Calcium signals and Calpain-dependent Necrosis are essential for Release of
Coxsackievirus B from Polarized Intestinal Epithelial Cells
Rebecca A. Bozym4, Kunal Patel1, Carl White2, King-Ho Cheung3, Jeffrey M. Bergelson1,
Stefanie A. Morosky4, and Carolyn B. Coyne4*
From the 1Division of Infectious Diseases, Children’s Hospital of Philadelphia, Philadelphia, PA,
the 2Department of Physiology & Biophysics, Rosalind Franklin University of Medicine and
Science, North Chicago, IL, 3Department of Physiology, University of Hong Kong, and
4Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh,
*Address correspondence: Carolyn B. Coyne, 518 Bridgeside Point II, 450 Technology Drive,
Pittsburgh, PA 15219, Phone (412) 383-5149, Fax (412) 624-1401 Email:email@example.com
Running Title: Calcium signals in coxsackievirus release
Coxsackievirus B (CVB), a member of the enterovirus family, targets the polarized
epithelial cells lining the intestinal tract early in infection. Although the polarized epithelium
functions as a protective barrier, this barrier is likely exploited by CVB in order to promote viral
entry and subsequent egress. Here we show that in contrast to non-polarized cells, CVB-infected
polarized intestinal Caco-2 cells undergo non-apoptotic necrotic cell death triggered by inositol
1,4,5-trisphosphate (IP3) receptor (IP3R)-dependent calcium release. We further show that CVB-
induced cellular necrosis depends on the Ca2+-activated protease calpain-2 and that this protease
is involved in CVB-induced disruption of the junctional complex and rearrangements of the actin
cytoskeleton. Our study illustrates the cell signaling pathways hijacked by CVB, and perhaps
other viral pathogens, to promote their replication and spread in polarized cell types.
Enteroviruses, including coxsackievirus B (CVB), are lytic viruses that destroy the host
cell membrane for progeny release. Lytic viruses often develop highly efficient strategies to
tightly regulate host cell death pathways in order to avoid killing the host cell prematurely (and
terminating viral replication). Studies on the mechanism(s) by which CVB induces host cell
death have identified apoptotic signaling, mediated by pro-apoptotic caspase-3, following CVB
infection in HeLa cells (Carthy et al., 1998; Carthy et al., 2003; Yuan et al., 2003). CVB
encounters the polarized epithelium lining the gastrointestinal tract early in infection and viral
replication in the mucosa is likely followed by epithelial cell lysis and subsequent viremia
(Morens, 1995). However, the precise mechanism(s) by which CVB induces cell death in
polarized epithelial cells remains unclear.
Viruses can trigger both apoptotic (programmed cell death) and non-apoptotic (necrosis,
autophagy) pathways during the course of infection (Agol et al., 1998; Barco et al., 2000; Lopez-
Guerrero et al., 2000). Apoptosis is a tightly controlled process of ‘cell-suicide’ that is associated
with well-characterized morphological and biochemical changes that include fragmentation of
DNA, activation of the caspase family of cysteine proteases, and the translocation of
phosphatidylserine from the inner to the outer membrane leaflet. Necrosis generally lacks the
changes associated with apoptosis and is instead characterized by irreversible swelling of the
cytoplasm and organelles, and ultimate lysis of the plasma membrane. Necrosis results in the
release of cellular material (often including degradative enzymes) from the cell into the
surrounding area that can induce cellular damage to neighboring non-necrotic cells [reviewed in
(Festjens et al., 2006; Zong and Thompson, 2006)].
Many pathogens target intracellular signaling molecules to disrupt normal cell function to
facilitate many aspects of their infectious life cycles. The link between alterations in Ca2+
homeostasis and cell death pathways has been well-established (Trump and Berezesky, 1996;
Lee et al., 1999; Mattson, 2000; Sattler and Tymianski, 2000; Xu et al., 2001; Orrenius et al.,
2003) and has been used by several viruses to promote their infections [reviewed in (Zhou et al.,
2009)]. Modulation of intracellular Ca2+ ([Ca2+]i) signaling has been implicated in both pro- and
anti-apoptotic pathways and is a key component in the activation of necrotic cell death and
autophagy (Cardenas et al.; Hoyer-Hansen et al., 2007; Hoyer-Hansen and Jaattela, 2007). Thus,
levels of Ca2+ are tightly regulated by the cell as a means to control both the induction and
inhibition of numerous types of cell death. The release of Ca2+ by inositol 1,4,5-trisphosphate
(IP3) receptor (IP3R)-dependent mechanisms can induce apoptosis through mitochondrial transfer
of Ca2+ (and the subsequent release of pro-apoptotic factors into the cytoplasm) or through the
activation of Ca2+-sensitive pro-apoptotic enzymes within the cytoplasm. In some cases, the
expression of IP3R is involved for the induction of apoptosis: reducing IP3R expression in both
chicken lymphoma (Sugawara et al., 1997) and human T-cells (Jayaraman and Marks, 1997)
renders them resistant to apoptosis.
The link between Ca2+ release and the activation of necrotic cell death is also striking.
Like apoptosis, necrotic cell death is often initiated by dramatic alterations in mitochondrial Ca2+
homeostasis resulting from ER-localized Ca2+ release and the concomitant activation of the Ca2+-
activated calpain family of cysteine proteases. Members of the calpain family can be categorized
into two subfamilies: μ-calpains (or calpain-1, are activated by micromolar concentrations of
Ca2+) and m-calpains (or calpain-2, are activated by millimolar concentrations of Ca2+) [reviewed
in (Liu et al., 2004)]. Once activated by autocatalytic hydrolysis, calpains translocate from the
cytosol to intracellular membranes where they are primed to cleave a number of diverse
substrates. Calpain substrates can include cytoskeletal proteins, adhesion molecules, membrane
proteins, kinases, phosphatases, ion transporters, and phospholipases (Rami, 2003). The
mechanisms by which the cell ‘decides’ to undergo either apoptosis or necrosis may depend on
the nature of the insult and/or on the signaling pathways involved in modulating the various cell
Here we show that in contrast to non-polarized cells, polarized intestinal Caco-2 cells do
not undergo apoptosis in response to CVB infection and instead undergo a caspase-independent
form of necrotic cell death. Whereas release of progeny virus from non-polarized cells occurred
by a caspase-dependent process, CVB release from Caco-2 cells occurred in a caspase-
independent manner and was instead mediated specifically through calpain-2. We further show
that CVB infection of Caco-2 cells results in an early [~2 hrs post-infection (p.i.)] increase in
[Ca2+]i that is followed by [Ca2+]i depletion occurring between 2-3 hrs p.i. CVB-induced Ca2+
release required the activity of phospholipase C (PLC) and the expression of IP3Rs and inhibiting
the activity or expression of these factors prevented CVB progeny release. Taken together, these
findings indicate that the host cell factors manipulated by CVB in order to induce cell death and
subsequent progeny release may differ among cell types, and that CVB exploits PLC-dependent
Ca2+ release to promote its escape from polarized intestinal cells.
