Virology 462-463 (2014) 135–148

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Virology
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Nitazoxanide, an antiviral thiazolide, depletes ATP-sensitive

intracellular Ca2 þ stores
Omodele Ashiru, Jonathon D. Howe, Terry D. Butters n
Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, Oxfordshire OX1 3QU, UK

art ic l e i nf o a b s t r a c t

Article history: Nitazoxanide (NTZ) inhibits influenza, Japanese encephalitis, hepatitis B and hepatitis C virus replication
Received 27 February 2014 but effects on the replication of other members of the Flaviviridae family has yet to be defined. The
Returned to author for revisions pestivirus bovine viral diarrhoea virus (BVDV) is a surrogate model for HCV infection and NTZ induced
1 April 2014
PKR and eIF2α phosphorylation in both uninfected and BVDV-infected cells. This led to the observation
Accepted 14 May 2014
Available online 25 June 2014
that NTZ depletes ATP-sensitive intracellular Ca2 þ stores. In addition to PKR and eIF2α phosphorylation,
consequences of NTZ-mediated Ca2 þ mobilisation included induction of chronic sub-lethal ER stress as
Keywords: well as perturbation of viral protein N-linked glycosylation and trafficking. To adapt to NTZ-mediated ER
ER stress stress, NTZ treated cells upregulated translation of Ca2 þ -binding proteins, including the ER chaperone
N-linked glycans
Bip and the cytosolic pro-survival and anti-viral protein TCTP. Depletion of intracellular Ca2 þ stores is
Flaviviridae
the primary consequence of NTZ treatment and is likely to underpin all antiviral mechanisms attributed
PKR
Intracellular Ca2 þ to the thiazolide.
TCTP & 2014 Elsevier Inc. All rights reserved.

Introduction eIF2B thereby inhibiting the replacement of the inactive eIF2-GDP

Nitazoxanide (NTZ) is a thiazolide with antiviral properties.
It was licensed, as Alinias, in the USA for the treatment of
diarrhoea and enteritis caused by Cryptosporidium spp. and Giardia
lamblia in 2002. Serendipitously, NTZ was suspected of having
antiviral activity when used to treat cryptosporidial diarrhoea in
acquired immune deficiency syndrome (AIDS) patients co-infected
with hepatitis B virus (HBV) or the Flaviviridae family member
hepatitis C virus (HCV) (Rossignol and Keeffe, 2008). Since then
NTZ has been shown to inhibit the replication of HBV, HCV (Korba
et al., 2008), Japanese encephalitis virus (Shi et al., 2014) and
influenza virus (Rossignol et al., 2009) in cells. Furthermore, the
mechanism underlying the anti-HCV activity of NTZ is reported to
involve phosphorylation of PKR (Elazar et al., 2009).
PKR is a serine/threonine kinase that is constitutively expressed
at low levels in all differentiated cells (Clemens, 1997). This kinase
is activated by dsRNA, which is generated during virus infections.
Binding of dsRNA to PKR promotes dimerisation and autopho-
sphorylation. The best-characterised substrate of PKR is the α
subunit of the translation initiation factor eIF2 (eIF2α). Phosphory-
lated eIF2α sequesters the guanine nucleotide exchange factor
n
Corresponding author. Tel.: þ 44 1865 275725; fax: þ 44 1865 275216.
E-mail addresses: Omodele.Ashiru@nibsc.org (O. Ashiru),
jonathon.howe@bioch.ox.ac.uk (J.D. Howe),
terry.butters@bioch.ox.ac.uk (T.D. Butters).
with eIF2-GTP, which is required for the formation of active 43S
pre-initiation complexes that are recruited to mRNA 50 cap
structures (Rowlands et al., 1988). Consequently, activation of
PKR results in global inhibition of cap-dependent translation
initiation. However, HCV translation, which is mediated by an
internal ribosome entry site (IRES), is insensitive to phosphory-
lated eIF2α (Robert et al., 2006).
The effect of NTZ on the replication of another Flaviviridae
family member was investigated in this study. HCV is a member of
the Hepacivirus genus of the Flaviviridae family, whilst Bovine viral
diarrhoea virus (BVDV) is a member of the Pestivirus genus of the
Flaviviridae family. BVDV is an enveloped virus possessing a single
strand, positive sense RNA genome. The genome of non-cytopathic
BVDV strains is approximately 12.3 kb in length, whilst that of
cytopathic BVDV strains varies greatly due to genomic duplications
and cellular RNA inserts. In addition to being an important
surrogate model for HCV infection (Buckwold et al., 2003), BVDV
is also an important veterinary pathogen and can cause severe
economic loss. As its name suggests, BVDV is primarily a pathogen
of cattle, however it can also infect most ruminant species. Out-
comes of BVDV infection can range from subclinical symptoms to
severe acute haemorrhagic syndrome (Neill, 2013).
In this study, we show that NTZ treatment significantly reduces
the replication of both cytopathic and non-cytopathic BVDV, via a
mechanism that is likely to involve the phosphorylation of PKR
and eIF2α. Unexpectedly, NTZ also induced PKR and eIF2α

