JOURNAL OF VIROLOGY, Sept. 2010, p. 8446–8459
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 17
Coxsackievirus B3 Infection Activates the Unfolded Protein Response
and Induces Apoptosis through Downregulation of p58IPKand
Activation of CHOP and SREBP1?
Huifang M. Zhang,1Xin Ye,1Yue Su,1Ji Yuan,1Zhen Liu,1
David A. Stein,2and Decheng Yang1*
Department of Pathology and Laboratory Medicine, University of British Columbia, The Providence Heart and Lung Institute,
St. Paul’s Hospital, Vancouver, British Columbia, Canada,1and Vaccine and Gene Therapy Institute,
Oregon Health and Science University, Beaverton, Oregon 970062
Received 9 July 2009/Accepted 7 June 2010
Cardiomyocyte apoptosis is a hallmark of coxsackievirus B3 (CVB3)-induced myocarditis. We used car-
diomyocytes and HeLa cells to explore the cellular response to CVB3 infection, with a focus on pathways
leading to apoptosis. CVB3 infection triggered endoplasmic reticulum (ER) stress and differentially regulated
the three arms of the unfolded protein response (UPR) initiated by the proximal ER stress sensors ATF6a
(activating transcription factor 6a), IRE1-XBP1 (X box binding protein 1), and PERK (PKR-like ER protein
kinase). Upon CVB3 infection, glucose-regulated protein 78 expression was upregulated, and in turn ATF6a
and XBP1 were activated via protein cleavage and mRNA splicing, respectively. UPR activity was further
confirmed by the enhanced expression of UPR target genes ERdj4 and EDEM1. Surprisingly, another UPR-
associated gene, p58IPK, which often is upregulated during infections with other types of viruses, was down-
regulated at both mRNA and protein levels after CVB3 infection. These findings were observed similarly for
uninfected Tet-On HeLa cells induced to overexpress ATF6a or XBP1. In exploring potential connections
between the three UPR pathways, we found that the ATF6a-induced downregulation of p58IPKwas associated
with the activation of PKR (PERK) and the phosphorylation of eIF2?, suggesting that p58IPK, a negative
regulator of PERK and PKR, mediates cross-talk between the ATF6a/IRE1-XBP1 and PERK arms. Finally, we
found that CVB3 infection eventually produced the induction of the proapoptoic transcription factor CHOP
and the activation of SREBP1 and caspase-12. Taken together, these data suggest that CVB3 infection activates
UPR pathways and induces ER stress-mediated apoptosis through the suppression of P58IPKand induction/
activation of CHOP, SREBP1, and caspase-12.
Coxsackievirus B3 (CVB3) is a single-stranded positive-
sense RNA virus of the genus Enterovirus in the family Picor-
naviridae and can cause acute or chronic viral myocarditis.
Epidemiological studies reveal that viral myocarditis is one of
the major heart diseases worldwide, particularly in infants,
children, and adolescents (17). Further, CVB3-induced myo-
carditis can result in dilated cardiomyopathy, a condition for
which the only treatment is heart transplantation (12). CVB3
infection has been studied for decades in various systems, but
the mechanisms of pathogenesis underlying CVB3-induced
myocarditis in humans remain poorly defined. Cumulative ev-
idence suggests that both direct viral injury and subsequent
inflammatory responses contribute to the damage of cardiac
myocytes, and that the extent of such damage determines
the severity of late-stage heart dysfunction (10, 35). Previous
studies have documented that apoptosis in cardiomyocytes
can result in damaged myocardial tissue and is a hallmark of
CVB3-induced myocarditis (1, 45). Although it has been
shown that apoptosis facilitates the release of viral progeny
during CVB3 infection (9, 48), the molecular events leading
to apoptosis in CVB3-infected cells have not been well char-
The endoplasmic reticulum (ER) system, a primary site for
protein synthesis and folding, is a major site of signal initiation
and transduction in response to a variety of stimuli, including
virus infections (24, 66). Endogenous imbalances in cells, such
as the overproduction of proteins, the accumulation of mutant
proteins, or the loss of calcium homeostasis, can cause a mal-
function of cellular processes and stress to the ER system (26).
In response to ER stress, a coordinated adaptive program
called the unfolded protein response (UPR) is activated and
serves to minimize the accumulation and aggregation of mis-
folded proteins by increasing the capacity of the ER machinery
to fold proteins correctly and activate the degradation of ab-
errant proteins. The UPR program represents a network of
signal transduction from the ER to various locations within the
cytoplasm and the nucleus, resulting in either the enhancement
of cell survival or the induction of apoptosis (5). Glucose-
regulated protein 78 (GRP78) functions as a master regulator
of the UPR, and its upregulation indicates the activation of the
UPR program (18, 27, 44). Under normal conditions, GRP78
is associated with stress sensor proteins in the ER luminal
domain. Under stress conditions, GRP78 is released and binds
to misfolded proteins, resulting in the activation of stress sen-
The UPR network of interactions is considered to have
* Corresponding author. Mailing address: The iCapture Center,
Heart and Lung Institute, University of British Columbia, St. Paul’s
Hospital, 1081 Burrard St., Vancouver, British Columbia, Canada V6Z
1Y6. Phone: (604) 682-2344, ext. 62872. Fax: (604) 806-9274. E-mail:
?Published ahead of print on 16 June 2010.
three major arms, each activated by a characteristic sensor,
PERK (PKR-like ER protein kinase), IRE1 (inositol-requiring
enzyme 1), and ATF6a (activating transcription factor 6a) (59).
The activation of the PERK pathway results in the phosphor-
ylation of the eukaryotic translation initiation factor 2?