CVB-induced cell death in polarized intestinal epithelial cells is caspase-independent
Pro-apoptotic signals mediated by activated caspase-3 have been associated with CVB-
induced cell death in non-polarized cells types (Carthy et al., 1998; Yuan et al., 2003).
Consistent with this, we found that CVB infection of HeLa cells led to the activation of caspase-
3 (as determined by the appearance of cleaved caspase-3 by immunoblotting) within 3-4 hrs
post-infection (p.i.) (Figure 1A). In contrast, we found no enhancement in cleaved caspase-3 in
CVB-infected polarized intestinal epithelial Caco-2 cells (Figure 1A). Both CVB–infected HeLa
and Caco-2 cells died within 7-10 hrs after infection, showing gross morphologic changes and
loss of membrane integrity as detected by uptake of propidium iodide (PI) (Figure 1B). However,
whereas CVB-infected HeLa cells displayed changes consistent with apoptosis including
externalization of phosphatidylserine (detectible by Annexin V binding), CVB-infected Caco-2
cells did not exhibit any detectable Annexin V binding (Figure 1B). This effect was not specific
to Caco-2 cells as we observed the lack of Annexin V binding to other CVB-infected polarized
epithelial cells [HCT-116 and HT-29, (Supplemental Figure 1)]. Moreover, we also found that
CVB-infected Caco-2 cells did not exhibit apoptosis-associated DNA fragmentation as assessed
by TUNEL assay whereas infected HeLa cells did (Supplemental Figure 2A). We confirmed
that Caco-2 cells were capable of undergoing apoptosis by incubating cells with the apoptosis
inducing agent staurosporine which led to enhanced Annexin V binding (Supplemental Figure
We also found that whereas the caspase inhibitor Z-VAD-FMK prevented Annexin V
binding and PI uptake in CVB-infected HeLa cells (Figure 1C), it had no effect on PI uptake in
CVB-infected Caco-2 cells (Figure 1D). Furthermore, we found that whereas Z-VAD-FMK
inhibited viral egress from CVB-infected HeLa cells, it had no effect on CVB release from
infected Caco-2 cells (Figure 1E, 1F). Taken together, these data indicate that in contrast to non-
polarized cells, the loss of membrane integrity in CVB-infected Caco-2 cells occurs via a
Calpains are involved in CVB-induced necrosis in Caco-2 cells
Necrotic cell death fails to display many of the classical features (such as
phosphatidylserine externalization) associated with apoptotic cell death, but is still associated
with the loss of plasma membrane integrity. The activation of Ca2+-sensitive calpain proteases
often contributes to the onset of necrosis (Wang, 2000; Liu et al., 2004). Because CVB appeared
to kill Caco-2 cells by a non-apoptotic mechanism, we tested whether calpain-dependent necrosis
might be important. We found that treatment of Caco-2 cells with the calpain inhibitor Z-Val-
Phe-CHO inhibited the increase in membrane permeability associated with cell death induced by
CVB infection (as assessed by PI uptake) (Figure 2A), but had no effect on CVB replication
(Figure 2A, 2B). Furthermore, we found that Z-Val-Phe-CHO prevented the release of CVB
particles from infected Caco-2 cells but had minimal effect on virus titers (Figure 2C).
To identify the time period during which calpain activity was required to induce CVB-
mediated increases in membrane permeability, we performed a time-of-addition experiment in
which Z-Val-Phe-CHO was added at various times p.i. We found that Z-Val-Phe-CHO exerted
its most potent inhibitory effects when added to CVB-infected cultures prior to 2-3 hrs p.i.
(Figure 2D). While Z-Val-Phe-CHO partially inhibited CVB-induced PI uptake at 4 hrs p.i., it
had no effect when added at >5 hrs p.i. (Figure 2D). Consistent with this, we found a slight
elevation in overall calpain activity in cells infected with CVB that peaked at 2-3hrs p.i., but
returned to control levels by 4hrs p.i. (Supplemental Figure 4). Taken together, these data
indicate that calpains are likely activated early in CVB infection (~2-3 hrs p.i.).
Calpain-2 is specifically involved in CVB-induced necrosis
We found that calpains were required for alterations in membrane permeability required
for CVB egress in Caco-2 cells (Figure 2A, 2C). Members of the calpain family can be
categorized into two subfamilies: μ-calpains (or calpain-1) and m-calpains (or calpain-2) based
upon their sensitivity to Ca2+ [reviewed in (Liu et al., 2004)]. To determine which member of
the calpain family was involved in CVB-induced necrosis, we downregulated either calpain-1 or
-2 expression in Caco-2 cells by RNAi and determined the effects of this on CVB-induced
membrane permeability. We found that whereas downregulation of calpain-1 had no effect on PI
uptake in CVB infected cells, calpain-2 siRNA significantly reduced PI uptake while having no
effect on CVB replication (Figure 2E). Furthermore, we found that downregulation of calpain-2
expression by siRNA transfection significantly inhibited CVB release from infected cells (Figure
2F). These data point to a role for calpains in CVB-mediated escape from infected Caco-2 cells.