http://dx.doi.org/10.1016/j.virol.2014.05.015
0042-6822/& 2014 Elsevier Inc. All rights reserved.
136 O. Ashiru et al. / Virology 462-463 (2014) 135–148

phosphorylation in uninfected Madin Darby Bovine Kidney
(MDBK) cells. This led to the observation that NTZ depletes ATP-
sensitive intracellular Ca2 þ stores in both MDBK and Huh-7 cells
(which are used for the HCV cell culture model). NTZ-mediated
Ca2 þ mobilisation resulted in mild ER stress, which perturbed
N-linked glycosylation of BVDV structural proteins. Despite inhi-
biting cell proliferation, NTZ treatment also elicited strong pro-
survival signals. Furthermore, NTZ upregulated expression of the
cellular protein TCTP, which is likely to play a role in the antiviral
activity of NTZ.
(vehicle) for 24 h following virus adsorption. Treatment with
10 μM NTZ had no significant effect on the titre of Pe515 and
NADL progeny, compared with DMSO treated controls (Fig. 1a).
However, 25 μM NTZ treatment resulted in a  99% reduction in
the titre of Pe515 and NADL progeny (Fig. 1a). In addition, 25 μM
NTZ also significantly reduced the proportion of MDBK cells
expressing the BVDV NS2-3 proteins 24 h post-infection (Fig. 1b).
NTZ mediates PKR and eIF2α phosphorylation in uninfected
MDBK cells

Due to the relatedness of HCV and BVDV, it was possible that

Results
NTZ inhibits BVDV replication
NTZ has been reported to mediate anti-viral effects via several
mechanisms, including the phosphorylation of both PKR and eIF2α
in HCV infected and replicon-harbouring Huh-7 cells (Elazar et al.,
2009) resulting in global inhibition of cap-dependent translation.
However, translation of HCV proteins is mediated by a viral IRES
that is insensitive to phosphorylated eIF2α (Robert et al., 2006).
To investigate the effect of NTZ on Flaviviridae IRES-mediated
translation, and on replication of Flaviviridae family members in
general, BVDV was employed. BVDV is a surrogate model for HCV
infection, and uses a functionally similar IRES for translation
(Frolov et al., 1998). BVDV strains exist as two biotypes: non-
cytopathic and cytopathic. Importantly, non-cytopathic BVDV
strains inhibit PKR activation, whilst cytopathic BVDV strains
activate PKR (Gil et al., 2006). Consequently, the effect of NTZ
treatment on Pe515 (non-cytopathic BVDV) and NADL (cytopathic
BVDV) replication was investigated. Pe515- and NADL-infected
MDBK cells were treated with NTZ (10 μM and 25 μM) or DMSO
NTZ inhibited the replication of both viruses via similar mechan-
isms. Western blots were carried out to determine whether NTZ
treatment resulted in PKR and eIF2α phosphorylation during
BVDV infection. Treatment with 25 μM NTZ resulted in phosphor-
ylation of PKR and eIF2α in Pe515-infected MDBK cells whilst
10 μM NTZ had little or no effect (Fig. 2a). NADL infection, as
previously reported (Gil et al., 2006), induced PKR and eIF2α
phosphorylation in MDBK cells. Consequently, any effect of NTZ
treatment on PKR and eIF2α in NADL-infected MDBK cells could
not be readily detected by Western blot. Unexpectedly, treatment
of uninfected MDBK cells with 25 μM NTZ also resulted in
significant phosphorylation of PKR and eIF2α. This suggested that
NTZ activated MDBK PKR in the absence of viral dsRNA.
To investigate the effect of NTZ on cellular and viral translation,
a translation reporter assay was carried out using MDBK cells
transfected with a bicistronic plasmid encoding firefly and renilla
luciferases expressed by HCV IRES- and cap-mediated translation.
NTZ treatment resulted in a dose-dependent increase in the ratio
of IRES- vs cap-mediated translation (as determined by luciferase
activity) in transfected, uninfected MDBK cells (Fig. 2b); thus
indicating that NTZ downregulated cellular cap-dependent

