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LYMPHOID NEOPLASIA
Endoplasmic reticulum stress causes EBV lytic replication
Gwen Marie Taylor,1 Sandeep K. Raghuwanshi,1 David T. Rowe,2 Robert M. Wadowsky,1 and Adam Rosendorff1
1Department of Pathology, University of Pittsburgh and Children’s Hospital of Pittsburgh, Pittsburgh, PA; and 2University of Pittsburgh Graduate School of Public
Health, Pittsburgh, PA
Endoplasmic reticulum (ER) stress triggers a homeostatic cellular response in
mammalian cells to ensure efficient folding, sorting, and processing of client proteins. In lytic-permissive lymphoblastoid
cell lines (LCLs), pulse exposure to the
chemical ER-stress inducer thapsigargin
(TG) followed by recovery resulted in the
activation of the EBV immediate-early
(BRLF1, BZLF1), early (BMRF1), and late
(gp350) genes, gp350 surface expression, and virus release. The protein phosphatase 1 a (PP1a)–specific phosphatase
inhibitor Salubrinal (SAL) synergized with
TG to induce EBV lytic genes; however,
TG treatment alone was sufficient to activate EBV lytic replication. SAL showed
ER-stress–dependent and –independent
antiviral effects, preventing virus release
in human LCLs and abrogating gp350
expression in 12-O-tetradecanoylphorbol13-acetate (TPA)–treated B95-8 cells.
TG resulted in sustained BCL6 but not
BLIMP1 or CD138 expression, which is
consistent with maintenance of a germinal center B-cell, rather than plasma-cell,
phenotype. Microarray analysis identified
candidate genes governing lytic replication in LCLs undergoing ER stress.
(Blood. 2011;118(20):5528-5539)
Introduction
EBV is the causative agent of acute infectious mononucleosis
and most posttransplantation lymphomas. The virus is also
associated with a variety of lymphoid and epithelial malignancies, such as Burkitt lymphoma, nasopharyngeal carcinoma, and
gastric lymphomas. The latent form of infection is observed in
rare circulating B lymphocytes from patients who have recovered from acute EBV infection (latency I) and during initial
infection of B lymphocytes (latency III), whereas lytic EBV
gene expression occurs during passage of B lymphocytes
(mostly plasma cells and tonsillar B lymphocytes) within epithelial tissue.1
The conversion of B lymphocytes from latent to lytic gene
expression is controlled by the EBV DNA-binding, immediateearly transactivator proteins, ZTA (Z) and RTA (R). During lytic
reactivation, Z is initially expressed, binding to and activating
expression of R, via 3 Z-response elements in the R promoter.
Together, Z and R are capable of activating the entire set of EBV
lytic gene promoters.2-4 However, the cellular events responsible
for the initiation of lytic replication remain unknown. The
histone deacetylase inhibitors butyric and valproic acid induce
lytic replication, as does the protein kinase C (PKC) activator
12-O-tetradecanoylphorbol-13-acetate (TPA) in certain cell lines.
Recently, there has been growing interest in the effects of
various chemotherapeutic drugs on EBV replication.5-7
Data derived from the use of these chemicals, however, provide
limited insight regarding the specific pathways required for conversion of EBV-immortalized lymphoblastoid cell lines (LCLs) from
latency to the lytic phase. For example, histone deacetylase
inhibitors result in histone rearrangements and gene activation at
multiple loci, many of which may be unrelated to physiologic lytic
cues, and therefore cannot be used to study EBV lytic gene
activation and replication.
Studies using reporter assays show that ZTA and RTA promoters are very weakly activated by the unfolded protein response
(UPR)–activated form of XBP1, XBP1(s), and more robustly in
combination with protein kinase D in EBV-positive epithelial and
LCLs,8,9 suggesting the possibility that the UPR is a trigger for
EBV lytic reactivation. XBP1 is required for both plasma cell
differentiation and UPR induction.10
EBV may mount a UPR during initial infection because of the
buildup of viral proteins in the ER or via direct eIF2␣ phosphorylation
through PKR activation secondary to viral RNA (including EBVencoded RNA) accumulation, resulting in a “lytic burst” of viral
amplification. During lytic replication in the oropharynx, the UPR may
occur when infected lymphocytes are exposed to bacterial TLR agonists
such as lipopolysaccharides or methylated CpG nucleotides produced
by colonizing bacteria,9,11-15 or during expansion of the ER in the course
of activated B lymphocyte to plasma-cell differentiation in the germinal
center reaction. The latter hypothesis is supported by studies suggesting
that the lytic pool of B lymphocytes may consist mainly of memory
B and plasma cells.11
To investigate the effects of the UPR on the lytic cycle in vitro,
we used the UPR inducer thapsigargin (TG), a noncompetitive
inhibitor of the endoplasmic reticulum Ca2⫹ ATPase (SERCA2),
which depletes luminal ER calcium stores and is thought to induce
ER stress by disabling Ca2⫹-dependent resident ER chaperone
proteins such as calnexin and calreticulin. We also used tunicamycin (TM), which induces the UPR by preventing protein glycosylation in the Golgi and secondarily causes protein accumulation in
the ER. Salubrinal (SAL), a protein-phosphatase 1 inhibitor
identified in a chemical screen for agents that prevent TG-induced
apoptosis,16 was used to antagonize ER stress in our experiments.
We conducted a series of experiments with these agents,
measuring: (1) expression of immediate-early, early, and late EBV
Submitted April 8, 2011; accepted August 11, 2011. Prepublished online as Blood
First Edition paper, August 17, 2011; DOI 10.1182/blood-2011-04-347112.
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.
The online version of this article contains a data supplement.
© 2011 by The American Society of Hematology
5528
BLOOD, 17 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 20
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BLOOD, 17 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 20
lytic mRNAs; (2) cell-surface gp350 expression; and (3) EBV
copies in supernatants of permissive LCLs and EBV particles in
electron micrography (EM) sections after chemical induction of ER
stress by TG or protection against ER stress by SAL.
To specifically identify genes involved in ER-stress–induced lytic
replication, we performed gene-expression profiling at early (before
viral lytic protein expression) and late time points, resulting in the
identification of previously characterized and novel cellular proteins.
Methods
Cell lines
LCLs derived from B95-8 infection of PBMCs, were contributed by David
Rowe (Graduate School of Public Health, University of Pittsburgh) and
cultured in RPMI 1640 plus GlutaMax (Invitrogen), supplemented with
10% FBS. B95-8 marmoset B cells were acquired from ATCC. The
experiments shown in Figure 3A through D were conducted with LCL-6,
which contains ⬎ 400 copies of episomal DNA per cell.
Chemical treatments
LCLs were split at 2 ⫻ 105 cells/mL in complete medium 24 hours before
treatment with 50␮M TG (Sigma-Aldrich), 5 ␮g/␮L of TM (SigmaAldrich), 40␮M SAL (Thermo Fisher Scientific), or 22.5 ␮g/mL of
acyclovir (ACY; Sigma-Aldrich). T24 and T72 cells were treated with TG
for 3 or 6 hours, washed with complete medium, and cultured in fresh
complete medium for an additional 18 or 66 hours for a total of 24 (T24) or
72 (T72) hours. In dual treatments with either TG and SAL (TS24 and
TS72) or TM and SAL, the cells were treated with both chemicals for 6
hours, then washed in complete medium, and subsequently cultured in
growth medium supplemented with SAL for an additional 18 or 66 hours.
S24 and S72 cells were treated with SAL continuously for either 24 or 72
hours. In control experiments, cells were cultured in ACY continuously for
24 or 72 hours.
Western blot analysis
LCLs were harvested 24 hours after chemical treatment and lysed in 100 ␮L
of RIPA buffer supplemented 1⫻ with protease and phosphatase inhibitors.
Samples were run on 8% SDS-PAGE gels with equal protein loading and
transferred to PVDF membranes. Membranes were blocked in 5% (wt/vol)
BSA and probed with either eIF2a (total), eIF2␣ pS51, (Cell Signaling
Technology), anti-ZTA (Santa Cruz Biotechnologies), or anti-LMP1 Abs
(Dako) and anti–rabbit IgG-HRP secondary Abs (Jackson ImmunoResearch
Laboratories).