Calpains mediate tight junction and actin cytoskeletal reorganization in CVB-infected
Polarized epithelial cells are characterized by the presence of distinct apical and
basolateral domains separated by junctional complexes composed in part by the apical tight
junction (TJ) complex. We found that CVB infection of Caco-2 cells led to a loss of TJ integrity
as assessed by decreased transepithelial resistances (TER) within 4 hrs p.i. (Figure 3A) and
relocalization of the TJ-associated component zonula occludens-1 (ZO-1) (Figure 3B). Activated
calpains target a number of molecules associated with maintaining polarized cell architecture
such as components of apical TJ complexes and components associated with actin cytoskeletal
stability (Rios-Doria et al., 2003; Franco et al., 2004; Lebart and Benyamin, 2006). We found
that incubation of cells with Z-Val-Phe-CHO inhibited CVB-induced decreases in TER and ZO-
1 relocalization (Figure 3A, 3B), indicating that this process requires calpain activity. We also
found pronounced alterations in the actin cytoskeleton in CVB-infected cells that were
characterized by loss of junction-associated actin and increased stress fiber formation (Figure
3C). CVB-induced alterations in actin cytoskeleton architecture were also inhibited by Z-Val-
Phe-CHO (Figure 3C), indicating that calpains also mediate the modulation of actin cytoskeletal
Calpains target components of junctional complexes in CVB-infected Caco-2 cells
Calpains regulate a variety of actin-dependent cellular processes and have been
implicated in the maintenance of cell adhesion and in the control of cellular motility. The direct
proteolysis of actin- and junction-associated cellular components is likely central to calpain-
dependent regulation of these processes. Calpains have been shown to cleave (either in vitro
and/or in vivo) the cytoskeletal proteins talin, vinculin, and paxillin and the junction-associated
proteins occludin, E-cadherin, and β-catenin (Rios-Doria et al., 2003; Franco et al., 2004; Lebart
and Benyamin, 2006; Chun and Prince, 2009). Because we observed calpain-dependent
alterations in TER and rearrangements of the actin cytoskeleton, we investigated the expression
pattern of known calpain substrates in CVB-infected Caco-2 cells. We found that many calpain
substrates were unaffected by CVB infection including Ezrin, paxillin, talin, vinculin, and ZO-1
(Figure 4A). However, we observed the appearance of a distinct cleavage fragment of the TJ-
associated transmembrane protein occludin in CVB-infected cultures that could be blocked by
treatment of cells with Z-Val-Phe-CHO (Figure 4A). In addition, we found that CVB infection
elicited significant decreases in the expression of both β-catenin and E-cadherin that were
restored when cells were infected in the presence of Z-Val-Phe-CHO (Figure 4A). We also found
that both occludin cleavage and E-cadherin downregulation occurred in a time-dependent
manner in CVB-infected cells and that both events were evident by 3-4hrs p.i., but most
prominent at 6hrs p.i. (Figure 4B). In contrast, the expression of ZO-1 did not exhibit any
significant decreases nor did we detect any cleavage fragments in response to CVB infection
(Figure 4A, 4B). Taken together, these data indicate that CVB infection elicits the cleavage
and/or downregulation of several junction-associated membrane components and that calpains
likely play a central role in these processes.
We also observed extensive rearrangements of occludin and E-cadherin (not shown)
localization including a loss of junctional association and corresponding appearance of
intracellular occludin-containing vesicles in CVB-infected cells (Figure 4C). The redistribution
of occludin was inhibited in CVB-infected cells treated with Z-Val-Phe-CHO, indicating that
calpains likely mediate some event in the cleavage and subsequent relocalization of occludin.
As we observed alterations in two components of the junctional complex in CVB-
infected Caco-2 cells, we next examined the subcellular localization of calpains in infected
cultures. Calpains localize predominantly to the cytosol but upon exposure to Ca2+, translocate
to a variety of intracellular membranes where they undergo autolysis and subsequent activation
(Michetti et al., 1996; Glading et al., 2001; Leloup et al., 2010). Relocalization of activated
calpains thus serves to facilitate substrate cleavage. We found that CVB infection of Caco-2 cells
induced the cleavage and/or downregulation of β-catenin, E-cadherin, and occludin, all
components known to localize to cell-cell junctions. Interestingly, we observed the
relocalization of calpain-2 to sites of cell-cell contact and to intracellular vesicles where it
colocalized with occludin within 4 hrs following CVB infection (Figure 4D). We also found that
Z-Val-Phe-CHO lost its inhibitory effect when added 2-3 hrs p.i. (Figure 2D). Taken together,
these data indicate that calpain-2 is likely activated prior to 4 hrs p.i. and is then translocated to
cellular junctions where it cleaves several components of the junctional complex thus disrupting
junctional architecture, cell-cell contacts and cell polarity.
Inhibitors of ER-derived Ca2+ release block CVB-induced necrosis
Because alterations in [Ca2+]i homeostasis are essential for calpain activation, we next
determined the effect of a panel of pharmacological inhibitors known to modulate intracellular
calcium signaling on CVB-induced necrosis and identified a number of inhibitors that prevented
cell death but had no effect on CVB infection (Figure 5A). These included Bapta-AM, an
intracellular Ca2+ chelator; caffeine, which causes release of Ca2+ stores by activating ryanodine
receptors; cyclopiazonic acid (CPA), an inhibitor of sarco/endoplasmic reticulum Ca2+-ATPase
(SERCA)-mediated Ca2+ uptake; and thapsigargin, a specific inhibitor of SERCAs which results
in depletion of ER-derived Ca2+ stores. In contrast, cyclosporine A (CSA), an inhibitor of the
mitochondrial permeability transition pore, had no effect on either necrosis or virus replication
These results point to a role for ER-derived release of Ca2+ in CVB-induced necrosis
and suggest that the release of mitochondrial Ca2+ stores did not play a significant role in this
process. Consistent with this, we found that 2-APB, an inhibitor of IP3Rs, and U73122, an
inhibitor of PLC, also prevented CVB-induced necrosis (Figure 5B).
Release of ER Ca2+ stores was also required for alterations in actin cytoskeletal and TJ
integrity, as we found that thapsigargin inhibited CVB-induced reorganization of ZO-1 (Supp
Fig 3), and occludin (Figure 5C). Furthermore, thapsigargin U73122, and 2-APB all inhibited
CVB release from infected Caco-2 cells (Figure 5D). These data point to a role for PLC-
dependent activation and subsequent release of Ca2+ via ER-localized IP3R channels in
alterations in membrane integrity required for CVB release from Caco-2 cells.