(eIF2?) subunit, leading to translation attenuation (18). PERK
also activates the expression of ATF4, a transcription factor,
leading to an upregulation of the proapoptotic genes CHOP
(c/EBP homologous protein) and GADD34 (growth arrest and
DNA damage-inducible protein-34) (38). IRE1 is a bifunc-
tional ER transmembrane protein with both serine-threonine
kinase and RNase activities (52, 56). Upon activation, IRE1
can remove a 26-nucleotide (nt) intron from unspliced X box
binding protein 1 (XBP1) mRNA (XBP1u) by RNase activity,
resulting in a translational frameshift. The spliced form of
XBP1 mRNA (XBP1s) encodes a protein with a novel C ter-
minus and acts as a potent transcriptional activator of many
genes involved in the UPR (42). ATF6a is an ER transmem-
brane protein residing in the cytosol under normal physiolog-
ical conditions. Upon the accumulation of misfolded proteins
in the ER, ATF6a migrates to the Golgi apparatus, where it is
cleaved by S1P and S2P proteases, releasing a soluble fragment
that enters the nucleus and activates the transcription of ER
chaperones and other genes responsible for correct protein
A number of viruses have been shown to trigger ER stress
upon infection. However, the pattern of molecular interactions
that occurs within the UPR program differs depending on virus
identity and type of host cell. Many viruses apparently activate
only one or a subset of UPR pathways, and interestingly, some
viral infections activate one pathway yet suppress others. For
example, the expression of hepatitis C virus (HCV) proteins
activates the PERK- and ATF6a-initiated pathways (4, 8, 40)
yet suppresses the IRE1-XBP1 pathway (51). Similarly, human
cytomegalovirus (CMV) activates PERK and IRE1-XBP1 but
suppresses the ATF6a pathway (23, 53).
In this study, with the use of mouse cardiomyocytes and
unmodified HeLa cells, as well as HeLa cell lines engineered to
inducibly express genes integral to the UPR, we focus on the
mechanisms of linkage between the ER stress response to
CVB3 infection and the induction of apoptosis. We found that
CVB3 infection activates ER stress effectors and differentially
regulates the three arms of the UPR. CVB3 infection pro-
duced a downregulation of p58IPKand associated enhance-
ment of PKR (PERK) phosphorylation activity, and these
alterations affected the other two arms of the UPR. Subse-
quently, the proapoptotic proteins CHOP, SREBP1 (sterol
regulatory element binding protein 1), and caspase-12 can be-
come induced. Taken together, these activities appear to par-
ticipate in a coordinated shifting of the ER stress response to
an apoptotic program in CVB3-infected cells.
MATERIALS AND METHODS
Virus, cells, siRNAs, plasmids, and transfections. CVB3 (CG strain) was
obtained from Charles Gauntt (University of Texas Health Science Center) and
propagated in HeLa cells (ATCC). Virus stock was isolated from cells by three
freeze-thaw cycles followed by centrifugation to remove cell debris and was
stored at ?80°C. The titer of virus stock was determined by plaque assay as
described previously (67) and below. HeLa cells were grown in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 100 ?g/ml penicillin, 100
?g/ml streptomycin, 2 mM glutamine, and 10% fetal bovine serum (FBS) (Clon-
tech). The HL-1 cell line, a mouse cardiac muscle cell line established from a
cardiomyocyte tumor lineage, was a gift from William C. Claycomb (Louisiana
State University Health Science Center). HL-1 cells were maintained in Clay-
comb medium (11) supplemented with 10% FBS (JRH Biosciences), 100 ?g of
penicillin-streptomycin/ml, 0.1 mM norepinephrine (Sigma), and 2 mM L-
glutamine (Invitrogen). The individual short interfering RNAs (siRNAs)
targeting human or mouse XBP1 and human ATF6a and GRP78 were pur-
chased from Santa Cruz Biotech and transfected according to the manufac-
turer’s instructions. Briefly, 2 ? 105HeLa cells were grown at 37°C overnight
to 60 to 70% confluence in 6-well plates, washed with phosphate-buffered
saline (PBS), and overlaid for 24 h with transfection complex containing
siRNA and Oligofectamine (Invitrogen). The transfection medium then was
replaced with DMEM containing 10% FBS, and the incubation was continued
for 48 h. The transfection of plasmid pcDNA1/neo-p58IPK(a gift from Mi-
chael Katze, University of Washington) and pcDNA3.1-ATF6(171-373) (a
gift from K. Mori, Kyoto University, Kyoto, Japan) into HeLa cells was
conducted by the same procedures as those for siRNA described above,
except Lipofectamine (Invitrogen) was the transfection reagent.
Construction of pTRE-HA-XBP1u-GFP and pTRE-HA-ATF6a and production
of double-stable Tet-On/HeLa cell lines. cDNA of XBP1u fused with GFP
(green fluorescence protein) was amplified from the plasmid pHA-XBP1u-
GFP (a gift from Yi-Ling Lin, Academia Sinica, Taiwan, Republic of China)
by PCR using primers listed in Table 1. The fragments were digested by
HindIII and XbaI and ligated into the pTRE-HA vector (Clontech). The
recombinant plasmid was confirmed by DNA sequencing. The full-length
cDNA of ATF6a was amplified by PCR from the plasmid pCMV-ATF6a
(OriGene, Rockville, MD) using primers listed in Table 1. The pTRE-HA-
ATF6a expression plasmid was constructed by using the same strategy as that
for constructing pTRE-HA-XBP1u-GFP.
The double-stable Tet-On cell line inducibly expressing ATF6a or XBP1 was
established by plasmid transfection. Briefly, 105Tet-On HeLa cells were cotrans-
fected with either pTRE/HA-ATF6a or pTRE/HA-XBP1u-GFP, along with
pTK-Hyg (encoding hygromycin resistance), using Lipofectamine (Invitrogen)
according to the manufacturer’s instructions. After 8 h of incubation with 4 ?g of
plasmid and Lipofectamine, the mixture was replaced with DMEM containing
10% FBS and hygromycin or G418 at a concentration of 200 or 400 ?g/ml,
respectively, and incubation was continued for 2 days before the initial se-
lection of double-stable cells. Doubly resistant clones were picked 3 weeks
later and further screened for ATF6a or XBP1 protein expression by Western
blotting using the hemagglutinin (HA) antibody (Covance). Cells transfected
with the empty vectors pTRE and pTK-Hyg also were selected by hygromycin
and G418 in parallel for use as controls. To induce the expression of ATF6a
or XBP1, cells were cultured for 12 to 72 h in medium containing 1 ?g/ml
RNA preparation and RT-PCR analysis. Total RNA from cultured cells was
isolated with the RNeasy Mini kit (Qiagen) by following the supplier’s instruc-
tions. First-strand cDNAs were reverse transcribed (RT) with 3 ?g of RNA as
the template, and the target genes were amplified by PCR by following the
procedures described in the one-step RT-PCR kit (Qiagen), with minor modi-
fications. Briefly, 2 ?g of first-strand cDNA and 100 ng of each primer (Table 1)
were used for PCR under standard conditions for 30 cycles, a point confirmed as
falling within the exponential phase of amplification.