RT-PCR and qPCR
Cell pellets were harvested at 24 or 72 hours after chemical treatment. RNA was
isolated from the cell pellets using the RNeasy Mini kit (QIAGEN) following the
manufacturer’s instructions. cDNA was synthesized using 1-5 ␮g of purified
RNA, the SuperScript II First Strand Synthesis Kit (Invitrogen), and random
hexamers. Quantitative PCR (qPCR) was performed in triplicate for each data
point shown using an ABI 7500, Maxima SYBR green/ROX mix (Thermo
Fisher Scientific) and 1 ␮L of cDNA with the following primers: BRLF1
5⬘-AATTTACAGCCGGGAGTGTG-3⬘ (sense) and 5⬘-AGCCCGTCTTCTTACCCTGT-3⬘ (antisense), BZLF1 5⬘-CATGTTTCAACCGCTCCGACTGG-3⬘
(sense) and 5⬘-GCGCAGCCTGTCATTTTCAGATG-3⬘ (antisense), BMRF1
5⬘-CTAGCCGTCCTGTCCAAGTGC-3⬘ (sense) and 5⬘-AGCCAAACGCTCCTTGCCCA-3⬘ (antisense), gp350 5⬘-GTCAGTACACCATCCAGAGCC-3⬘
(sense) and 5⬘-TTGGTAGACAGCCTTCGTATG-3⬘ (antisense), BLIMP1 5⬘ TCT TTGGGACATTCTTTGGG-3⬘ (sense), and 5⬘ - CGGAGAGCTGACAATGATGA-3⬘ (antisense), BCL6 5⬘ - CAATGCCTTGCTTCACAGTC-3⬘ (sense)
and 5⬘ - TGGGGTTCTTAGAAGTGGTGA-3⬘ (antisense), CD138 5⬘ - AGCCATCTTGATCTTCAGGG-3⬘ (sense) and 5⬘ - CTCTGGCTCTGGCTGTGC-3⬘
(antisense), and GAPDH 5⬘ - GAGTCAACGGATTTGGTCGT-3⬘ (sense) and
5⬘ - TTGATTTTGGAGGGATCTCG-3⬘ (antisense). Relative mRNA abundance
ER STRESS AND EBV REPLICATION
5529
was calculated using GAPDH as the internal control using the 2⌬⌬CT method. For
each experiment, DMSO was normalized to 1 and the mRNA changes were
represented relative to DMSO.
Affymetrix data analysis
cDNA synthesis, labeling, hybridization to Affymetrix U133 plus 2.0 chips,
and signal-intensity analysis was performed by the University of Pittsburgh
Genomics and Proteomics Core Laboratories (24- and 72-hour samples) or
by Asuragen (3-hour samples). Data from each treatment condition were
derived from independent experiments, independent labeling, and hybridization steps, and the fluorescence intensity was averaged over 2 replicates.
Median normalization for fold change was used throughout, filtering for
signal intensity ⬎ 20. For early (3-hour) gene-expression analysis, LCL-03
and LCL-06 cells were treated in duplicate with either TG or DMSO.
Clustering was performed filtering on signal intensity ⬎ 20 and an absolute
fold change ⬎ 2, and considering coordinately regulated gene sets at 3, 24,
or 72 hours using the Venn diagram utility in GeneSpring GX software
(Agilent Technologies).
EBV copy number determinations
DNA was extracted from 200-␮L portions of cell culture supernatants using
the QIAsymphony instrument (QIAGEN), and eluted into 50-␮L aliquots
for downstream PCR. EBV copy number was normalized per milliliter of
cell culture supernatant. A primer-probe set for a 90-bp target within the
EBV DNA polymerase gene (BALF-5) was used in this TaqMan PCR assay.
Details of the assay have been described previously.17
Determination of EBV-gp350 expression by FACS analysis
EBV-positive, permissive marmoset B cells (B95-8) were plated at 0.5 ⫻ 106
cells/well in 6-well tissue-culture plates 24 hours before treatment. B958
cells were treated with DMSO (0.01%), SAL (40␮M), and ACY
(22.5 ␮g/mL) with or without TPA (50 ng/mL). Cells were also treated with
SAL 0, 24, 48, and 72 hours after TPA treatment. LCLs were treated with
DMSO (0.01%), TG (50␮M), SAL (40␮M), or TG ⫹ SAL. Cells were
harvested 5 days after chemical treatment, counted, and washed with FACS
medium (DMEM with 1% FBS, 20mM HEPES, penicillin 10 U/mL, and
streptomycin 10 ␮g/mL), and incubated with monoclonal anti-gp350/250
Ab (clone 2L10; Millipore) for 60 minutes on ice with constant shaking.
Cells were washed twice with FACS medium, stained with goat anti–mouse
FITC-conjugated secondary Abs for 45 minutes on ice in the dark, washed
3 times, fixed, and analyzed by flow cytometry (FACSCalibur; BD
Biosciences).
Transmission EM
Cells treated with DMSO or TG were fixed in 2.5% glutaraldehyde,
processed, and sectioned.42 Sections were observed on a JEM 1210 electron
microscope (JEOL).
Results
Effects of TG and SAL on eIF2␣ phosphorylation and
expression of lytic and latent genes
ER stress causes translational arrest via protein kinase RNA-like
endoplasmic reticulum kinase (PERK)–mediated phosphorylation of
eIF2␣. To determine the effects of TG and SAL on this pathway in
LCLs, we applied short (6-hour) exposure to TG (50␮M), followed by
18 hours of washout with medium. This series of treatments resulted in
increased eIF2␣ phosphorylation (Figure 1A lane 3 vs lane 1). Likewise,
continuous (24-hour) exposure to SAL (40mM) also resulted in eIF2␣
phosphorylation (Figure 1A lane 2 vs lane 1). Short exposure to TG in
the continuous presence of SAL also resulted in eIF2␣ phosphorylation,
with little synergy observed with the combination of treatments (Figure
1A lane 4 vs lane 1). These data indicate that in LCLs, continuous
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5530
TAYLOR et al
BLOOD, 17 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 20
promoters, we performed qRT-PCR from total RNA purified after
24 or 72 hours from LCLs that had been exposed to TG, SAL, or
both (Figure 2B-C). TG was applied to cells for 6 hours, and the
medium was replaced with either DMSO or with SAL for the
remaining 18 or 66 hours of the assay, as indicated in Figure 2A.
Cells maintained ⬎ 80% viability by trypan blue exclusion with all
chemical treatments except TS24 and TS72 (cell viability, 50%).
TG reproducibly increased mRNA abundance relative to DMSO of
immediate-early (BRLF1 ⬃ 7.3-fold), early (BMRF1 ⬃ 3.2-fold),
and late mRNAs (gp350 ⬃ 7.6-fold) 24 hours after treatment,
whereas DMSO alone or SAL alone had little effect. When cells
were allowed to recover for 24 hours in the presence of SAL, EBV
lytic gene activation was reduced: BRLF1 activation decreased
from ⬃ 7.3-fold to ⬃ 2.6-fold; BZLF1 from ⬃ 7.3-fold to ⬃ 2.7fold; BMRF1 from ⬃ 3.2-fold to ⬃ 1.4-fold; and gp350 from
⬃ 7.6-fold to ⬃ 3.0-fold (Figure 2B).
When cells were harvested 72 hours after the start of TG
pulse, a pattern of EBV lytic gene activation similar to the
24-hour TG treatment was observed (Figure 2C). An ⬃ 7-fold
increase in the mRNA abundances of BRLF1, BZLF1, and
gp350 and a 3-fold increase in the mRNA abundance of BMRF1
was observed relative to DMSO. However, in contrast to the
24-hour data series, SAL augmented TG-induced EBV lytic
gene activation. Relative to TG alone, combined treatment with
SAL further increased the mRNA abundance of BRLF 7.3-fold
to 6.5-fold; BZLF1 7.3-fold to 21.4-fold; BMRF1 3.2-fold to
4.8-fold; and gp350 7.6-fold to 15.6-fold (Figure 2C). Remarkably, TG activation and TG ⫹ SAL synergy were maintained as
long as 5 days after the 6-hour TG pulse (Figure 3E). The 3-hour
TG pulse resulted in a similar pattern of EBV lytic gene
expression (data not shown).