CVB Infection Depletes ER-Derived Ca2+ stores early in infection
To better define the time period during which Ca2+ signaling was required to induce
necrosis, we performed a time-of-addition experiment in which thapsigargin, U73122, and 2-
APB were added at various times p.i. We found that all three drugs maintained their inhibitory
effects when added to cells 2-3hrs p.i., but lost their effectiveness when added at 4hrs p.i. (Figure
5E). Interestingly, these kinetics were similar to the pattern we had observed with a calpain
inhibitor (Figure 2D). Taken together, these data suggest that alterations in cellular Ca2+
signaling likely occur early in CVB infection, by 3-4 hrs p.i.
To more precisely measure the effect of CVB infection on ER Ca2+ stores, we monitored
the level of Ca2+ release in response to the addition of thapsigargin at various times p.i. Whereas
uninfected control cells exhibited pronounced release of Ca2+ stores upon exposure to
thapsigargin (Figure 6A, 6B), cells infected with CVB for 3 hrs displayed a marked decrease in
Ca2+ release in response to thapsigargin (Figure 6A, 6B). At 2 hr p.i. we also observed a
decrease in Ca2+ release compared to uninfected controls (Supplemental Figure 5). After 3 hrs
p.i., we were unable to measure Ca2+ levels in CVB-infected cells because necrosis-induced
increases in membrane permeability made it impossible to load cells with calcium-sensitive dye.
Next we monitored real-time Ca2+ signaling in cells infected with CVB. As we had
observed a reduction in thapsigargin-sensitive Ca2+ release in CVB infected cells at 3hr p.i., we
monitored real-time changes in [Ca2+] over the course of infection over one hour (spanning from
2-3 hrs p.i.). We found that whereas Ca2+ levels remained stable in uninfected cells over the
course of one hour, there was a pronounced decrease in Ca2+ in CVB-infected cells between 2-3
hrs p.i. (Figure 6C, 6D). These data indicate that alterations in ER Ca2+ stores occur early in
CVB infection (between 2-3 hrs p.i).
CVB-mediated Depletion of ER-Derived Ca2+ Requires PLC and IP3Rs
To determine whether CVB-induced depletion of ER Ca2+ stores involved IP3 production
and/or IP3Rs, we tested the effects of pharmacological inhibitors of Phospholipase C (U73122)
and IP3R function (2-APB). Cells were treated with inhibitors and infected with CVB for 3 hrs,
then thapsigargin was added and ER-derived Ca2+ release was measured. In the absence of
inhibitors, CVB-infected cells exhibited significantly smaller changes in [Ca2+] in response to
thapsigargin than did uninfected controls (Figure 7A, 7B). In contrast, after treatment with
U73122 or 2-APB, the response to thapsigargin was the same in both CVB-infected and
uninfected control cells (Figure 7A, 7B).
To further define the role of IP3Rs in CVB-induced alterations in [Ca2+]i, we transfected
cells with siRNAs targeted against IP3R1 and 3 which are expressed in Caco-2 cells (there were
no detectable levels of IP3R2 found in these cells, not shown) and measured the effect of
combined IP3R1 and 3 depletion on thapsigargin-sensitive Ca2+ stores in CVB-infected cells.
Similar to our findings with 2-APB, we found that transfection of cells with IP3R1 and IP3R3
siRNAs prevented the depletion of thapsigargin-sensitive ER Ca2+ stores in CVB-infected cells
(Figure 7C, 7D). Moreover, we found that depletion of IP3R1 and IP3R3 expression by RNAi
prevented the release of CVB progeny from infected Caco-2 cells but had no effect on virus titers
(Figure 7E, F).
Although pathogens likely hijack multiple host cell signaling cascades during infection,
the precise cascade of signals that culminate in the release of enteroviruses from host polarized
cells have remained unclear. Here we show that in polarized intestinal epithelial cells, CVB
infection leads to the release of ER-derived Ca2+ in order to induce a cascade of events resulting
in the destruction of the host cell membrane as a means to promote progeny release. CVB
specifically exploits IP3R-dependent Ca2+ release in order to induce non-apoptotic necrotic cell
death and involves the activation of the Ca2+-activated protease calpain-2. Activated calpain-2
are involved in the cleavage and/or downregulation of β-catenin, E-cadherin, and occludin, all
components of epithelial tight junctions and cell-cell contacts, which likely contributes to CVB-
induced loss of cell polarity and adhesion. These findings point to a novel role for [Ca2+]i
signaling in the release of CVB from polarized epithelia and suggest that the signals necessary
for multiple steps in the virus life cycle may differ between polarized and non-polarized cells
Alterations in cellular Ca2+ homeostasis play a fundamental role in the response of many
tissue types to injury or assault. We found that CVB infection induced the depletion of
thapsigargin-sensitive Ca2+ stores by 3 hrs p.i. and several pharmacological inhibitors targeting
intracellular Ca2+ signaling lost their inhibitory effects between 2 and 3 hrs p.i. Other
investigators have reported that the CVB protein 2B perturbs Ca2+ homeostasis in HeLa cells by
directly modifying ER membrane permeability in order to promote virus release (van Kuppeveld
et al., 1997). The effects we observe on Ca2+ homeostasis occured by 2-3hrs p.i., a time prior to
the detection of newly synthesized viral RNA and protein (which generally occurs at ~4hrs p.i. in
Caco-2 cells, Figure 1A and not shown) which would indicate that very low levels of 2B are
likely present at these times. Moreover, our observations suggest that the depletion of ER Ca2+
stores induced by CVB infection of Caco-2 cells involves the initiation of PLC-dependent
signaling cascades, as we observed that pharmacological inhibitors of PLC, an IP3R antagonist
(2-APB), and RNAi-mediated downregulation of IP3R1 and IP3R3 inhibited CVB-induced Ca2+
release. Although CVB 2B may contribute to further Ca2+ alterations induced at later stages of
CVB replication, our data indicate that PLC-dependent activation of IP3Rs contributes primarily
to the release of Ca2+ required to initiate necrosis and facilitate calpain activation in Caco-2 cells.