Fig. 1. NTZ has anti-BVDV activity. MDBK cells were infected with non-cytopathic BVDV strain Pe515 or cytopathic BVDV strain NADL, then treated with DMSO or NTZ for
24 h following virus adsorption. The titre of progeny virus within each culture supernatant was determined by fluorescent focus assay (a), whilst infection efficiency (b), i.e.
the proportion of cells expressing BVDV NS2-3 proteins, was determined by immunofluorescence microscopy.
 O. Ashiru et al. / Virology 462-463 (2014) 135–148 137

Fig. 2. NTZ induces PKR phosphorylation in uninfected MDBK cells. (a) Lysates of DMSO- or NTZ-treated MDBK cells were analysed, by Western blot, for expression of
phosphorylated PKR, total PKR, phosphorylated eIF2α and total eIF2α. MDBK cells were infected with BVDV at an MOI of 1, and treated with DMSO or NTZ for 24 h. (b) MDBK
cells were transfected with psiCHECK2-HCV IRES, a bicistronic plasmid encoding HCV IRES-translated firefly luciferase (FLuc) as well as renilla luciferase (RLuc) expressed by
cap-dependent translation. At 20 h post-transfection, cells were treated with DMSO or NTZ for 24 h. Ratio of FLuc to RLuc activity was calculated to determine the IRES:Cap
translation ratio. (c) DMSO- or NTZ-treated MDBK cells were analysed, by Western blot, for expression of TCTP following 24-h DMSO or NTZ treatment. GAPDH expression
was used as a loading control. (d) Fura-2-labelled MDBK and Huh-7 cells were treated with 0.1% DMSO or 0.1–50 μM NTZ. Fura-2 emission were measured and normalised to
average baseline emissions to determine cytosolic Ca2 þ levels following compound treatment. (e) DMSO or BAPTA-AM pretreated (p.t.) Fura-2-labelled MDBK cells were
treated with 0.1% DMSO or 25 μM NTZ.
138 O. Ashiru et al. / Virology 462-463 (2014) 135–148

translation but had no effect on viral IRES-mediated translation.

Interestingly, NTZ treatment of uninfected MDBK cells also
resulted in increased expression of translationally controlled
tumour protein (TCTP) (Fig. 2c), which is a cellular protein
encoded by a highly structured mRNA that activates PKR
(Bommer et al., 2002).
Thapsigargin, an inhibitor of HCV replication (Nakagawa et al.,
2005), also induces PKR phosphorylation in the absence of dsRNA
by depleting ER-sequestered calcium ions (Ca2 þ ) (Prostko et al.,
1995). In order to determine whether NTZ perturbs intracellular
stores of Ca2 þ , cytosolic Ca2 þ of MDBK and Huh-7 cells was
measured using Fura-2 AM, which is a cell permeable ratiometric
fluorescent Ca2 þ indicator. Fura-2 AM emits at wavelength 510 nm
when excited at wavelengths 340 nm and 380 nm. The 340/
380 nm ratio directly correlates to the intracellular Ca2 þ concen-
tration. Addition of 1–50 μM NTZ to Fura-2 AM-labelled MDBK
and Huh-7 cells immediately resulted in a dose-dependent
increase in cytosolic Ca2 þ (Fig. 2d). Pretreatment with the cell
permeable Ca2 þ chelator BAPTA-AM prevented the NTZ-mediated
increase in cytosolic Ca2 þ (Fig. 2e). This phenomenon was
observed for both 10 mM and 25 mM NTZ.