Figure 1. ER stress induces phosphorylation of eIF2␣ and expression of ZTA
and LMP1. (A) SAL and ER stress result in increased levels of eIF2␣ S51
phosphorylation. LCLs were treated with SAL (40␮M for 24 hours), TG (50␮M for
6 hours), or SAL and TG as described. Western blot analysis for total eIF2␣ and pS51
eIF2␣ were performed 24 hours after treatment. (B) ER stress induces ZTA
expression in LCLs. LCLs were treated with TG, SAL, or TG ⫹ SAL as described and
at 6 or 24 hours after treatment, the cells were lysed in 1⫻ RIPA buffer. Equal
amounts of protein were analyzed by Western blot for ZTA. (C) Time-course
experiment of ZTA and LMP1 protein expression after the induction of ER stress.
LCLs were treated with TG and cells were lysed at the indicated times after
TG treatment in 1⫻ RIPA buffer. Equal amounts of protein were analyzed by Western
blot for ZTA and LMP1.
protein phosphatase 1 a (PP1a) activity is required to maintain dephosphorylated eIF2␣, and that both SAL and TG exposure result in
increased levels of eIF2␣ serine 31 phosphorylation with little
cooperativity.
To determine the effects of these treatments on EBV genes, we
selected candidate lytic (ZTA) and latency III (LMP1) proteins.
Twenty-four hours after the start of TG exposure, ZTA protein
levels were increased 5- to 6-fold (Figure 1B lane 2 bottom panel).
SAL alone had little effect on ZTA levels (Figure 1B lane 3 top and
bottom panels), but antagonized TG induced ZTA induction
(Figure 1B lane 4 bottom panel). A time-course experiment
indicated that ZTA protein levels were minimally increased at 3, 6,
and after TG exposure, but increased dramatically starting at
24 hours. Levels were also elevated as early as 3 hours after TG
exposure, and continued to increase over the course of the
experiment (Figure 1C).
ER stress induces EBV lytic genes at 24 and 72 hours and SAL
has both inhibitory and additive effects
Our preliminary data indicated that ER stress could increase ZTA
protein levels. To determine whether TG activates EBV lytic gene
EBV lytic gene activation is achieved with a different class of
UPR inducer
Because a TG pulse induces ER stress by transiently altering
calcium concentrations in the ER, we used TM, a drug that induces
ER stress by a different mechanism (inhibition of protein
N-glycosylation), to determine whether ER stress per se could
cause lytic gene activation. Pulse TM treatment (for 6 hours) also
caused consistent increases in lytic gene activation with moderate
additive effects of SAL over a 72-hour experiment (P ⬍ .05 for all
comparisons, Figure 2D).
Plasma cell marker expression in cells exposed to TG
Because plasma cell differentiation has been implicated in lytic
replication, we measured the levels of mRNAs encoding the
plasma cell differentiation proteins BLIMP1 and BCL6 and the
plasma cell marker CD138 in LCLs, activating lytic genes 1, 3, 6,
and 72 hours after the TG pulse. At 1 hour (Figure 4A), BLIMP1
and BCL6 mRNA levels were increased 2- to 3-fold in TG- and
TG ⫹SAL–treated cells relative to DMSO-treated cells; however,
by 3 hours (Figure 4B) and 6 hours (Figure 4C), only BCL6
remained elevated. Seventy-two hours after the TG pulse (Figure
4D), we observed no effect on BLIMP1 mRNA levels, slightly
increased BCL6 mRNA levels, and decreased mRNA levels for the
plasma cell marker CD138/Syndecan (P ⬍ .05 for TG vs DMSO
for BCL6 and CD138 only). However, these effects were not
observed with TG ⫹ SAL under conditions that result in synergistic up-regulation of EBV lytic mRNAs (Figure 2C). These data
indicate that TG and TG ⫹ SAL treatments result in transcriptional
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BLOOD, 17 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 20
ER STRESS AND EBV REPLICATION
5531
Figure 2. ER stress induces EBV lytic genes in LCLs.
(A) Schematic representation of drug treatments in
LCLs. Cells were treated with DMSO (panel 1) or 40␮M
SAL (panel 3) for the total length of the experiment; with
a 6-hour pulse of 50␮M TG and then cultured in media
lacking TG for the remaining length of the experiment
(panel 2); or with a 6-hour pulse of 50␮M TG as in panel
2 but in the presence of 40␮M SAL for the total length of
the experiment (panel 4). (B) Induction of EBV lytic
genes 24 hours after chemical treatment. At 24 hours
after drug treatment, LCLs were harvested, RNA isolated, and gene expression of immediate-early (BRLF1
and BZLF1), early (BMRF1), and late (gp350) EBV lytic
genes was evaluated by real-time qPCR. Comparison of
DMSO versus TG, DMSO versus TG ⫹ SAL, and TG
versus TG ⫹ SAL were all statistically significant at
P ⬍ .05. (C) Induction of EBV lytic genes 72 hours after
drug treatment, when LCLs were harvested and treated
as in panel B. Comparison of DMSO versus TG, DMSO
versus TG ⫹ SAL, and TG versus TG ⫹ SAL were all
statistically significant at P ⬍ .05. (D) TM treatment also
induces EBV lytic genes. LCLs were treated with a
4-hour pulse of TM (5 ␮g/mL) or TM ⫹ SAL and then
cultured for a total of 72 hours. At 72 hours after drug
treatment, LCLs were harvested as described and EBV
lytic gene expression was analyzed by real-time qPCR.
All comparisons were statistically significant.
responses more consistent with germinal center B cells, rather than
a postgerminal center B- or plasma-cell phenotype.
Effects of UPR induction and UPR protection on cell-surface
gp350 staining and EBV episome copy number in cell-culture
supernatants
To exclude the possibility that the UPR induced a transient
activation of EBV lytic genes without true viral assembly and
release, we monitored for the appearance of surface gp350 by
FACS in LCLs treated with a variety of agents (Figure 3A-B), as
well as the appearance of EBV DNA in cell supernatants of high
(⬎ 400 episomes/cell) and low (⬍ 50 episomes/cell) copy number
LCLs by qPCR (Figure 3C-D). In high-copy-number LCLs, we
observed an ⬃ 5% rate of spontaneous gp350 expression in both
DMSO-treated and -untreated cells (Figure 3A DMSO and NEG).
Remarkably, 5 days after the 3- or 6-hour TG pulse, gp350
expression increased from 5% to 26% (Figure 3A-B). Although
SAL strongly increased TG activation of lytic genes in this assay,
doubling or tripling mRNA abundances of lytic genes relative to
TG alone (Figure 3E), this did not translate into increased gp350
expression, which remained at 25%-26% under TG ⫹ SAL conditions, suggesting that, despite increased lytic mRNAs, other factors
were limiting for virus maturation or assembly. TM treatment did
not result in increased gp350 membrane staining, likely because of
inhibition of EBV glycoprotein glycosylation, indicating that this
method of UPR induction is incompatible with viral production. As
expected, by blocking viral DNA synthesis and late gene expression, ACY blocked spontaneous gp350 membrane expression
(Figure 3A).