Western blot analysis. Western immunoblotting was performed by standard
protocols as previously described (32). Briefly, cells were washed with cold
PBS before the addition of an appropriate volume of lysis buffer (0.025 M
Tris-HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 1 mM EDTA, 1 mM EGTA,
1% Triton X-100, and proteinase inhibitor cocktail). After incubation for 20
min on ice, the cell lysates were centrifuged at 13,000 ? g for 15 min at 4°C,
and protein-containing supernatant was collected. For protein isolation, the
NE-PER nuclear and cytoplasmic extraction reagents (Pierce) were employed
per the manufacturer’s instructions. The isolated proteins were separated by
10% SDS-PAGE and transferred onto nitrocellulose membranes. Mem-
branes were blocked with 5% skim milk in PBS and incubated with one of the
following primary antibodies: monoclonal anti-human GRP78 (Transduction
Laboratory), monoclonal anti-mouse actin (Sigma), monoclonal mouse anti-
HA.11 (Covance), monoclonal mouse anti-VP1 (Novocastra), rabbit poly-
clonal anti-human caspase-7, polyclonal anti-human p-eIF2?, monoclonal
anti-human p58IPKand polyclonal anti-mouse p-PERK (Cell Signaling),
monoclonal anti-mouse caspase-12, polyclonal anti-human p-PKR, polyclonal
anti-mouse ATF6a, polyclonal anti-mouse XBP1, polyclonal anti-human
SREBP1, monoclonal anti-human caspase-3, monoclonal anti-human CHOP,
and polyclonal anti-mouse histone H1 (Santa Cruz). After several washes
with PBS, each blot was further incubated with an appropriate secondary
VOL. 84, 2010 CVB3-INDUCED ER STRESS AND APOPTOSIS8447
antibody (goat anti-mouse or donkey anti-rabbit) conjugated to horseradish
peroxidase (Amersham). Detection was carried out by enhanced chemilumi-
nescence (Amersham) and the manufacturer’s instructions.
Cell viability assay. To determine the effect of ATF6a expression on cell
viability, HeLa cells were left uninduced or were induced with Dox for 72 h and
then infected with CVB3 at a multiplicity of infection (MOI) of 10 for 10 h. Cell
viability was analyzed with a 3-(4-5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-
phenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay kit (Promega) by fol-
lowing the manufacturer’s instructions. Briefly, cells were incubated with MTS
solution for 4 h, after which the absorbance of formazon at 492 nm was measured
using an enzyme-linked immunosorbent assay (ELISA) plate reader. The absor-
bance of sham-infected/not induced cells was defined as 100% survival (control),
and the remaining data were converted to a percentage of the control.
Cell immunofluorescence assay. HeLa cells grown in 6-well plates to 80%
confluence were transfected with plasmid pHA-XBP1u-GFP for 24 h and then
infected with CVB3 at an MOI of 10 for 1 h by following methods described
previously (68). After 8 h of incubation in fresh growth medium, the cells were
washed with PBS and fixed with 2% paraformaldehyde. Cell morphology and
GFP expression were observed and photographed under an inverted fluorescent
Viral plaque assay. Viral titers were determined by plaque assay as described
previously (67). Briefly, samples of cells and medium from plates receiving the
various treatments were freeze-thawed and then centrifuged (4,000 ? g) to
isolate viruses. HeLa cells were seeded into 6-well plates (8 ? 105cells/well) and
incubated at 37°C for 20 h to a confluence of approximately 90% and then
washed with PBS and overlaid with 500 ?l of virus-containing samples serially
diluted in cell culture medium. After a viral adsorption period of 60 min at 37°C,
the supernatant was removed and the cells overlaid with 2 ml of sterilized soft
Bacto-agar-minimal essential medium, cultured at 37°C for 72 h, fixed with
Carnoy’s fixative for 30 min, and stained with 1% crystal violet. The plaques were
counted and viral PFU per ml calculated.
Statistical analysis. The Student’s t test was employed to analyze data. The
results are expressed as means ? standard deviations (SD) of three indepen-
dent experiments. A P value of less than 0.05 was considered statistically
CVB3 infection induces ER stress, and silencing of GRP78
sensitizes cells to virus-induced death yet inhibits viral protein
production. GRP78 upregulation is a major indicator of ER
stress. To study the effect of CVB3 infection on ER homeosta-
sis, we first determined the expression of GRP78 in both HeLa
cells and HL-1 cardiomyocytes at various time points postin-
fection (pi). Figure 1A and B show that GRP78 expression
gradually increased and reached a high level at 12 h pi in both
cell lines, although the upregulation of GRP78 expression was
more robust in HeLa cells than in HL-1 cells. The upregulation
of GRP78 indicates that CVB3 infection induces ER stress.
As the activation of ER stress response pathways is a pro-
tective mechanism against aberrant protein production, we
were interested to explore the effect of GRP78 silencing on
CVB3 replication and CVB3-induced cell death. Figure 1C
shows higher cell death in CVB3-infeceted cells transfected
with GRP78 siRNA (image d) than in infected cells transfected
with a negative control (scrambled sequence) siRNA (image b)
or in sham-infected cells transfected with GRP78 siRNA (im-
age c). This result indicates that reduced GRP78 expression
sensitizes the cells to CVB3-induced cell death, and this was
further confirmed by the decreased viability of cells treated
with GRP78 siRNA compared to that of cells treated with
scrambled siRNA (Fig. 1D). In CVB3-infected cells, the in-
creased activation of caspase-3, along with reduced viral rep-
lication, as indicated by the relative levels of the major CVB3
structural protein VP1, were apparent in GRP78 siRNA-
TABLE 1. Primer sequences used for RT-PCR to detect gene expression and for cloning to construct Tet-On plasmids
Target geneDirectionPrimer sequence (5? to 3?) Application
XBP1 (human) Forward
ERdj 4 (human) Forward
ERdj 4 (mouse)Forward
8448ZHANG ET AL. J. VIROL.
treated cells but not in those treated with scrambled siRNA
CVB3 infection activates the XBP1 pathway but suppresses
p58IPKexpression. It has been established that under ER
stress conditions, IRE1 can become activated and direct the
splicing of a 26-nucleotide (nt) intron from XBP1u, resulting in
a translational frameshift of XBP1 mRNA (7). The gene prod-
uct of XBP1s acts as a potent transcriptional activator of sev-
eral genes involved in the UPR. To determine whether CVB3
infection can trigger XBP1 mRNA splicing, we employed the
reporter plasmid pHA-XBP1u-GFP. This plasmid encodes an
N-terminal HA-tagged XBP1u, with the C terminus fused to
GFP at the second open reading frame (ORF) of XBP1u. The
GFP is expressed only after XBP1u is spliced, thereby remov-
ing a 26-nt intron containing a PstI site (66). The confirmation
of GFP expression was performed by fluorescence microscopy
after the CVB3 infection of reporter plasmid-transfected cells.