Calpains are ubiquitously expressed Ca2+-dependent proteases that are categorized based
upon the Ca2+ concentration required for their activation. Our results indicate that calpain-2
plays a key role in promoting CVB-induced host cell necrosis, as inhibition of calpain
activity or RNAi-mediated silencing of calpain-2 prevented CVB egress from polarized
epithelial cells. Interestingly, apicomplexan parasites, including Plasmodium falciparum and
Toxoplasma gondii, have also been shown to utilize calpains to facilitate their egress from
infected cells, although the mechanism by which they activate these proteases remains
unclear (Chandramohanadas et al., 2009). Our data indicate that calpain activity is required
to induce a loss of polarity and junctional integrity that accompanies CVB infection of Caco-
2 cells, indicating that activated calpains target key components of epithelial architecture
during the course of CVB infection. Although a recent study has implicated a role for
calpains in echovirus replication (Upla et al., 2008), we found no evidence that calpains were
specifically involved in CVB replication in Caco-2 cells as evidenced by our finding that
calpain inhibitors and calpain depletion with siRNAs did not decrease titers of newly
replicated virus. While we have previous shown that calpain 2 regulates CVB trafficking in
polarized endothelial cells (Bozym et al., 2010), we did not observe any effects of calpain
inhibition on CVB entry into Caco-2 cells, suggesting that the role of calpains in facilitating
CVB infection may differ between polarized cells types.
Polarized epithelial cells regulate the flow of ions and macromolecules across the
epithelium by the presence of junctional complexes located at their apicolateral poles
(Schneeberger and Lynch, 2004). We found that CVB infection induced a loss of junctional
integrity (as assessed by decrease in TER) within 4 hrs p.i., and that the change in TER that
required the activity of calpains. We found that CVB infection led to the relocalization of ZO-1,
occludin, and several other junctional-associated components such as β-catenin and E-cadherin
(not shown). In addition, we found that occludin undergoes cleavage in CVB-infected cells and
that both β-catenin and E-cadherin expression levels are diminished by CVB infection. These
events require calpain activity as they were all blocked by a pharmacological inhibitor of
calpains. Cleavage of occludin by calpains is also important in leukocyte transmigration across
polarized airway epithelium (Chun and Prince, 2009) and calpains target both β-catenin (Benetti
et al., 2005) and E-cadherin (Rios-Doria et al., 2003; Chun and Prince, 2009).
The relocalization of calpains from the cytoplasm to intracellular membranes has been
speculated to play an important role in their activation (Leloup et al., 2010). Consistent with
this, attachment of a farnesyl anchor to calpain-2 triggers the strong induction of calpain activity
(Leloup et al., 2010). We found that calpain-2 relocalized from the cytoplasm to both the
junctional complex and to internalized occludin-containing vesicles within 3 hrs following CVB
infection. Interestingly, this time point corresponded with a significant decrease in [Ca2+]i levels
in CVB-infected cells and the loss of an inhibition of necrosis by small molecule inhibitors of
calpains and those that disrupt [Ca2+]i signaling. Taken together, these data suggest that calpains
are activated within 2-3 hrs p.i. in CVB-infected cells that likely initiate a cascade of events,
including cleavage of cell-cell contacts and junctional components, that ultimately leads to loss
of membrane integrity and virus escape.
The mechanism of cell death we observed in several polarized intestinal epithelial cell
lines differs markedly from the apoptotic cell death induced by CVB in non-polarized cells [such
as HeLa cells (Carthy et al., 1998; Carthy et al., 2003; Yuan et al., 2003), pancreatic β cells
(Rasilainen et al., 2004), and neurons (Joo et al., 2002; Feuer et al., 2003)]. The reasons for the
induction of divergent cell death pathways between polarized and non-polarized cells are likely
complex and may be due to inherent differences in Ca2+ regulation and/or signaling [such as in
differences in the expression and cellular localization of IP3Rs (Colosetti et al., 2003)], in the
expression, localization, and/or activation of innate immune-associated components (Hershberg,
2002; Cario et al., 2007), or in the expression of signaling molecules that are associated with the
regulation of cell death. Another possibility is that the composition and/or regulation of
paracellular junctions of polarized epithelia play key roles in the regulation of cell death
pathways in response to microbial assault. For example, as enterocytes display high rates of
turnover, they have developed highly regulated means of cell death regulation, which have been
linked to the integrity of their cell-cell contacts and most notably to the presence of E-cadherin.
For example, loss of E-cadherin localization at epithelial junctions induces anoikis (Fouquet et
al., 2004; Lugo-Martinez et al., 2009)}, a form of apoptosis induced by loss of cell-matrix
interactions. Interestingly, the expression of a calpain-mediated cleavage fragment of E-cadherin
potentiates cell death in epithelial cells (Rios-Doria and Day, 2005) and cleavage of the β-
catenin binding domain of E-cadherin is associated with the induction of apoptosis (Vallorosi et
al., 2000). We observed that cleavage of occludin and decreases in E-cadherin and β-catenin
expression occurred in a calpain-dependent manner in CVB-infected Caco-2 cells. Although we
do not know whether these events are responsible for the induction of necrosis in CVB-infected
polarized epithelia, or are merely nonspecific events associated with calpain activation, it is
attractive to speculate that the divergent mechanism of cell death observed in polarized versus
non-polarized epithelial cells may be the result of calpain-mediated cleavage of junctional-
associated components that serve to induce alternative mechanisms of cell death.