NTZ depletes ATP-sensitive Ca2 þ stores

Thapsigargin depletes ER Ca2 þ stores by inhibiting sarcoplasmic/

endoplasmic reticulum calcium ATPase (SERCA) (Thastrup
et al., 1990), which pumps Ca2 þ into the ER lumen thereby main-
taining the Ca2 þ gradient between the ER (mM Ca2 þ ) and the cytosol
( 100 nM Ca2 þ ). To determine whether NTZ also depletes ER Ca2 þ
stores, the effect of NTZ on thapsigargin-pretreated MDBK cells was
investigated. Addition of thapsigargin induced an immediate rise in
cytosolic Ca2 þ of MDBK cells (Fig. 3a). Subsequent addition of 25 μM
NTZ resulted in no further rise in cytosolic Ca2 þ in the thapsigargin-
pretreated cells (Fig. 3a), thereby indicating that NTZ depletes Ca2 þ
sequestered in the ER. NTZ treatment induced an increase in cytosolic
Ca2 þ even in the absence of extracellular Ca2 þ (Fig. 3b). Furthermore,
NTZ induced a similar fold increase in cytosolic Ca2 þ in the presence
and absence of extracellular Ca2 þ ; this indicated that all Ca2 þ
released into the cytosol by NTZ treatment were from one or more
intracellular Ca2 þ stores.
In addition to the ER, the Golgi, lysosomes and mitochondria
are also intracellular Ca2 þ stores. To identify NTZ-depleted intra-
cellular Ca2 þ store(s), the effect of NTZ on ionomycin-pretreated
MDBK cells was investigated. Ionomycin complexes with Ca2 þ
between pH 7 and 9.5, therefore it can only deplete Ca2 þ
sequestered in the ER (pH 7.2) and mitochondrial matrix
(pH 7.8), but not in the Golgi (pH 6–6.7) or lysosomes (pH 5.5)
(Paroutis et al., 2004). NTZ increased the cytosolic Ca2 þ levels of
MDBK cells pretreated with ionomycin in the presence (data not
shown) and absence of extracellular Ca2 þ (Fig. 3b). It was
necessary to carry out the assay in the absence of extracellular
Ca2 þ because depletion of ionomycin sensitive stores triggers an
influx of extracellular Ca2 þ , via Ca2 þ -release-activated Ca2 þ
(CRAC) channels in the plasma membrane (Hoth and Penner,
1992). The effect of NTZ on cells pretreated with ionomycin
indicated that the thiazolide depletes acidic Ca2 þ store(s), i.e.
the Golgi and/or lysosome. In concordance with this, NTZ raised
the cytosolic Ca2 þ levels of MDBK cells pretreated with CCCP,
which breaks down the mitochondrial membrane potential neces- Fig. 3. NTZ mobilises Ca2 þ from ATP-sensitive stores. Cytosolic Ca2 þ levels were
sary for Ca2 þ uptake (Fig. 3c). However, NTZ also increased the measured in Fura-2-labelled MDBK cells treated with two consecutive compounds:
cytosolic Ca2 þ in MDBK cells pretreated with GPN (a compound (a) DMSO or thapsigargin followed by DMSO or NTZ; (b) DMSO or ionomycin
followed by DMSO or NTZ; (c) DMSO or CCCP followed by DMSO or NTZ; (D) DMSO
whose hydrolysis by cathepsin C results in osmotic lysis of or GPN followed by DMSO or NTZ; or (e) water or ATP followed by DMSO or NTZ.
lysosomes) (Fig. 3d); thus ruling out the involvement of lysosomal Filled and open arrows denote the point at which the first or second compound
Ca2 þ stores in NTZ-mediated Ca2 þ mobilisation. These phenom- was added, respectively. Black and grey arrows denote assays carried out in the
ena were observed for both 10 mM and 25 mM NTZ. presence or absence of extracellular CaCl2, respectively.
 O. Ashiru et al. / Virology 462-463 (2014) 135–148 139

The ability of NTZ to raise cytosolic Ca2þ levels in the absence of
extracellular Ca2 þ (Fig. 3b) pointed to the involvement of intracellular
channels that release Ca2 þ into the cytosol. Since the involvement of
mitochondrial and lysosomal Ca2þ stores had been ruled out, the
effect of NTZ on Ca2þ release channels in ER and Golgi membranes
was investigated. Ryanodine receptor (RyR) and inositol 1,4,5-trispho-
sphate receptor (IP3R) are Ca2þ release channels present on the ER
and Golgi. Importantly, MDBK cells do not express RyRs (Schubert et
al., 2005). Consequently, treatment of MDBK cells with ryanodine
elicited no rise in cytosolic Ca2þ (data not shown). Interestingly,
addition of NTZ to MDBK cells pretreated with ATP, which allosterically
potentiates IP3R activity (Mak et al., 1999), resulted in no increase in
cytosolic Ca2þ (Fig. 3e). This phenomenon was observed for both
10 mM and 25 mM NTZ. Altogether, these data suggested that NTZ
depleted ATP-sensitive (IP3R-containing) intracellular Ca2þ stores, i.e.
the ER and Golgi.
NTZ induces ER stress
The ER is an IP3R-containing, ATP-sensitive Ca2 þ store. Ca2 þ is
essential for correct protein folding and transport within the ER,
such that its depletion leads to accumulation of misfolded proteins
and subsequent ER stress (Lodish and Kong, 1990). Notably,
depletion of intracellular Ca2 þ stores by both thapsigargin

