by National Institutes of Health grants
P50 CA96888 and P01CA1539627 (to R.F.A.)
Contribution: C.M.S. and J.C. carried out the experiments; C.M.S.,
M.S., and R.A. designed the experiments; H.L., C.A.Z., and S.D.H.
contributed plasmids and cell lines and critiqued the experimental
design; and C.M.S. and R.F.A. analyzed the data and wrote the
Conflict-of-interest disclosure: The authors declare no compet-
ing financial interests.
Correspondence: Richard F. Ambinder, Department of Oncol-
ogy, Johns Hopkins University School of Medicine, 389 CRB1,
1650 Orleans, Baltimore, MD 21287; e-mail: email@example.com.
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