To determine the effects of these agents on cell-free virus in
high-copy-number LCLs, EBV DNA was measured by PCR in
cell supernatants (Figure 3C) and the effects on EBV lytic genes
were assayed by qRT-PCR in parallel (Figure 3E). Induction of
the UPR by TG resulted in increased titers of free virus, with
EBV DNA copies increasing from 66 ⫻ 106/mL to 171 ⫻ 106/mL
(Figure 3C), which is consistent with TG transcriptional effects
on lytic genes (Figure 3D). Whereas SAL alone had little
activity in activating EBV lytic genes, it potently reduced EBV
replication. In the representative experiment shown, incubation
with SAL alone resulted in a reduction of EBV copies from
⬃ 66 ⫻ 106/mL to ⬃ 35 ⫻ 106/mL (an ⬃ 50% reduction), indicating a lytic inhibitory effect even in the absence of exogenous
ER stress. For comparison, under the same conditions, ACY
treatment reduced copy number from ⬃ 66 ⫻ 10 6/mL to
15 ⫻ 106/mL (a 78% reduction). Furthermore, whereas the TG
pulse resulted in a near doubling of EBV copies (66 ⫻ 106/mL
to 171 ⫻ 106/mL), this effect was completely abrogated when
SAL was continuously present in the culture medium after the
TG pulse, resulting in a decrease of EBV copies from
171 ⫻ 106/mL to 28 ⫻ 106/mL (an 84% reduction). Therefore,
SAL inhibited both basal and ER-stress–induced lytic replication. Similar results were not observed in low-copy-number
LCLs (Figure 3D), in which UPR induction by TG did not result
in increased EBV DNA copies (1.6 ⫻ 106/mL to 1.2 ⫻ 106/mL).
However, SAL treatment did result in a reduction of EBV copies
from ⬃ 1.5 ⫻ 106/mL to ⬃ 0.7 ⫻ 106/mL (an ⬃ 50% reduction), as was observed in high-copy-number LCLs.
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BLOOD, 17 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 20
Figure 3. ER stress induces late events in EBV lytic production. (A-B). TG treatment results in gp350 expression at the
plasma membrane. Five days after chemical treatment, LCLs
were stained for gp350 and analyzed by flow cytometry. The
percentage of gp350-positive cells is plotted (A). The shift in
fluorescence intensity of gp350-positive cells from DMSO-treated
(pink trace) to TG-treated (green trace) LCLs can be seen
(B). The background fluorescence of LCLs is shown as the filled
trace. (C) TG treatment results in increased EBV copy number in
high-copy-number LCLs (⬎ 400 episomes/cell). Five days after
chemical treatment of LCLs, supernatant was collected and EBV
copy number determined by qPCR. (D) TG treatment does not
increase EBV copy number in low-copy-number LCLs
(⬍ 50 episomes/cell). Five days after chemical treatment of LCLs,
supernatant was collected and EBV copy number determined by
qPCR. (E) Induction of EBV lytic genes 5 days after chemical
treatment, when LCLs were harvested, RNA isolated, and gene
expression of immediate-early (BRLF1 and BZLF1), early
(BMRF1), and late (gp350) EBV lytic genes was evaluated by
real-time qPCR.
SAL prevents lytic replication independently of ER stress
induction
Treatment of B95-8 marmoset B cells with TPA results in robust lytic
replication.18 To confirm that SAL could inhibit lytic replication under
non–ER-stress conditions (Figure 3C-D), we induced lytic replication
with an alternative method (TPA-induced PKC activation) in B95-8
cells and applied SAL at different time points after TPA treatment
(Figure 5). When SAL was applied at the same time as TPA, lytic
Figure 4. ER stress effects on germinal center B- and
plasma-cell markers. Gene expression of plasma cell markers
Blimp1, CD138, and BCL6 were evaluated by real-time qPCR.
LCLs were treated with TG and SAL for 1 hour (A), 3 hours (B), or
6 hours (C) before gene expression of plasma cell markers was
evaluated by real-time qPCR. (D) LCLs were treated with TG and
SAL as described and plasma cell markers were evaluated at
72 hours after drug treatment by real-time qPCR.
replication was inhibited by ⬎ 90% with an efficacy comparable to
ACY. Inhibition was reduced when SAL was applied at later time points.
When SAL was applied 24 hours after TPA, inhibition was reduced to
⬃ 65%, and only a ⬃ 25% inhibition was observed when SAL was
applied at 48 or 72 hours. These data suggest an immediate-early or
early effect of SAL on viral replication in TPA-stimulated B95-8 cells.
The ability of SAL to inhibit lytic replication in B95-8 cells exposed to
TPA indicates an effect independent of ER stress.
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BLOOD, 17 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 20
Figure 5. SAL prevents TPA-induced virus production in B95-8 cells. The
permissive EBV-positive marmoset B-cell line B95-8 was treated with SAL 0, 24, 48,
and 72 hours after TPA treatment or with TPA, ACY, or DMSO alone. Cells were
incubated for a total of 5 days after TPA treatment and assayed for gp350 expression,
as described in “Methods.”
TG treatment of LCLs results in the production of infectious
viral particles
Whereas TG treatment causes increases in EBV DNA in supernatants of treated LCLs, the possibility that these supernatants consist
of naked viral DNA due to cell lysis of noninfectious particles
remained. We performed EM on these cells 5 days after the TG
pulse (Figure 6A-D). Exhaustive searching of multiple EM fields
failed to show any nucleocapsid or viral particles (Figure 6A), with
largely intact nuclear structure in DMSO-treated cells. In contrast,
Figure 6. ER stress induces virus production. LCLs
were treated with DMSO (A) or TG (B-D) and at 5 days
after treatment the cells were processed for EM.
(B) Accumulation of nucleocapsid cores near the nuclear
membrane with an inset of a higher magnification of the
nucleocapsid cores. (C) EBV-enveloped virus transport
and budding at the plasma membrane. Arrow indicates
the occurrence of a double-membrane vesicle, a hallmark
of autophagy. (D) Higher magnification of the boxed area
in panel C. Thick arrow shows an enveloped virion being
transported to the plasma membrane and the thin arrow
shows a budding virus.
ER STRESS AND EBV REPLICATION
5533
in TG-treated cells, we observed massive accumulation of nucleocapsids (Figure 6B boxed area and inset) adjacent to the disrupted
inner and outer nuclear laminae. Numerous fields indicated enveloped, cell-free virus that was visible because it had been trapped
within intercellular regions (Figure 6C-D). To determine whether
TG treatment resulted in the production of infectious virus, we
infected EBV-negative BJAB cells with the supernatants of TGtreated LCLs, induced lytic replication in BJABs, isolated RNA,
and quantified expression of lytic genes BZLF1, BRLF1, and
BMRF1 (supplemental Figure 1, available on the Blood Web site;
see the Supplemental Materials link at the top of the online article).
These genes failed to amplify with mock-infected BJABs. The
mRNA abundance relative to supernatants from DMSO-treated
LCLs (which have an ⬃ 5% rate of lytic activity by gp350 staining)
was measured and indicated production of lytic viral RNAs in
infected BJAB cells. These data indicate that TG treatment results
in the production of infectious viral particles.
Differential gene-expression analysis of ER stress effects in LCLs
Microarray data are available at the Gene Expression Omnibus
(www.ncbi.nlm.nih.gov/geo/) under accession number GSE 31447.
We performed microarray analysis using Affymetrix HG133U_Plus
2 chips containing 53 613 probe sets derived from ⬎ 30 000 unique
genes and control probe sets. We used mRNA from independent
cell lines (LCL-3 and LCL-6) to determine the early (3-hour)
effects of TG on gene expression, and compared these patterns with
cells harvested at 24 and 72 hours in LCL-6. Because ZTA protein
is expressed at 24 but not 6 hours after TG treatment (Figure 1B),
early transcriptional changes are kinetically upstream of ZTA.
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5534
BLOOD, 17 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 20
TAYLOR et al
Figure 7. Venn diagram-based clustering method: T3,
T24, and T72 hours.
To prevent the bias that results from hierarchical cluster analysis
and curating of multiple gene clusters, we identified regulation
clusters by Venn diagram analysis. We filtered on absolute fold
change ⬎ 2 (ie, fold change ⬎ 2 or fold change ⬍ ⫺2) to identify 6
gene clusters of interest: (1) sets of mRNAs with absolute fold
change ⬎ 2 at 3, 24, and 72 hours (sustained activation and
sustained repression, Figure 7A-C and Table 1); (2) mRNAs with
absolute fold change ⬎ 2 at 3 hours but absolute fold change ⬍ 2 at
24 and 72 hours (early activation and early repression, Figure
7A-B,D, and Table 2); and (3) mRNAs with absolute fold change
⬍ 2 at 3 hours and ⬎ 2 absolute fold change at 24 and 72 hours (late
activation and late repression, Figure 7A-B,E, and Table 3).