The results of our study indicate that cell death in CVB-infected Caco-2 cells occurs by a
caspase-independent mechanism that instead depends on the activity of PLC, subsequent release
of [Ca2+]i from IP3Rs, and the activation of calpains. These findings illustrate the unique
mechanisms by which enteroviruses, and perhaps other viral pathogens, co-opt intracellular
signaling pathways in polarized cell monolayers to promote their entry, replication and eventual
Cell culture and Viruses
Caco-2 (ATCC) and HeLa (CCL-2) cells were cultured in MEM supplemented with 10%
fetal bovine serum, nonessential amino acids, sodium pyruvate, and pen/strep. Caco-2 (BBE
clone) were grown in DMEM-H supplemented with 10% FBS and pen/strep. CVB3-RD was
expanded and purified as described previously (Coyne and Bergelson, 2006). Unless otherwise
stated, all infections were performed with a multiplicty of infection of 1-5 particle forming
Mouse anti-enterovirus VP1 (Ncl-Entero) was obtained from Novocastra Laboratories
(New Castle upon Tyne, UK). Alexa fluor-conjugated secondary antibodies, propidium iodide,
phalloidin, mouse or rabbit anti-ZO-1,-occludin, or -β−catenin, and Annexin V were purchased
from Invitrogen (Carlsbad, CA). Rabbit anti-caspase-3 and E-cadherin antibodies were
purchased from Cell signaling. Mouse anti-paxillin antibody was purchased from Abcam;
mouse anti-talin and mouse anti-vinculin antibodies were purchased from Sigma; and goat anti-
calpain -1 and -2 antibodies were purchased from Santa Cruz.
Cyclosporine A (CSA, 5μM), Z-Val-Phe-CHO (5μM), and Z-VAD-FMK (20μM) were
purchased from Calbiochem (Gibbstown, NJ); U73122 (1μM), Bapta-AM (10μM), 2-APB
(30μM), caffeine (1mM), CPA (10μM), ruthenium red (5μM) and thapsigargin (3μM) were
purchased from Sigma (St. Louis, MO).
Transepithelial Resistance Measurements
Transepithelial resistance (TER) measurements were performed using an ohmmeter
(EVOM; World Precision Instruments) on cells grown on Transwell-COL inserts for a minimum
of 3 days. Once cells exhibited RT values of at least 600 Ω-cm2, monolayers were infected with
CVB (5-10 PFU/cell) at 37°C. At the indicated times, cells were removed and TER measured.
All measurements were background corrected using a blank insert without cells.
Caco-2 monolayers grown in collagen-coated chamber slides (BD Biosciences, San Jose,
CA) were exposed to CVB at the indicated MOIs for 8 hours (or the indicated time) at 37°C. The
cells were then washed and fixed with ice-cold methanol or paraformaldehyde and permeabilized
with Triton X-100. Monolayers were incubated with primary antibody, washed, and incubated
with Alexa Fluor–488 or -594-conjugated secondary antibodies, washed and then mounted with
Vectashield containing DAPI. Propidium iodide and annexin V staining were performed
following the manufacturer’s protocol. Briefly, cells were washed with PBS, annexin V-488
conjugate and/or PI were added in annexin binding buffer (10mM HEPES, 140mM NaCl,
2.5mM CaCl2, pH 7.4) for 15 min at room temperature. Cells were rinsed in annexin binding
buffer and then fixed with methanol and mounted with Vectashied containing DAPI. Images
were captured with an Olympus IX81 inverted microscope equipped with a motorized stage or
with an Olympus Fluoview 1000 laser scanning microscope. Images of infected cells (PI and
annexin V) were taken using an Olympus Pan Apo 10x/0.28 NA dry objective whereas all other
images were taken with an Olympus PlanApo 60x/1.42 NA oil objective. Quantification of
percent positive cells was performed using ImageJ (NIH) analysis, where a minimum of 3 fields
per condition were counted (at least 600 cells total). Cells were counted via DAPI staining, and
positive cells were counted in the appropriate channel for the condition.
Ratiometric Calcium Imaging
Cells grown on collagen-coated glass bottom 35mm dishes (MatTek Corp., Ashland,
MA) were loaded with Fura-2 AM (1μM - Invitrogen) for 30 min at 37°C. Cells were rinsed 3
times and bathed in a final volume of 1 mL Ca2+- and Mg2+-free PBS. Images were captured on
an Olympus IX81 motorized inverted microscope equipped with a Hamamatsu Orca-R2 CCD
camera, Sutter Lambda 10-3 High Speed filter wheel system, and an Olympus UApo/340 20x
objective with an N.A. of 0.75. Images were acquired using Slidebook 5.0 advanced imaging
software. Selected cells were chosen (60 regions of interest (ROI)/dish) and images captured at
both excitation 340nm and 380nm every 10 seconds for 10 minutes (experiments were
performed a minimum of three times). Thapsigargin was added to dishes once baseline was
established (t=50sec). Intensity ratios for selected ROIs were calculated using Slidebook 5.0,
representative traces for each experiment were plotted as a function of time (Figures 6A, 7A, and
7C). Images were pseudocolored (using Slidebook 5.0) in order to better visualize [Ca2+]i
mobilization with blue = low Cai2+ and red = high Cai2+. Overall changes in fluorescence
intensity ratio (340/380) were calculated by subtracting the resting intensity ratio from the max
intensity ratio achieved after thapsigargin addition.
Live Cell Calcium Imaging
Caco-2 cells were grown to confluence on collagen-coated glass bottom 35mm dishes as
stated above. For virus experiments, CVB was added to the dish at an MOI=5 and placed at 37°C
for 1.5 hrs. At this time, Fura-2 AM was added to the dish and infection was allowed to continue
for 30min at 37oC. Media was then removed and cells washed three times and placed in a final
volume of 1mL Ca2+/Mg2+-free PBS. Dishes were placed on a 37oC temperature-controlled stage
insert (Bioptechs) mounted over an Olympus IX81 microscope (described in detail above).
Images were captured (from ~60 ROI’s per experiment) at both 340nm and 380nm every 10 min
for 1 hr. Intensity ratios were calculated using Slidebook 5.0, with representative traces plotted
as a function of time (Figure 6C). For uninfected control experiments, cells were incubated for
1.5 hrs in the absence of CVB, incubated with Fura-2AM for 30 min at 37oC, rinsed, and imaged
as described above.
siRNAs against calpain-1 (5’-GGCAGCUUUCGCUUGUUCCtt-3’) and calpain-2 (5’-
GGCAGCUUUCGCUUGUUCCtt-3’) were synthesized by Integrated DNA Technologies (IDT).
siRNAs to IP3R-1, and -3 have been described (Bozym et al., 2010). Cells were transfected using
HiPerfect (Qiagen), according to the manufacturer's protocol or were delivered by nucleofection
with an Amaxa nucleofection device (solution T, program B-24).