Figure 2A shows that GFP signal was apparent in both CVB3-
infected HeLa cells and tunicamycin (Tu)-treated control cells
transfected with reporter plasmids, but not in sham-infected or
vector-transfected cells, indicating that XBP1 was correctly
spliced. XBP1 splicing was further confirmed by RT-PCR de-
tecting the XBP1u and XBP1s forms, as well as by the expres-
sion of downstream genes responsive to XBP1s. The data in
Fig. 2B indicate the splicing of the PstI-containing intron, as
the digestion of the PCR product with PstI did not produce the
FIG. 1. CVB3 infection upregulates GRP78 expression, and the silencing of GRP78 enhances CVB3-induced cell death and reduces VP1
synthesis. Mouse cardiomyocyte HL-1 cells (A) and HeLa cells (B) were infected with CVB3 at an MOI of 100 and 10, respectively. The cell lysates
were prepared at the indicated time points pi and subjected to Western blot analysis using the indicated antibodies. HeLa cells treated with
tunicamycin (Tu) (2 mg/ml) to induce ER stress were included as a positive control. Actin is detected as a loading control. HeLa cells were
transfected with GRP78 siRNA or scrambled (Scr) siRNA and then infected with CVB3. At 12 h pi, cell morphology was observed by
phase-contrast microscopy (C), and cell viability was measured by MTS assay and converted to a percentage of a control receiving no siRNA
transfection and sham infection (D). Error bars represent means ? SD. P ? 0.05. (E) Relative levels of CVB3 VP1 protein and procaspase-3
cleavage were detected by Western blot analysis.
VOL. 84, 2010CVB3-INDUCED ER STRESS AND APOPTOSIS 8449
two fragments (291 and 307 nt) characteristic of unspliced
RNA isolated from HeLa and HL-1 cells. The spliced XBP1s
gene product further upregulated the expression of its target
genes ERdj4 (ER-localized DnaJ4) and EDEM1 (ER degra-
dation-enhancing ?-mannosidase-like protein). To our sur-
prise, p58IPK, a cellular inhibitor of the eIF2? kinases PKR and
PERK, which is often upregulated in association with the
UPR, was downregulated in the setting of CVB3 infection.
Silencing XBP1 expression enhances CVB3-induced cell
death and inhibits CVB3 VP1 synthesis. To further explore the
function of XBP1 in the UPR, the effect of reduced XBP1
expression on cell survival and viral replication was investi-
gated. The siRNA targeting of XBP1 significantly reduced the
level of XBP1 mRNA and that of its downstream target genes
EDEM1 and ERdj4 (Fig. 3A). In addition, HeLa cell death
induced by CVB3 infection was greatly enhanced in XBP1
siRNA-transfected cells (Fig. 3B, image d) compared to that of
controls (Fig. 3B, images b and c). The enhanced death of
infected cells containing lowered levels of XBP1 mRNA was
confirmed by cell viability assays (Fig. 3C), increased pro-
caspase-3 cleavage (Fig. 3D), and decreased CVB3 VP1 pro-
tein synthesis (Fig. 3E).
Overexpression of XBP1 enhances UPR and promotes
CVB3 VP1 protein synthesis. To further semiquantitatively
determine the effect of XBP1 expression levels on cell survival
and CVB3 replication efficiency, we established a Tet-On/
FIG. 2. CVB3 infection induces XBP1 mRNA splicing and alters the expression of its responsive genes. (A) Demonstration of XBP1 splicing
by a GFP-based reporter in HeLa cells. Cells were transfected with pHA-XBP1u-GFP and then infected with CVB3, or sham infected with
DMEM, or treated only with Tu at a final concentration of 2 mg/ml. Vector-transfected/CVB3-infected cells were included as an additional control.
Reporter expression after XBP1 splicing was determined by fluorescent microscopy. (B) RT-PCR analysis. Both HeLa cells and HL-1 cells were
infected with CVB3 or sham infected with DMEM. Total cellular RNAs were prepared at the indicated time points pi, and RT-PCRs were
performed using specific primers to determine the level of XBP1 splicing and the expression of each indicated target gene. For detecting XBP1
splicing, PCR products were digested with PstI and electrophoresed. The XBP1u (291/307 bp) and XBP1 (572 bp) bands are indicated.
8450 ZHANG ET AL. J. VIROL.
XBP1-inducible cell line to examine the effect of XBP1 expres-
sion on the upregulation of ER chaperone in the absence of
CVB3 infection. Figure 4A shows that XBP1 expression was
increased beginning at 12 h after Dox induction (pdi), and that
this increase coincided with the upregulation of GRP78 and
downregulation of p58IPK. Further, the downregulation of
p58IPKcorrelated with the increased phosphorylation of PKR
and eIF2? (Fig. 4A). Second, we examined the effect of XBP1
expression on CVB3 replication at different time points after
infection. Tet-On/XBP1 HeLa cells were induced for 72 h with
Dox to promote XBP1 expression and then infected with
CVB3. VP1 protein synthesis, viral plaque formation, and cell
viability were evaluated at several time points pi. We found
that the expression of GRP78 and CVB3 VP1 were signifi-
cantly enhanced 6 h pi in Dox-induced cells compared to that
in uninduced cells at the corresponding time points (Fig. 4B).
However, CVB3 plaque formation data exhibited a converse
pattern compared to that observed in the VP1 production data,
with Dox-induced cells producing less infectious virus than
uninduced cells (Fig. 4C). We speculate that this may be be-
cause XBP1 expression suppresses a later stage of the CVB3
life cycle, such as particle assembly, resulting in a decrease in
infectious virus production. This speculation is further sup-
ported, at least in part, by cell viability data (Fig. 4D) showing
that cells induced to express XBP1 and then infected with
CVB3 have higher viability than uninduced and infected cells.
Such higher viability may result from XBP1 expression sup-
pressing CVB3 particle formation and thereby benefiting the
health of host cells.