To prevent cell-line–specific changes, only genes differentially
expressed (using a fold cutoff of 2.0) in both cell lines, LCL-03 and
LCL-06, were included in the 3-hour analysis. Genes induced or
repressed ⬎ 2.0-fold in LCL-06 at 3 hours and in LCL-06 at 24 and
72 hours (Figure 7A set m) were overlapped with genes induced or
repressed ⬎ 2.0-fold in LCL-03 at 3 hours and LCL-06 at 24 and
72 hours (Figure 7B set q). The intersection of sets m and q (plotted in
Figure 7C) therefore represents sustained activation or repression over
Table 1. Sustained gene expression changes in stressed LCLs
Gene
LCL-6, 3 h
LCL-3, 3 h
LCL-6, 24 h
LCL-06, 72 h
Average
RefSeq transcript ID
Sustained activation
(3, 24, and 72 hours)
TAC1
2.8
18.3
25.1
27.5
18.4
NM_003182
21.1
30.1
8.7
11.1
17.8
NM_000584
NR4A2
6.9
18.0
3.2
4.4
8.1
NM_006186
INHBE
4.4
5.4
9.1
12.0
7.7
NM_031479
KLF4
9.7
3.4
5.5
6.8
6.4
NM_004235
IL8
NR4A3
4.4
9.9
3.6
6.3
6.1
NM_006981
PTHLH
3.2
9.0
3.6
3.0
4.7
NM_002820
EMP1
3.9
2.1
6.5
5.9
4.6
NM_001423
NAMPT
4.3
4.9
2.1
3.4
3.7
NM_005746
NM_032717
AGPAT9
3.1
3.5
3.9
3.3
3.5
CCL4
3.6
3.0
3.1
4.1
3.4
NM_002984
PHLDA1
3.1
3.3
3.2
3.3
3.2
NM_007350
STC2
2.4
2.5
3.1
3.8
2.9
NM_003714
RAB11FIP1
3.0
3.0
2.1
3.4
2.9
NM_001002233
MAFF
2.9
2.2
2.3
4.0
2.9
NM_012323
CTH
3.6
3.1
2.2
2.4
2.8
NM_001902
CEBPB
2.7
3.1
2.5
2.8
2.8
NM_005194
NFIL3
2.0
3.4
2.2
3.0
2.7
NM_005384
VLDLR
3.2
2.5
2.2
2.2
2.5
NM_001018056
AHNAK
3.1
2.2
2.4
2.3
2.5
NM_001620
CD55
2.3
3.0
2.2
2.4
2.5
NM_000574
ANKRD28
2.3
2.2
2.3
2.3
2.3
NM_015199
CHAC1
2.5
2.0
2.3
2.1
2.2
NM_001142776
2.3
2.3
2.2
3.6
2.6
NM_005615
Sustained repression
(3, 24, and 72 h)
RNASE6
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BLOOD, 17 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 20
ER STRESS AND EBV REPLICATION
Table 2. Early gene expression changes in stressed LCLs
Table 3. Late gene expression changes in stressed LCLs
Gene
Gene
LCL-6, 3 h
Early activation
(3 h only)
EGR3
MYADM
FOS
TC2N
PTGS2
SIAH2
OTUD1
RGS2
FAM62B
KLF6
TUBE1
FOSB
PSAT1
TMEM87A
CSDA
TEX9
JUN
SCHIP1
ARL4C
B3GNT2
HIPK1
LONRF1
FOSL2
TMEM70
JUND
NAB1
CDC14B
EXOC5
IFRD1
ATP1B3
ANKRD28
LETM2
WSB1
RELL1
PARL
TRIB1
NFKBID
NUDT4
MNS1
ATP2B1
PCTK2
PIP5K1B
EIF5B
TFG
SDCBP
SQLE
ST8SIA4
PCGF5
RSPH10B2
SLC25A13
ANKRD12
SLC4A7
ARL2BP
SESN2
ZMYM6
ZFP36L1
SLC3A2
AMMECR1
GNG2
FAM107B
TRIB3
CSNK2A1
NIPSNAP3B
PPP1R2
PANK2
Early repression
(3 h only)
FAM46C
LCP2
NOTCH2
AICDA
LCL3, 3 h
Average
Refseq transcript ID
LCL-06, 24 h
LCL-06, 72 h
Average
5535
RefSeq Transcript ID
Late activation
(24, 72 h only)
6.7
5.6
3.2
5.9
3.1
2.8
3.6
2.0
2.9
2.9
2.8
2.8
2.7
2.6
3.1
2.0
2.4
2.8
3.3
3.1
2.9
2.5
2.1
2.7
2.3
2.2
2.1
2.6
2.4
2.4
2.4
2.0
2.2
2.5
2.3
2.1
2.6
2.3
2.1
2.3
2.7
2.3
2.2
2.5
2.3
2.3
2.0
2.3
2.4
2.3
2.3
2.0
2.1
2.3
2.1
2.3
2.2
2.0
2.3
2.0
2.1
2.1
2.1
2.1
2.0
⫺3.3
⫺2.3
⫺2.2
⫺2.2
6.4
4.7
6.3
2.3
4.3
4.2
3.2
4.7
3.7
3.2
3.3
3.3
3.3
3.2
2.6
3.6
3.2
2.8
2.3
2.5
2.6
3.0
3.3
2.6
2.8
2.9
2.9
2.4
2.5
2.5
2.5
2.8
2.7
2.3
2.5
2.7
2.2
2.5
2.7
2.4
2.1
2.4
2.5
2.1
2.3
2.3
2.5
2.2
2.1
2.2
2.1
2.4
2.3
2.1
2.2
2.0
2.1
2.3
2.0
2.2
2.2
2.1
2.1
2.0
2.0
⫺2.8
⫺2.3
⫺2.1
⫺2.0
6.6
5.2
4.8
4.1
3.7
3.5
3.4
3.4
3.3
3.0
3.0
3.0
3.0
2.9
2.9
2.8
2.8
2.8
2.8
2.8
2.8
2.7
2.7
2.6
2.5
2.5
2.5
2.5
2.5
2.5
2.4
2.4
2.4
2.4
2.4
2.4
2.4
2.4
2.4
2.4
2.4
2.4
2.3
2.3
2.3
2.3
2.3
2.2
2.2
2.2
2.2
2.2
2.2
2.2
2.2
2.2
2.2
2.2
2.1
2.1
2.1
2.1
2.1
2.1
2.0
⫺3.0
⫺2.3
⫺2.1
⫺2.1
NM_004430
NM_001020818
NM_005252
NM_001128595
NM_000963
NM_005067
NM_001145373
NM_002923
NM_020728
NM_001160124
NM_016262
NM_001114171
NM_021154
NM_001110503
NM_001145426
NM_198524
NM_002228
NM_014575
NM_005737
NM_006577
NM_152696
NM_152271
NM_005253
NM_001040613
NM_005354
NM_005966
NM_001077181
NM_006544
NM_001007245
NM_001679
NM_015199
NM_144652
NM_015626
NM_001085399
NM_001037639
NM_025195
NM_139239
NM_019094
NM_018365
NM_001001323
NM_002595
NM_003558
NM_015904
NM_001007565
NM_001007067
NM_003129
NM_005668
NM_032373
NM_001099697
NM_001160210
NM_001083625
NM_003615
NM_012106
NM_031459
NM_007167
NM_004926
NM_001012661
NM_001025580
NM_053064
NM_031453
NM_021158
NM_001895
NM_018376
NM_006241
NM_024960
NM_017701
NM_005565
NM_024408
NM_020661
IL2
5.39
11.35
8.37
NM_000586
DGKI
4.25
12.48
8.37
NM_004717
FGFBP2
3.79
12.85
8.32
NM_031950
CTHRC1
6.77
8.23
7.50
NM_138455
SMPDL3A
8.45
5.79
7.12
NM_006714
GZMB
2.78
10.55
6.66
NM_004131
IFNG
2.29
10.58
6.43
NM_000619
GBP2
5.09
7.05
6.07
NM_004120
F5
5.16
6.77
5.97
NM_000130
SERPINA1
3.63
7.99
5.81
NM_000295 127704
PALLD
4.90
6.40
5.65
NM_016081
SORBS1
2.10
9.02
5.56
NM_001034954
GLYATL2
7.37
3.73
5.55
NM_145016
SERPINB2
5.84
5.14
5.49
NM_001143818
HS3ST1
4.70
6.02
5.36
NM_005114
MYOF
4.59
6.11
5.35
NM_013451
MAP1B
6.50
4.15
5.32
NM_005909
TNFRSF9
2.10
8.36
5.23
NM_001561
ASS1
4.07
5.96
5.01
NM_000050
XCL1
3.29
6.21
4.75
NM_002995
GPR56
4.08
5.32
4.70
NM_001145770
FILIP1L
4.12
4.96
4.54
NM_001042459
ETV7
3.42
5.45
4.43
NM_016135
PRF1
2.26
6.55
4.41
NM_001083116
CD72
4.01
4.54
4.27
NM_001782
PDZK1
4.31
4.08
4.19
NM_002614
DHRS9
3.76
4.49
4.12
NM_001142270
TMEM100
2.94
5.05
3.99
NM_001099640
HLF
2.21
5.72
3.97