Statistical Analysis. Data are presented as mean ± standard deviation. One-way analysis of
variance (ANOVA) and Bonferroni’s correction for multiple comparisons were used to
determine statistical significance (p < 0.05 or <0.001).
We are grateful to Dr. Kevin Foskett for helpful advice and suggestions. This work was
supported by funding from the NIH [R01AI081759 (CBC) and [R01AI52281 (JMB)].
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Figure 1: Caco-2 cells infected with CVB do not undergo apoptosis (A) Western blot analysis
of caspase-3 (top) and VP1 (bottom) in HeLa and Caco-2 cells at the indicated times following
CVB infection. (B) Annexin V (green) binding and propidium iodide (red) uptake in HeLa (left)
or Caco-2 (right) cells infected with CVB for 8hrs. (C, D) Quantification of immunofluorescence
images of annexin V binding, propidium iodide (PI) uptake, and infection (as determined by VP1
immunofluorescence) in CVB-infected HeLa (C) or Caco-2 (D) cells incubated with no inhibitor
(NoI) or with the caspase-3 inhibitor Z-VAD-FMK, or in uninfected (NoV) controls. Data are
presented as the percent positive cells over the total number of cells. (E, F) Virus titers (shown
as pfu/mL) determined by plaque assays from the media versus lysed cells of HeLa (E) or Caco-
2 (F) cells infected with CVB for 12hrs in the absence [no inhibitor (NoI)] or presence of Z-
VAD-FMK. In (C-F), data are presented as mean + SD from experiments performed in triplicate
a minimum of three times, *p<0.001.
Figure 2: Calpains mediate CVB-induced necrosis in Caco-2 cells. (A) Propidium iodide (PI)
uptake and VP1 staining in Caco-2 cells infected with CVB for 8hrs in the absence of inhibitor
(NoI) or in the presence of the calpain inhibitor Z-Val-Phe-CHO or in uninfected (NoV)
controls. Representative images of DAPI (blue) and PI (red) are shown below. (B) Western blot
analysis for VP1 in Caco-2 cells infected with CVB for the indicated time in the absence of
inhibitor (left) or in the presence of Z-Val-Phe-CHO (right). GAPDH is shown at bottom as a
loading control. (C) Virus titers (shown as pfu/mL) determined by plaque assays from the media
versus lysed cells of Caco-2 cells infected with CVB for 12hrs in the absence [no inhibitor (NoI)]
or presence of Z-Val-Phe-CHO. (D) Percent PI uptake and VP1 staining in Caco-2 cells infected
with CVB in the absence (NoI) or presence of Z-Val-Phe-CHO added to cells at the indicated
times p.i. or in uninfected (NoV) controls. Shown are the percentage of PI or VP1 positive cells
over the total number of cells. (E) Percent PI uptake and VP1 staining in Caco-2 cells transfected
with control (Con) or calpain-1 or -2 siRNAs (by Amaxa nuclefection) and infected with CVB
(for 8hrs) 48hrs post-transfection. Shown are the percentage of PI or VP1 positive cells. Bottom,
immunoblot analysis for calpain-1 (left) or -2 (right) in siRNA-transfected cells. GAPDH is
included as a loading control. (F) Virus titers (shown as pfu/mL) determined by plaque assays
from the media versus lysed cells of Caco-2 cells transfected with control siRNA or calpain-2
siRNA (by Amaxa nucleofection) and infected with CVB for 12 hrs. In (C-F), data are presented
as mean + SD from experiments performed in triplicate a minimum of three times, *p<0.05 and
Figure 3: Loss of junctional integrity in CVB-infected Caco-2 cells. (A) Transepithelial
electrical resistance (TER) measurements in Caco-2 cells infected with CVB in the absence (No
Inh) or presence of Z-Val-Phe-CHO (Calpain Inh) for the indicated times or in uninfected (no
virus) controls. (B) Confocal micrographs of Caco-2 cells infected with CVB for 8hrs in the
absence (No Inh) or presence of Z-Val-Phe-CHO and stained for DAPI (blue), ZO-1 (green), and
VP1 (red) compared to uninfected (no virus) controls. (C) Immunofluorescence microscopy
staining for actin in Caco-2 cells infected with CVB for 8hrs in the absence (No Inh) or presence
of Z-Val-Phe-CHO (Calpain Inh) or in uninfected controls.
Figure 4: Several junction-associated components are modulated by calpains during CVB
infection. (A) Western blot analysis for the indicate proteins from lysates of Caco-2 cells
infected with CVB for 12hrs in the absence (NoI) or presence of Z-Val-Phe-CHO (CalpI) or in
uninfected controls (NoV). GAPDH is shown as a loading control and VP1 production is shown
as a control for infection levels. Arrow (grey) denotes a cleaved occludin fragment. (B) Western
blot analysis for occludin, E-cadherin, and ZO-1 from lysates of Caco-2 cells infected with CVB
for the indicated times. VP1 production is shown as a control for infection levels. Arrow (grey)
denotes a cleaved occludin fragment. (C) Confocal micrographs of Caco-2 cells stained for VP1
(blue), occludin (green), and ZO-1 (red) in the absence of virus or in cells infected with CVB for
8hrs in the absence or presence of calpain inhibitor Z-Val-Phe-CHO. (D) Confocal micrographs
of Caco-2 cells stained for DAPI (blue), occludin (green), or calpain-2 (red) in the absence and
presence of CVB 3hrs p.i.