CVB3 infection activates the ATF6a pathway. In response to
ER stress, ATF6a is cleaved in the Golgi apparatus by trans-
membrane proteases and translocated to the nucleus, where it
activates genes responsible for UPR (20, 47). To assess
whether CVB3 infection activates the ATF6a pathway, ATF6a
cleavage was evaluated by the Western blot analysis of HeLa
and HL-1 cell lysates prepared at various time points after
FIG. 3. Silencing of XBP1 enhances CVB3-induced cell death and reduces VP1 synthesis. (A) HeLa cells at 90% confluence were transfected
with XBP1 siRNA or scrambled siRNA, and the expression of XBP1 as well as its responsive genes was determined by RT-PCR using primers listed
in Table 1. siRNA-transfected HeLa cells were infected with CVB3 or sham infected with DMEM. (B) Cell morphology changes were observed
by microscopy at 12 h pi. (C) Cell viability was measured by MTS assay and converted to a percentage of the control as described for Fig. 1. Error
bars represent means ? SD. P ? 0.05. In addition, procaspase-3 cleavage (D) and CVB3 VP1 production (E) were detected by Western blot
analysis using the same amount of cell lysate for each time point. Actin was used as a loading control.
VOL. 84, 2010CVB3-INDUCED ER STRESS AND APOPTOSIS 8451
CVB3 infection. Figure 5A shows an intact form of ATF6a (90
kDa) detected in sham-infected cells, while in infected cells the
cleavage of ATF6a, producing a 50-kDa band, was detectable
at 8 h pi and almost complete at 12 h pi, indicating that CVB3
infection activates the ATF6a pathway in the two cell lines
evaluated. To further evaluate the effect of ATF6a activation
on cell viability and viral replication, we first used siRNAs
targeting ATF6a and then measured the expression levels of
ATF6a and its target gene XBP1 by Western blotting and
RT-PCR, respectively. As shown in Fig. 5B, ATF6a expression
was strongly reduced by its specific siRNA in uninfected cells.
Cells that had been treated with ATF6a-siRNA were then
infected with CVB3 and evaluated for viability, VP1 protein
levels, infectious virus production, and changes in morphology.
Figure 5C shows that, at each time point tested, VP1 protein
synthesis was reduced in cells transfected with ATF6a siRNA
compared to cells transfected with scrambled siRNA. Figure
5D shows that more cell death was observed in infected cells
transfected with ATF6a siRNA (image d) than in controls
(images b and c). This result was confirmed by the decreased
cell viability (Fig. 5E) and increased cleavage of procaspase-3
(Fig. 5F) observed in ATF6a-siRNA treated cells compared to
that in controls. We note that, as found in the experiments
producing Fig. 1E and 3D, the cleavage product of pro-
caspase-3 was difficult to detect by immunoblotting. It may
be that the siRNA silencing of target genes contributed to
CVB3-induced proteosome-mediated degradation of the
procaspase form, precluding its detection (33, 50). To con-
firm the occurrence of cleavage, we performed additional
Western blot analysis on 17% acrylamide resolving gels,
loading 80 ?g of total protein (instead of the usual 30 ?g),
and then were better able to detect the cleaved form (17 to
19 kDa) of caspase-3 (Fig. 5G).
Overexpression of ATF6a upregulates XBP1 and alters
chaperone expression. To further determine whether tran-
scription factor ATF6a regulates XBP1 and the expression of
other downstream genes, we established a Tet-On/HA-ATF6a
cell line that expresses ATF6a upon induction with Dox. To
FIG. 4. Overexpression of XBP1 differentially alters target gene expression in the absence or presence of CVB3 infection. (A) Overexpression
of XBP1 upregulates GRP78 but downregulates p58IPKin the absence of CVB3 infection. Tet-On/HA-XBP1 HeLa cells were induced (?Dox) or
not induced (?Dox) for XBP1 expression. Cell lysates were subjected to Western blot analysis to detect XBP1 and its target genes, GRP78 and
p58IPK. (B) Overexpression of XBP1 promotes CVB3 VP1 protein synthesis. Tet-On/HA-XBP1 HeLa cells were left uninduced or were induced
with Dox and then infected with CVB3. At the indicated time points pi, cell lysates were prepared for Western blot analyses of CVB3 VP1 and
GRP78 proteins. Actin expression was detected in parallel as a loading control. (C) Viral plaque assay. CVB3 titer was determined at 12 h pi. An
uninduced sample was included as a control. (D) Cell viability assay. An MTS assay was performed on the cells described above at 12 h pi. The
data are presented as percentages of the uninduced/uninfected control. Error bars represent means ? SD. P ? 0.05.
8452 ZHANG ET AL.J. VIROL.
ensure that the overexpressed HA-ATF6a protein was func-
tional, we performed Western blot analysis to detect the acti-
vated form of ATF6a and found that the p50 cleavage product
was present in Dox-induced/CVB3-infected cells but not in the
three controls, including noninduced/CVB3-infected cells,
noninduced/sham-infected cells, and Dox-induced/sham-in-
fected cells, that were incubated for 10 h (Fig. 6A). Since
AFT6a is an ER membrane protein, we wanted to rule out the
possibility that the activation of the UPR was nonspecifically
generated by the overexpression of an ER membrane protein
in our experimental system. We therefore detected GRP78
expression levels in HeLa cells transfected with a plasmid
pcDNA3.1-ATF6(171-373) overexpressing a dominant-nega-
tive (DN) ATF6a and found that the overexpression of DN-
ATF6a decreased GRP78 production compared to that of the
controls (Fig. 6B). These data indicate that the overexpressed
HA-ATF6a is functional and can specifically induce UPR.
With the Tet-On/HA-ATF6a cell line, we first evaluated
whether ATF6a overexpression could induce the activation of
ER chaperones in the absence of CVB3 infection. As shown in
Fig. 6C, ATF6a expression was dramatically increased by 12 h
pdi. The increased expression of ATF6a, like the increased
expression of XBP1 shown in Fig. 2 and 4, induced an overall
upregulation of ER chaperone GRP78, although a transient
downregulation at 24 to 48 h pdi was observed. The increased
ATF6a expression also apparently induced the downregulation
of p58IPKand increased the phosphorylation of eIF2?. As
expected, the overexpression of ATF6a also upregulated XBP1
FIG. 5. CVB3 infection induces cleavage of ATF6a, and silencing of ATF6a enhances CVB3-caused cell death and reduces VP1 synthesis. Both
HL-1 and HeLa cells were cultured and infected with CVB3 as described for Fig. 1. (A) Cell lysates were prepared and subjected to Western
blotting to determine the pattern of ATF6a cleavage. Histone detection serves as a loading control. (B) HeLa cells were transfected with ATF6a
siRNA or scrambled siRNA (control). The expression of ATF6a was detected by Western blotting and RT-PCR, as indicated. (C) HeLa cells were
infected with CVB3 after siRNA transfection. Cell lysates collected at the indicated time points pi were used to detect VP1 production. (D) Cell
morphology was observed by phase-contrast microscopy, and (E) cell viability was measured by MTS assay. The data are presented as a percentage
of the control (as described for Fig. 1D). Error bars represent means ? SD. P ? 0.05. (F) Western blot of cell lysates collected at 12 h pi, detecting
procaspase-3 cleavage. (G) The cleavage of caspase-3 was further confirmed on high-percentage gels on which 80 ?g of total protein was loaded.