NM_002126
S100A10
5.75
2.17
3.96
NM_002966
GSTA4
4.21
3.58
3.89
NM_001512
LAG3
2.78
4.83
3.80
NM_002286
LBH
2.28
5.24
3.76
NM_030915
CD109
3.73
3.69
3.71
NM_001159587
IL1R2
2.08
5.26
3.67
NM_004633
CCDC144A
3.55
3.77
3.66
NM_014695
SHC4
2.01
5.24
3.62
NM_203349
ENC1
2.44
4.68
3.56
NM_003633
ATP9A
2.02
5.01
3.51
NM_006045
ABAT
3.43
3.40
3.41
NM_000663
RUNDC3B
4.35
2.32
3.34
NM_001134405
GABRB2
4.48
2.13
3.30
NM_000813
COL5A2
3.71
2.86
3.29
NM_000393
ITK
2.35
4.15
3.25
NM_005546
AMY1A
3.11
3.39
3.25
NM_000699
ALDH1L2
3.43
3.01
3.22
NM_001034173
NKG7
2.40
4.05
3.22
NM_005601
DKK4
3.57
2.81
3.19
NM_014420
IGF2R
2.33
3.95
3.14
NM_000876
COL9A3
2.73
3.55
3.14
NM_001853
CAV1
2.14
4.11
3.12
NM_001753
PLEKHA7
3.96
2.25
3.10
NM_175058
CBS
3.24
2.96
3.10
NM_000071
CD160
3.44
2.74
3.09
NM_007053
SYNPO2L
2.45
3.70
3.07
NM_001114133
FYN
2.96
3.13
3.05
NM_002037
ITPRIPL2
2.58
3.45
3.01
NM_001034841
FNIP1
3.73
2.25
2.99
NM_001008738
RASSF6
3.54
2.39
2.97
NM_177532
GLT8D2
3.00
2.86
2.93
NM_031302
UGT2B4
3.71
2.14
2.93
NM_021139
ABCB4
2.60
3.16
2.88
NM_000443
GPT2
3.21
2.55
2.88
NM_001142466
PABPC1L
2.54
3.22
2.88
NM_001124756
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TAYLOR et al
Table 3. Continued
Gene
Table 3. Continued
LCL-06, 24 h
LCL-06, 72 h
Average
RefSeq Transcript ID
Gene
LCL-06, 24 h
LCL-06, 72 h
Average
RefSeq Transcript ID
PDE4DIP
2.40
3.35
2.87
NM_001002810
FRMD6
2.17
2.07
2.12
NM_001042481
LRRC50
2.31
3.40
2.85
NM_178452
ZNF79
2.05
2.20
2.12
NM_007135
CACNA1A
2.49
3.18
2.84
NM_000068
TWSG1
2.11
2.11
2.11
NM_020648
BEND6
3.63
2.04
2.84
NM_152731
FCHO2
2.10
2.11
2.11
NM_001146032
DDR2
2.76
2.91
2.84
NM_001014796
ERMP1
2.09
2.12
2.10
NM_024896
ASZ1
3.06
2.58
2.82
NM_130768
CNR1
2.19
2.02
2.10
NM_001160226
MYLK
2.44
3.10
2.77
NM_053025
RAB3B
2.10
2.10
2.10
NM_002867
SERPINB1
2.06
3.48
2.77
NM_030666
MPZL3
2.03
2.16
2.09
NM_198275
TMEM149
2.73
2.78
2.76
NM_024660
GBP1
2.04
2.13
2.09
NM_002053
CACNA1D
3.16
2.17
2.67
NM_000720
DFNB59
2.05
2.12
2.09
NM_001042702
ANK2
2.83
2.50
2.67
NM_001127493
LOXL3
2.05
2.12
2.09
NM_032603
NDFIP1
2.58
2.75
2.66
NM_030571
NEU1
2.08
2.09
2.08
NM_000434
TNFRSF10D
2.60
2.70
2.65
NM_003840
RTTN
2.09
2.08
2.08
NM_173630
FRY
2.02
3.24
2.63
NM_023037
TESK1
2.11
2.05
2.08
NM_006285
ZDHHC11
2.69
2.53
2.61
NM_024786
RAPGEF2
2.00
2.13
2.07
NM_014247
TOX2
2.62
2.55
2.59
NM_001098796
PCGF5
2.06
2.06
2.06
NM_032373
BEX2
2.25
2.91
2.58
NM_032621
DNASE2
2.02
2.08
2.05
NM_001375
CCL5
2.93
2.17
2.55
NM_002985
PARP11
2.03
2.06
2.04
NM_020367
PAM
2.25
2.84
2.55
NM_000919
TWSG1
2.05
2.03
2.04
NM_020648
ANKRD29
2.67
2.38
2.53
NM_173505
CTTN
2.02
2.05
2.04
NM_005231
ZFYVE21
2.33
2.69
2.51
NM_024071
Late repression
ATF5
2.43
2.53
2.48
NM_012068
TSC22D1
2.47
2.48
2.47
NM_006022
MCM4
⫺2.61
⫺4.96
⫺3.78
NM_005914
TPBG
2.35
2.55
2.45
NM_006670
FCRL4
⫺3.33
⫺3.77
⫺3.55
NM_031282
SLC19A2
2.76
2.14
2.45
NM_006996
PRKCB
⫺3.88
⫺2.84
⫺3.36
NM_002738
DBNDD2
2.05
2.83
2.44
NM_001048221
STAT1
⫺3.31
⫺2.99
⫺3.15
NM_007315
ABCB1
2.18
2.67
2.42
NM_000443
CCNE2
⫺3.44
⫺2.57
⫺3.01
NM_057749
CCPG1
2.16
2.66
2.41
NM_004748
CT45A5
⫺3.98
⫺2.01
⫺2.99
NM_001007551
JAG1
2.27
2.54
2.41
NM_000214
LMNB1
⫺3.08
⫺2.71
⫺2.89
NM_005573
PLA1A
2.23
2.58
2.40
NM_015900
BUB1
⫺2.79
⫺2.86
⫺2.83
NM_004336
BSPRY
2.47
2.33
2.40
NM_017688
NUCKS1
⫺2.82
⫺2.83
⫺2.83
NM_022731
NLRP1
2.10
2.67
2.39
NM_001033053
RASSF5
⫺3.23
⫺2.39
⫺2.81
NM_182663
TM6SF1
2.46
2.31
2.38
NM_001144903
SFPQ
⫺2.79
⫺2.57
⫺2.68
NM_005066
LPAR5
2.21
2.56
2.38
NM_001142961
RAD51L3
⫺2.76
⫺2.34
⫺2.55
NM_001142571
TRIM2
2.29
2.46
2.37
NM_001130067
SLC29A1
⫺2.38
⫺2.62
⫺2.50
NM_001078174
PHLDA1
2.48
2.25
2.37
NM_007350
SFRS1
⫺2.82
⫺2.17
⫺2.50
NM_001078166
TMEM22
2.14
2.56
2.35
NM_001097599
TMPO
⫺2.25
⫺2.70
⫺2.48
NM_001032283
CCDC88A
2.61
2.05
2.33
NM_001135597
SCYL2
⫺2.52
⫺2.42
⫺2.47
NM_017988
SPAG1
2.40
2.24
2.32
NM_003114
WNT6
⫺2.42
⫺2.46
⫺2.44
NM_006522
B3GNT7
2.30
2.33
2.31
NM_145236
CDV3
⫺2.56
⫺2.25
⫺2.40
NM_001134422
ALDH8A1
2.48
2.14
2.31
NM_022568
ESCO2
⫺2.43
⫺2.30
⫺2.36
NM_001017420
GPR133
2.25
2.37
2.31
NM_198827
MRPL30
⫺2.14
⫺2.56
⫺2.35
NM_145212
SPAG9
2.36
2.25
2.30
NM_001130528
CXCR7
⫺2.34
⫺2.29
⫺2.31
NM_020311
PELI1
2.16
2.44
2.30
NM_020651
PDE7A
⫺2.50
⫺2.10
⫺2.30
NM_002603
CEACAM21
2.33
2.24
2.29
NM_001098506
EXOSC3
⫺2.13
⫺2.45
⫺2.29
NM_001002269
SNX18
2.32
2.23
2.27
NM_001102575
ATP6V0D2
⫺2.55
⫺2.03
⫺2.29
NM_152565
PCGF5
2.29
2.25
2.27
NM_032373
BAT2D1
⫺2.30
⫺2.25
⫺2.27
NM_015172
KIF1B
2.15
2.39
2.27
NM_015074
UNG
⫺2.10
⫺2.41
⫺2.26
NM_003362
UNC84A
2.46
2.09
2.27
NM_001130965
RFC3
⫺2.28
⫺2.20
⫺2.24
NM_002915
CRIP1
2.34
2.20
2.27
NM_001311
NEXN
⫺2.22
⫺2.22
⫺2.22
NM_144573
IFT57
2.24
2.30
2.27
NM_018010
POLH
⫺2.36
⫺2.08
⫺2.22
NM_006502
NINJ2
2.33
2.19
2.26
NM_016533
PKN2
⫺2.38
⫺2.01
⫺2.19
NM_006256
N4BP2L2
2.16
2.35
2.25
NM_014887
PNN
⫺2.32
⫺2.05
⫺2.18
NM_002687
LTBP3
2.43
2.06
2.25
NM_001130144
SKP2
⫺2.27
⫺2.07
⫺2.17
NM_005983
MLLT11
2.11
2.37
2.24
NM_006818
GART
⫺2.02
⫺2.13
⫺2.08
NM_000819
PCGF5
2.40
2.08
2.24
NM_032373
PHTF2
⫺2.07
⫺2.06
⫺2.06
NM_001127357
ZBTB32
2.27
2.18
2.23
NM_014383
POLE3
⫺2.06
⫺2.04
⫺2.05
NM_017443