Figure 5: Pharmacological inhibitors of calcium homeostasis or signaling prevent CVB-
induced necrosis in Caco-2 cells. (A) Propidium iodide (PI) uptake and VP1 staining (shown as
percent positive staining over the total number of cells) in Caco-2 cells infected with CVB for
8hrs in the absence of inhibitor (NoI) or in the presence of the indicated inhibitors [Bapta-AM,
caffeine (Caff), cyclopiazonic acid (CPA), cyclosporine A (CSA), or thapsigargin (Thap)] or in
uninfected (NoV) controls. (B) Propidium iodide (PI) uptake and VP1 staining (shown as percent
positive staining over the totoal number of cells) in Caco-2 cells infected with CVB for 8hrs in
the absence of inhibitor (NoI) or in the presence of the indicated inhibitors [2-APB or U73122
(U7)] or in uninfected (NoV) controls. (C) Immunofluorescent microscopy for VP1 (green) and
occluding (red) in Caco-2 cells infected with CVB for 8hrs in the absence (NoI) or presence of
thapsigargin (Thap), or in uninfected (NoV) controls. (D) Virus titers (shown as pfu/mL)
determined by plaque assays from the media versus lysed cells of Caco-2 cells infected with
CVB for 12hrs in the absence [no inhibitor (NoI)] or presence of thapsigargin (thap), U73122, or
2-APB. (E) Percent PI uptake in Caco-2 cells infected with CVB for 8hrs in the absence (NoI) or
presence of thapsigargin (thap), U73122, or 2-APB added to cells at the indicated times p.i., or in
uninfected (NoV) controls. Shown are the percentage of PI positive cells over the total number
of cells. Data in (A-B, D-E) are presented as mean + SD from experiments performed in
triplicate a minimum of three times, *p<0.001.
Figure 6: Intracellular calcium stores are depleted between 2-3hr following CVB infection
of Caco-2 cells. (A) Fluorescence intensity ratio of Fura-2 in response to thapsigargin (black
arrow) in Caco-2 cells infected with CVB for 3hrs or in uninfected (No Virus) controls. Shown
are representative traces per condition. (B) Representative images of Fura-2 AM loaded Caco-2
cells from (A). Time of thapsigargin addition is represented by a black arrow. Images are
pseudocolored for visual assessment with dark blue = low Ca2+ and red = high Ca2+. (C)
Fluorescence intensity ratio over the course of 1hr in Caco-2 cells infected with CVB for 2hrs (2-
3hrs p.i.) or in uninfected controls. (D) Change in intensity ratio from traces shown in (C). Data
in (D) are presented as mean + SD from experiments performed in triplicate a minimum of three
Figure 7: PLC activity and IP3R expression are required for CVB-induced alterations in
Ca2+. (A) Fluorescence intensity ratio over time in response to thapsigargin (black arrow) in
Caco-2 cells infected with CVB for 3hr p.i. in the absence (NoI) or presence of 2-APB or
U73122 or in uninfected (NoV) controls. (B) Overall change in fluorescence intensity ratio from
traces shown in (A). (C) Fluorescence intensity ratio over time in response to thapsigargin (black
arrow) in Caco-2 cells co-transfected with control or IP3R 1 and 3 siRNAs (using Hiperfect) and
infected (48hrs post-transfection) with CVB for 3hr. (D) Overall change in fluorescence intensity
ratio from traces shown in (C). (E) Virus titers (shown as pfu/mL) determined by plaque assays
from the media versus lysed cells of Caco-2 cells transfected with control (CON) or IP3R1-3
siRNAs (by Amaxa nucleofection) and infected with CVB for 12hrs. Data in (B), (D), and (E)
are presented as mean + SD from experiments performed in triplicate a minimum of three times,
*p<0.001. (F) Western blot analysis for IP3R-1 and IP3R-3 expression in Caco-2 cells transfected
with control siRNA, or siRNAs against IP3R-1 or IP3R-3. GAPDH is shown as a loading control.
HT-29 (human colorectal adenocarcinoma cells) and HCT-116 (human colorectal
carcinoma cells) were cultured in McCoy’s 5A medium with 10% FBS and pen/strep. Cells
were grown in collagen-coated chamber slides and infected with CVB (MOI=5) and then stained
with annexin V-488 as a marker for apoptosis.
Calpain activity assay
Caco-2 cells were cultured in collagen-coated 24-well plates. CVB was added to the virus
wells and the media was changed in no virus wells. Monolayers were infected at 37oC with CVB
(10 PFU/cell) for the indicated times and lysates were collected. Calpain activity was
determined using the Calpain-GLO Protease Assy (Promega) according to the manufacturers
protocol using a BioTek Synergy 2 luminesecnce microplate reader.
HeLa and Caco-2 cells were cultured in collagen-coated 8-well chamber slides and
infected with CVB for 8hrs. Following infection, TUNEL assay was performed using the
DeadEnd Fluorometric TUNEL System (Promega) according to the manufacturer’s protocol.
Supplemental Figure 1: Polarized intestinal epithelial cell lines undergo apoptosis-
independent cell death in response to CVB infection. Fluorescent micrographs of HT-29 and
HCT-116 cells stained for DAPI (blue), VP1 (green), and annexin V (red) in the absence (NoV)
of following infection with CVB for 8hrs.
Supplemental Figure 2: Caco-2 cells undergo apoptosis when exposed to staurosporin. (A)
Percent of cells undergoing apoptosis (as assessed by TUNEL assay) in HeLa or Caco-2 cells
infected with CVB (1-5PFU/cell) for 8hrs. Data are shown as the percent cells positive by
TUNEL divided by the total number of cells (mean ± SD) and are representative of
quantification of ~500 total cells. (B) Fluorescent micrographs of Caco-2 cells exposed to
staurosporin (1μM for 8hrs) and analyzed for propidium iodide uptake (red) and annexin V
Supplemental Figure 3: Thapsigargin prevents CVB-induced reorganization of ZO-1.
Immunofluoresecnce microscopy for VP1 (green) and ZO-1 (red) in Caco-2 cells infected with
CVB for 8hrs in the absence (No Inh) or presence of thapsigargin or in uninfected controls.
Supplemental Figure 4: Increase in calpain activity in CVB-infected Caco-2 cells at 2-3
hours post-infection. Calpain activity was measured in Caco-2 cells or in no virus (NoV)
controls and at the indicated times after CVB infection. *p<0.05.
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Supplemental Figure 5: Thapsigargin responsiveness in Caco-2 cells at 2hr p.i. Fluorescence
intensity ratios of Fura-2 in response to thapsigargin (black arrow) in Caco-2 cells infected with
CVB for 2hrs or in uninfected (No Virus) controls. Shown are representative traces per