Actin detection serves as a loading control.
VOL. 84, 2010 CVB3-INDUCED ER STRESS AND APOPTOSIS8453
by 12 h pdi, further confirming that XBP1 is inducible by
ATF6a. These data support previous reports that ATF6a can
induce XBP1 expression, and that the IRE1 and ATF6a path-
ways interact with each other and functionally intersect at
XBP1 (31, 63). Note that XBP1 protein levels declined at 48 h
pdi, which may be due, as reported for XBP1u (64), to a
negative regulation targeting the XBP1 protein for protea-
ATF6a overexpression benefits CVB3 replication. CVB3 in-
fection induces ATF6a activation (i.e., cleavage) and initiates
the UPR. The UPR affects cell fate by either inducing adap-
tation to ER stress or by inducing apoptosis (29). CVB3 infec-
tion is known to be persistent in the heart (2, 25), implying the
sustained activation of ATF6a in cardiomyocytes. To semi-
quantitatively evaluate the effect of ATF6a expression levels
on cell survival and CVB3 replication efficiency, we compared
uninduced and induced Tet-On/HA-ATF6a cells at various
time points after infection with CVB3. We then evaluated the
effect of ATF6a overexpression on (i) CVB3 replication by the
Western blot analysis of VP1, (ii) infectious viral particle for-
mation by plaque assay, and (iii) cell viability by MTS assay.
The expression of GRP78 and CVB3 VP1 was significantly
enhanced from 6 to 10 h pi in cells induced for ATF6a over-
expression compared to that of uninduced cells at correspond-
ing time points (Fig. 7A). Enhanced CVB3 VP1 production at
12 h pi was further confirmed by viral plaque assay (Fig. 7B).
Finally, Fig. 7C shows that during CVB3 infection, the induc-
tion of ATF6a decreased cell viability.
CVB3-induced suppression of p58IPKcorrelates with up-
regulation of PKR and phosphorylation of eIF2?. Since the
CVB3-activated IRE1-XBP1 pathway and the overexpression
of ATF6a was associated with the downregulation of p58IPK, a
negative regulator of eIF2? kinases PKR and PERK, we next
examined if CVB3 infection also could activate PERK or PKR
and increase the phosphorylation of eIF2?. HeLa cells were
infected with CVB3, and total proteins were analyzed by West-
ern blotting. As shown in Fig. 8A, p58IPKwas downregulated,
and by 6 h pi the phosphorylation of PKR (p-PKR) had in-
creased. However, we failed to detect an increase in the phos-
phorylation of PERK (data not shown). Phosphorylated eIF2?
(p-eIF2?) production was upregulated at each time point pi.
Overexpression of p58IPKbenefits CVB3 replication. p58IPK
is a cochaperone in the ER stress response (43). Its upregula-
tion enhances cell survival and, in turn, allows increased viral
replication (16). To explore this circuitry, we used the
pcDNA1/Neo-p58IPKplasmid to establish a stable HeLa cell
line, and we prepared lysates from cells at different time points
pi. The level of p58IPKexpression and its effect on the phos-
phorylation of PKR and eIF2? and on the production of CVB3
VP1 protein were evaluated by Western blot analysis. In addi-
tion, the resulting viral titers were measured by plaque assay.
Figure 8B shows that p58IPKoverexpression counteracted the
CVB3-induced downregulation of p58IPKand produced an
overall net increase in p58IPKproduction, decreased the phos-
phorylation of PKR and eIF2?, and increased the synthesis of
CVB3 VP1 protein compared to that of a control line of stable
cells made by vector-only transfection. Enhanced CVB3 repli-
cation was further confirmed by the increased viral titer in the
p58IPKstable cells (Fig. 8C).
CVB3 infection induces upregulation of proapoptotic CHOP
and activation of SREBP1, caspase-7, and caspase-12. To an-
alyze the ER stress-mediated signal transduction pathway lead-
ing to cell apoptosis, we evaluated downstream proapoptotic
gene expression in HeLa and HL-1 cells after CVB3 infection.
In HeLa cells, CHOP was upregulated, and another ER-asso-
FIG. 6. Overexpression of ATF6a alters target gene expression in
the absence of CVB3 infection. (A) CVB3-induced cleavage of HA-
ATF6a. Tet-On/HA-ATF6a cells were left uninduced or were induced
with Dox and then infected with CVB3 or sham infected with PBS. At
the indicated time points pi, the functional form of ATF6a (p50) was
detected by Western blot analysis. Actin expression was used as a
loading control. (B) The specific induction of the UPR in Dox-induced
Tet-On/HA-ATF6a cells. HeLa cells were transfected with either the
plasmid pcDNA3.1-ATF6(171-373), which overexpresses a dominant-
negative (DN) ATF6a, or with the pcDNA3.1 vector only (as a con-
trol), and GRP78 expression was detected by Western blot analysis at
48 h posttransfection. GRP78 detected in Tet-On/ATF6a cells after
Dox induction was included as an additional control. (C) Overexpres-
sion of ATF6a upregulates XBP1 and alters UPR-responsive gene
expression in the absence of CVB3 infection. Tet-On/HA-ATF6a cells
were induced or not induced for ATF6a expression. Cell lysates were
subjected to Western blot analysis to detect the expression levels of
ATF6a and other UPR-related genes using the indicated antibodies.
8454ZHANG ET AL. J. VIROL.
ciated transcription factor, SREBP1, an ER stress marker and
proapoptotic protein (15), was activated (Fig. 9A). Full-length
SREBP1 (125 kDa) was cleaved to produce an ?60-kDa acti-
vated product as early as 4 h pi. We also observed some bands
with molecular sizes greater than 60 kDa, which likely are
nonspecific products, as they did not show an increase in in-
tensity over time, as the SREBP1 cleavage product did. Finally,
we detected activated caspase-12, a key marker of ER stress-
mediated apoptosis (36), in CVB3-infected HL-1 cells (Fig.