MBD6
2.05
2.40
2.22
NM_052897
PRPF18
⫺2.02
⫺2.02
⫺2.02
NM_003675
WARS
2.05
2.38
2.22
NM_004184
CARS
2.22
2.20
2.21
NM_001014437
GCG
2.23
2.19
2.21
NM_002054
UBE2F
2.07
2.31
2.19
NM_080678
PEX5L
2.18
2.17
2.17
NM_016559
SUPT3H
2.30
2.03
2.17
NM_003599
FAM169A
2.04
2.26
2.15
NM_015566
ALG13
2.25
2.01
2.13
NM_018466
(24, 72 h only)
the entire time course of TG-induced lytic replication and consists of 79
probe sets. After eliminating uncharacterized open reading frames and
control and redundant probe sets, we determined that 23 genes were
up-regulated and 1 gene was down-regulated at all time points (Figure
7C and Table 1). Because Z and LMP1 protein levels are increased by
TG at 24 but not 6 hours after TG, these genes are all kinetically
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BLOOD, 17 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 20
upstream of Z and LMP1. The UPR chaperone CHOP/DDIT3 was
up-regulated 3.0-fold in LCL-6 and 1.8-fold in LCL-03.
Genes regulated in a sustained manner include 2 members of the
nuclear hormone receptor family (NR4A2 and NR4A3). NR4A1
and NR4A3 are induced by lytic replication secondary to BCR
cross-linking in AKATA cells before ZTA expression,19 a transcription factor (CEBP␤) induced by proteasome inhibition and involved in Z activation,5 as well as candidate transcription factors of
interest such as NFIL3, KLF4, MAFF, cytokines such as IL8 and
CCL4, and genes involved in adjustment to calcium stress
(eg, STC2). Only RNASE6 was repressed at all time points.
Early up-regulated genes included EGR3, a transcription factor
induced by BCR cross-linking, and early-response genes such as
c-Jun, JunD, FOS, FOSB, and KLF6.19 Tribbles 1 and 3, previously
reported to be induced by ER stress, were up-regulated 2- to 3-fold.
Notch2 was down-regulated 2-fold, suggesting a contribution of
Notch/EBNA2 signaling to the maintenance of latency. Activationinduced cytidine deaminase (AICDA) was also repressed. AICDA
is involved in the somatic hypermutation, gene conversion, and
class-switch recombination of immunoglobulin genes required for
several crucial steps of B-cell terminal differentiation that is
necessary for efficient Ab responses, which is consistent with our
observations that ER stress is associated with inhibition of the
plasma cell differentiation pathway.
Late-regulated genes included those known to be induced by
ER stress (eg, ASS1, ATF5, and DHRS9), antiviral genes (IFNG),
and calcium-binding proteins (eg, S100A). DHRS9, a retinolmetabolizing enzyme, is activated directly by ZTA binding during
lytic infection in epithelial cells.20 XCL1 was up-regulated 3.3- and
6.2-fold at 24 and 72 hours, respectively (Table 3), so it may be
important for B cells undergoing lytic infection to migrate to
epithelial surfaces for viral release into saliva.
As shown in Figure 4A, at 24 hours, SAL incubation reduced
the amplitude of EBV lytic gene induction by TG pulse. We
therefore hypothesized that genes that are induced by T24 but in
which the mRNA expression level is reduced to near DMSO levels
in TS24 experiments might be specifically associated with EBV
lytic gene expression, either causing or resulting from activation of
EBV lytic genes (Table 4 and supplemental Figure 2).
Individual sets of genes regulated at each time point under each
treatment condition, together with their J5 values, are shown in
supplemental Tables 1-6.
ER STRESS AND EBV REPLICATION
5537
Table 4. Unique gene expression changes in T24 versus TS24
Gene
-Fold change
RefSeq Transcript ID
Genes > 3-fold up in
T24 but not TS24
CTHRC1
7.6
NM_138455
EMP1
7.2
NM_001423
GLYATL2
6.5
NM_145016
MYLK
6.1
NM_053025
PALLD
5.3
NM_016081
SERPINB2
5.2
NM_001143818
GBP2
5.1
NM_004120
CD24
5.0
NM_013230
ANXA1
4.7
NM_000700
XCL1
4.7
NM_002995
MUC4
4.5
NM_004532
PLEKHA7
4.4
NM_175058
NM_017527
LY6K
4.4
GABRB2
4.3
NM_000813
DHRS9
4.3
NM_001142270
DKK4
4.3
NM_014420
CTNNA2
4.3
NM_004389
NM_001730
KLF5
4.2
FHL5
4.2
NM_020482
ARAP3
4.2
NM_022481
DCHS1
4.0
NM_003737
SFN
4.0
NM_006142
DHRS9
4.0
NM_001142270
CD72
3.8
NM_001782
FGFBP2
3.7
NM_031950
SERPIND1
3.6
NM_000185
TNS3
3.6
NM_022748
ANKRD29
3.5
NM_173505
FNIP1
3.5
NM_001008738
GAP43
3.5
NM_001130064
NR4A3
3.4
NM_006981
RAB3B
3.3
NM_002867
EVI2A
3.3
NM_001003927
RUNDC3B
3.3
NM_001134405
CITED2
3.3
NM_006079
IL2
3.3
NM_000586
IL4
3.2
NM_000589
DNAH12
3.2
NM_178504
CCDC88A
3.1
NM_001135597
MYADM
3.1
NM_001020818
TEX9
3.1
NM_198524
ENDOD1
3.1
NM_015036
MPEG1
3.0
NM_001039396
Genes > 3-fold down in
T24 but not TS24
Discussion
Our data indicate that the induction of ER stress by TG is sufficient
to trigger high-copy (⬎ 400 copies/cell) LCLs into lytic replication, including activation of EBV lytic genes, surface gp350
expression, and release of virus. A 3-hour exposure to TG pulse is
sufficient for this effect, resulting in a transcriptional and lytic
permissive state that occurs as long as 5 days after the initial
application of ER stress. A low-copy-number (⬍ 50 copies) LCL
did not show activation of EBV lytic promoters in response to TG.