9B). It was not possible to investigate caspase-12 activation in
HeLa cells, as no human orthologue of mouse caspase-12 has
been clearly identified (14). The activation of caspase-12 in
HL-1 cells was further indicated by the activation of caspase-7,
an upstream activator of caspase-12 (41) (Fig. 9B). Activated
caspase-7 also was apparent in infected HeLa cells (Fig. 9C).
The intensity of cleaved caspase-7 product was reduced by 10 h
pi in HL-1 cells, perhaps because of proteasome-mediated
CVB3 is a nonenveloped RNA virus, which replicates rap-
idly in double-layered membrane vesicles derived from the
intracellular membrane system (58). Thus, it is not surprising
that we found evidence that CVB3 infection induces ER stress
response measures. In this study, we explored CVB3-induced
ER stress responses and their effects on the induction of
apoptosis in an attempt to better understand the molecular
pathogenesis of CVB3. We directly examined CVB3-infected
cardiomyocytes and HeLa cells and employed inducible HeLa
cell lines engineered to overexpress the stress response gene
XBP1 or ATF6a. Overall, we found that CVB3 infection in-
duces ER stress and differentially regulates the three arms of
the UPR. Specifically, we observed the upregulation of
GRP78, the master regulator of the UPR, soon after the CVB3
infection of both HeLa cells and cardiomyocytes. Further, the
siRNA silencing of GRP78 enhanced CVB3-induced cell
death, indicating that the activation of the UPR early during
CVB3 infection promotes host cell survival. Following the up-
regulation of GRP78 in CVB3-infected cells, the IRE1-medi-
ated splicing of XBP1u was activated and produced XBP1s. As
in previous reports (28, 30), XBP1s in turn induced the up-
regulation of various genes encoding protein degradation and
folding factors, including EDEM1 and ERdj4. p58IPKis a neg-
ative regulator of PKR and PERK (43, 54) and has been
reported to be present at unchanged or heightened levels in
cells during infection by various viruses, including influenza
virus (43), flaviviruses (66), and mouse hepatitis virus (MHV)
(3). Surprisingly, we observed p58IPKto be downregulated at
both the mRNA and protein levels in CVB-infected HeLa and
HL-1 cells. Furthermore, the induced overexpression of p58IPK
increased CVB3 VP1 protein production, implying that p58IPK
serves to promote overall host cell health and thereby viral
In unperturbed cells, GRP78 is associated with the ER stress
sensor ATF6a. Upon perturbation they typically disassociate,
with ATF6a then invoking the initiation of UPR activities. We
explored ATF6a protein expression in both HeLa and HL-1
cells as well as in a Tet-On HeLa cell line inducibly expressing
ATF6a, and we found that CVB3 infection activated ATF6a,
producing a 50-kDa cleavage product in all three cell types. In
the inducible HeLa cells, we found that the increased expres-
sion of ATF6a was associated with an overall increase in
GRP78 production (Fig. 6C). Further, we observed that
ATF6a pathway activation also regulated other components of
the UPR. For instance, XBP1, the substrate of IRE1 (63), was
upregulated in response to increased ATF6a, in agreement
with previous reports suggesting cross-talk between the IRE1-
XBP1 and ATF6a pathways of the UPR (31, 63).
P58IPKis an ER luminal cochaperone associated with
GRP78 (43). It functions primarily as an inhibitor of the eIF2?
protein kinases PERK and PKR (61), thus facilitating transla-
tion under normal cellular conditions. Recent evidence shows
FIG. 7. ATF6a overexpression benefits CVB3 replication. (A) Tet-
On/HA-ATF6a HeLa cells were induced or not induced for ATF6a
expression and then infected with CVB3. At the indicated time points
pi, cell lysates were prepared for Western blot analysis of CVB3 VP1
and GRP78. Actin was detected in parallel as a loading control. (B) Vi-
ral plaque assay. The CVB3 titer was determined by plaque assay at
12 h pi. A sample of uninduced cells is included as a control. (C) Cell
viability assay. HeLa cells were left uninduced or were induced and
then infected with CVB3. Cell viability was determined by MTS assay
at 12 h pi. The data are presented as percentages of the uninduced,
uninfected control. Error bars represent means ? SD. P ? 0.05.
VOL. 84, 2010 CVB3-INDUCED ER STRESS AND APOPTOSIS8455
that p58IPKhas PERK-independent functions and mediates
the cytosolic degradation of misfolded proteins delayed at the
ER translocon. Thus, it has been suggested that p58IPKhas
multiple functions involved in protecting the cell from ER
stress (39). Here, p58IPKwas downregulated upon ATF6a ac-
tivation in CVB3-infected HeLa cells and in noninfected, Dox-
induced Tet-On/HA-ATF6a cells, further suggesting that
p58IPKis involved in cross-talk among the UPR pathways. Our
data support the notion that the IRE1-XBP1 pathway is reg-
ulated by ATF6a and connected to the PKR (PERK) pathway
via p58IPK. We note that our data showing the downregulation
of p58IPKare inconsistent with a previous report that utilized
uninfected, tunicamycin-treated cell cultures (54). The down-
regulation of p58IPKthat we observed in the Tet-On cell line
may be related to the specific cellular conditions in these cells,
where the induced expression of ATF6a produced the overex-
pression of XBP1u. It has been reported that XBP1u can form
a complex with, and thereby negatively regulate, the UPR-
specific transcription factors ATF6a and XBP1s. Once formed,
the complex can be sequestered from the nucleus and de-
graded by proteasomes (64, 65). Thus, the negatively regulated
ATF6a and XBP1s in the Tet-On cells may result in the down-
regulation of p58IPK. For this reason, the Tet-On cell system,
although useful, may not be as meaningful an experimental
system as nontransfected cells for analyzing the effect of
ATF6a or XBP1 on CVB3 replication.