These differences in ER stress effects on low- and high-copynumber LCLs may be related to epigenetic differences in LCLs
derived from different patient sources.21 Our results indicate that
ER stress is an important in vitro trigger for lytic replication, and
provide a chemical model for studying the cellular pathways
involved. Our approach is validated by the fact that many of the
same genes induced by lytic replication secondary to BCR
NOTCH2
⫺3.5
NM_024408
FCRL4
⫺3.3
NM_031282
HIST1H2BC
⫺3.2
NM_003526
TXNIP
⫺3.0
NM_006472
cross-linking (eg, NR4A2 and EGR-1) are also up-regulated by
ER-stress–induced lytic replication in our studies.
TG is a chemical that depletes luminal ER calcium. Therefore, it
is possible that some of the lytic effects might be due to increases in
cellular Ca2⫹ and PKC activation.18 However, recapitulation of
EBV lytic gene activation with TM makes this less likely. As
expected, attempts in our laboratory to induce lytic replication with
TPA in all LCLs used were unsuccessful. This raises the question of
whether ER stress signaling per se (eg, IRE1 signaling) is a cue for
lytic replication. More generally, stress-responsive chaperones
activated by ER stress and calcium depletion may be critical for
initiating the lytic program.
From www.bloodjournal.org by guest on June 16, 2017. For personal use only.
5538
BLOOD, 17 NOVEMBER 2011 䡠 VOLUME 118, NUMBER 20
TAYLOR et al
SAL had opposite effects in modulating TG activity, depending
on the length of the assay, repressing TG-induced lytic gene
activation when cells were harvested 24 hours after TG pulse and
synergizing with TG at 72 hours or at 5 days after TG pulse. SAL
had no effect on EBV lytic gene activation when used alone in
these assays. However, SAL prevented virus release, as measured
by accumulation of EBV viral DNA in supernatants, in both
unstressed and stressed conditions, while minimally affecting the
TG-induced appearance of gp350 on the LCL cell surface. These
results strongly suggest that SAL inhibits a key event in viral
budding and egress. Further EM experiments should clarify the
location of the SAL block to viral assembly and/or exit. In B95-8
cells, SAL prevented lytic replication stimulated by TPA, almost
completely inhibiting gp350 expression, a result that differed from
the inhibition by SAL of ER-stress induced lytic induction in
human LCLs, in which little effect of SAL was observed on gp350
expression. These experiments suggest ER-stress–dependent and
-independent mechanisms of SAL antiviral activity.
The production of viral RNA and viral protein are both potent
stimuli for initiating the UPR via both PERK and PKR activation.
Both kinases phosphorylate eIF2␣ to arrest m7GTP cap-dependent
translation. Interestingly, both TG and SAL resulted in eIF2␣
phosphorylation in LCLs; however, their effects were uncoupled at
the level of EBV lytic gene activation. Whereas TG alone was able
to activate lytic gene expression, SAL alone had no effect. Despite
equivalent effects on eIF2a phosphorylation, TG resulted in
activation of ATF transcription factors, whereas SAL did not.
Further, we were unable to reproducibly recapitulate ER stress
effects on EBV lytic gene activation by overexpressing a constitutively active mutant of eIF2␣ (S51D) in transient transfection
experiments in LCLs (data not shown). These data indicate that
eIF2a is not a control point for lytic replication.
Despite previous reports implicating the ER stress- and
plasma cell–specifying transcription factor XBP1 in Z promoter
activation, extremely weak XBP1 splicing by TG compared with
other cell types was seen (data not shown), suggesting a relative
resistance to this arm of the UPR in LCLs. Our previous data
indicated that the typical UPR response is blunted in LCLs, with
limited XBP1(s) production and ATF6 N-terminal cleavage that
increased when cells were stressed in the absence of EBNA3C.22
Despite these effects, TG nevertheless induced lytic replication.
However, such a robust induction of the UPR by TG may not
reflect physiologic UPR induction, and latent gene expression
(including EBNA3C) may be sufficient to limit UPR-induced
lytic replication in vivo.
Differentiation to plasma cell phenotype has been implicated in
EBV lytic replication; however, the TG-induced changes in the
levels of BLIMP1, BCL6, and CD138 mRNAs did not support a
role for UPR lytic induction mediated by plasma cell differentiation
in our assays. Furthermore, high levels of EBV lytic gene activity
were associated with repression rather than activation of plasma
cell–associated genes. T24, T72, TS24, and TS72 treatments led to
at least a 2-fold down-regulation of multiple probe sets derived
from immunoglobulin ␬ and light chains, as well as 1 gene for the
essential plasma cell ER protein 1 (pERp1; supplemental Tables 3
and 4 and data not shown).23
ER stress induction resulted in LMP1 mRNA and protein
expression and the mRNA for the chemokine IL-8, which is
induced by ER stress in an LMP1-dependent manner in nasopharyngeal carcinoma cell lines.12 Because IL8 mRNA was increased as
early as 3 hours after TG exposure, a time point before maximal
LMP1 induction, IL-8 regulation is most likely independent of
LMP1 in LCLs undergoing ER stress. IL-8 (CXCL8) is a CXC-type
cytokine that is mostly produced by macrophages and epithelial
and endothelial cells; however, IL-8 receptor expression has been
recorded on CD19-positive B cells, resulting in enhanced chemotaxis.24 IL-8 released from B lymphocytes undergoing ER stress
in the oropharynx of patients with acute infectious mononucleosis
may result in enhanced B-cell chemotaxis to the oropharyngeal
epithelium with release of virus.25,26
The type of prolonged ER stress applied in our experiments is
likely to have resulted in autophagy, as indicated by the formation
of double-membrane vesicles by EM (Figure 6C arrow). Further
experiments will investigate the roles of autophagy in EBV lytic
replication.
Acknowledgments
The authors thank James Lyons-Weiler and Haiwen Shi from the
University of Pittsburgh genomics and proteomics core laboratories for their assistance with Affymetrix interpretation. They also
gratefully acknowledge the assistance of Donna Beer-Stolz, University of Pittsburgh Center for Biologic Imaging, who performed the
EM studies.
This work was supported by a Physician-Scientist Early Career
Grant (57006750) from the Howard Hughes Medical Institute
startup and by competitive medical research foundation funding
from the University of Pittsburgh (to A.R.).
Authorship
Contribution: G.M.T. and S.K.R. performed the experiments and
prepared the figures; A.R. and G.M.T. formulated the hypotheses
and devised the experiments; D.T.R. provided helpful suggestions,
cell lines, and reagents; R.M.W. performed the EBV DNA quantitation; and A.R. wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Adam Rosendorff, Rangos Research Center,
3rd Floor Pathology, 530 45th St, Pittsburgh, PA 15224; e-mail:
[email protected].
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2011 118: 5528-5539
doi:10.1182/blood-2011-04-347112 originally published
online August 17, 2011
Endoplasmic reticulum stress causes EBV lytic replication
Gwen Marie Taylor, Sandeep K. Raghuwanshi, David T. Rowe, Robert M. Wadowsky and Adam
Rosendorff
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