We speculate that the lowered expression of p58IPKthat we
observed during CVB3 infection facilitates persistent PKR sig-
naling, thereby sustaining the enhanced phosphorylation of
eIF2?. We note also that p-PERK was not significantly up-
regulated during CVB3 infection (data not shown), and we
assume that p-PERK’s role in increasing the phosphorylation
of eIF2? during CVB3 infection is compensated for by upregu-
lated p-PKR. It is known that eIF2? phosphorylation can in-
hibit the translation of eukaryotic cellular mRNAs. However,
during CVB3 infection, we observed enhanced VP1 protein
production under conditions of increased eIF2? phosphoryla-
tion present in cells overexpressing XBP1 or ATF6a. We spec-
ulate that robust CVB3 translation under conditions in which
host translation is inhibited is enabled by the CVB3 internal
ribosome entry site (IRES), which mediates translation initia-
tion in a cap-independent manner (62) and can bypass the
eIF2a-dependent translation block (46, 49). In addition, the
reduction in the cap-dependent translation of cellular mRNAs
FIG. 8. CVB3-induced downregulation of p58IPKactivates PKR-mediated phosphorylation of eIF2?, and overexpression of p58IPKincreases
CVB3 RNA translation. (A) HeLa cells were cultured and infected with CVB3 as described for Fig. 1. Cell lysates were subjected to Western blot
analysis to detect p58IPKand the phosphorylation of PKR and e-IF2?. (B) HeLa cells stably transfected to express p58IPKand vector-transfected
cells (as a control) were infected with CVB3 at an MOI of 10. Cell lysates were used to detect p58IPK, CVB3 VP1, and phosphorylated PKR and
eIF2? at the indicated time point pi. Actin expression is included as a loading control. (C) The results of viral plaque assays measuring viral titers
in infected cell samples collected at 12 h pi are shown.
8456 ZHANG ET AL.J. VIROL.
that occurs during CVB3 infection likely results in the greater
availability of translation machinery for the IRES-driven trans-
lation of viral RNA (13). It has been reported that HCV,
another IRES-containing RNA virus, suppresses cellular
XBP1 trans-activating activity, leading to the elevated transla-
tion of its own mRNA (51). Here, in CVB3-infected cells
overexpressing p58IPK, the phosphorylation of eIF2a by PKR
was downregulated (Fig. 8B), allowing increased cellular
mRNA translation and thereby enhanced cellular health and
an environment more conducive to CVB3 replication.
In several other experimental systems, the phosphorylation
of eIF2? has been associated with an upregulation of ATF4
and the subsequent induction of the proapoptotic transcription
factor CHOP (19, 37). We were unable to detect the increased
expression of ATF4 during CVB3 infection (data not shown),
but we did observe a significant upregulation in the level of
CHOP. Since ATF4 and ATF6a work in concert to activate
CHOP expression (60), we speculate that the CHOP upregu-
lation we observed during CVB3 infection is modulated pri-
marily by ATF6a.
SREBP1 is another transcription factor associated with the
ER membrane. Its function in the regulation of lipid ho-
meostasis has been studied extensively. It was reported that in
response to a low level of sterol and other unidentified factors,
SREBP1 translocates from the ER to the Golgi complex,
where it is cleaved by site-1 and site-2 proteases. The fully
processed mature form of SREBP1 then enters the nucleus
and transactivates target gene expression (6). The activation of
SREBPs by cleavage also was reported to occur in HCV-
infected Huh cells (57). In addition, two recent studies re-
ported that SREBP1c activation plays a major role in ?-cell
glucolipotoxicity and apoptosis (55) and that an increased level
of active SREBPs in macrophages is involved in HIV protease
inhibitor-induced apoptosis via the depletion of ER calcium
stores and the activation of caspase-12 (69). We found that
during CVB3 infection SREBP1 was induced, and the level of
its cleaved form increased coincidently with the upregulation
of p-eIF2? and CHOP as well as with the activation of
caspase-7 and -12. A recent report indicated that caspase-7
expression is positively controlled by SREBP1 and SREBP2
(15), thus providing a prospective linkage between SREBP1
and caspase-7 in the apoptosis cascade.
Caspase-12 is an ER membrane-associated cysteine protease
that is induced and activated by caspase-7 in response to ER
stress (36, 41). It has been suggested that caspase-12 is a key
mediator of ER stress-induced apoptosis and is required for
the recruitment of other cytosolic caspases to the ER mem-
brane during the induction of the apoptotic program (41). Our
results show that caspase-12 was cleavage activated in HL-1
cells by 6 h postinfection, and that this event temporally coin-
cided with the induction of other apoptotic mediators, such as
CHOP and caspase-3 and -7, two caspases involved in the
FIG. 9. CVB3 infection induces upregulation of CHOP and activation of SREBP1, caspase-7, and caspase-12. HeLa cells or HL-1 cells were
cultured and infected with CVB3 as described in Fig. 1. Cell lysates were subjected to Western blot analysis to detect the induction and activation
(cleavage) of proapoptotic transcription factors CHOP and SREBP1, respectively (A), cleavage pattern (indicating activation) of caspases-7 and
caspase-12 in HL-1 cells (B), and caspase-7 in HeLa cells (C). Actin was detected in parallel as a loading control.
VOL. 84, 2010CVB3-INDUCED ER STRESS AND APOPTOSIS8457
cascade of caspase-12-mediated apoptosis (34). We note that
although the human genome contains no identified orthologue
of mouse casapse-12 (14), it has two similar speculative coun-
terparts, namely, caspase-4 and caspase-5. A recent study re-
ported that caspase-4 is involved in ER stress (21), but as yet
there is no related report on caspase-5. The question of
whether caspase-4 and/or caspase-5 plays a role in humans
equivalent to that of mouse caspase-12 in mediating apoptosis
induction requires further study.
In summary, our studies using both HeLa cells and cardio-
myocytes indicate that during CVB3 infection, UPR pathways are
induced but are carried out in a manner involving the atypical
expression of several UPR target genes, including p58IPK,
EDEM1, and ERdj4 (Fig. 10). Further, CVB3-induced alter-
ations of UPR pathways can result in the activation of proapop-
totic genes, engendering a shift from ER stress pathways to those
of apoptosis. Since cardiomyocyte apoptosis is a hallmark of viral
myocarditis, these data provide further insight into the molecular
pathogenesis of CVB3-induced myocarditis.
We thank Yi-Ling Lin, Michael Katze, and Kazutoshi Mori for
providing us the plasmids pHA-XBP1u-GFP, pcDNA1/neo-p58IPK,
and pcDNA3.1-AFT6(171-373), respectively, and our colleagues Al-
housseynou Sall and Kasinath Viswanathan for helpful discussions.
This work was supported by grants from the Canadian Institutes of
Health Research and the Heart and Stroke Foundation of BC and
Yukon. J.Y. is a recipient of the Doctoral Research Award from the
Canadian Institutes of Health Research and Michael Smith Founda-
tion of Health; X.Y. is a recipient of the UGF Award of the University
of British Columbia; Z.L. is a recipient of the Doctoral Research
Award from the Heart and Stroke Foundation of Canada.
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