Fig. 4. NTZ treatment promotes ER stress. (a) DMSO- or NTZ-treated MDBK cells were analysed, by Western blot, for expression of BiP following 24-h DMSO or NTZ
treatment. GAPDH expression was used as a loading control. MDBK cells were infected with BVDV at an MOI of 1, and treated with DMSO or NTZ for 24 h. (b) Transduced
MDBK or Huh-7 cells stably expressing Gaussia luciferase (GLuc) were treated with DMSO or NTZ for 24 h. GLuc activity in each culture supernatant was measured in relative
light units (RLU). (c) Culture supernatants of transduced MDBK or Huh-7 cells were assayed for GLuc activity 10 min to 6 h after addition of DMSO or NTZ. (d) Lysates of
DMSO- or NTZ-treated transduced MDBK cells were analysed, by Western blot, for intracellular GLuc. GAPDH expression was used as a loading control. (e) Transduced MDBK
cells were incubated with DMSO, NTZ and/or MG132. Culture supernatants were assayed for GLuc activity after 24-h treatments.
140 O. Ashiru et al. / Virology 462-463 (2014) 135–148
(Yoshida et al., 2006) and thiazolidinediones (Gardner et al., 2005)
have been shown to induce ER stress. Cells respond to ER stress by
triggering the unfolded protein response (UPR), which results in
inhibition of translation, upregulated expression of the ER chaper-
one protein BiP (also known as GRP78) and several other down-
stream effects. To determine whether NTZ-mediated perturbation
of intracellular Ca2 þ stores results in ER stress, the effect of NTZ on
BiP expression was investigated. Significant upregulation of BiP
was observed in uninfected and Pe515-infected MDBK cells treated
with 25 μM NTZ (Fig. 4a). Any effect of NTZ on NADL-infected
MDBK cells could not be definitively ascertained because the
cytopathic BVDV strain upregulated BiP expression, as has been
previously reported (Jordan et al., 2002).
Gaussia luciferase (GLuc) is a naturally secreted luciferase from the
copepod marine organism Gaussia princeps. Since the secretory path-
way is disrupted by ER stress, measurement of secreted GLuc activity
can be utilised as a sensitive reporter assay for monitoring ER stress.
MDBK and Huh-7 cells were transduced with the replication-deficient
lentivirus vector pCSCW-GLuc-IRES-CFP, which carries an expression
cassette for humanised GLuc. A 24-h treatment with 25 μM NTZ
significantly reduced the amount of GLuc secreted from transduced
MDBK and Huh7 cells (Fig. 4b). 24-h treatment with 10 μM NTZ
seemed to have no effect on GLuc secretion (Fig. 4b). However,
reduced GLuc secretion was observable in the first 2–4 h of 10 μM
NTZ treatment; this was more evident for Huh-7 cells than for
transduced MDBK cells (Fig. 4c). Thus, these data indicated that both
10 μM NTZ and 25 μM NTZ caused ER stress, but cells recovered from
10 μM NTZ treatment within 24 h. This might explain the apparent
inability of 10 μM NTZ to mediate an antiviral effect (Fig. 1b).
Brefeldin A (BFA), which inhibits ER to Golgi anterograde
transport but induces Golgi to ER retrograde transport, blocked
GLuc secretion throughout the 24-h treatment (Fig. 4b and c).
Western blot confirmed that GLuc was intracellularly retained in
BFA treated cells (Fig. 4d). No intracellular retention of GLuc was
observed in NTZ treated cells (Fig. 4d). NTZ-induced ER stress might
inhibit GLuc secretion by inhibiting GLuc translation and/or by
inducing degradation of GLuc. NTZ inhibits cap-dependent transla-
tion (Fig. 2a and b), hence the reduction in GLuc secretion might be
due to a reduction in GLuc translation. Therefore, the effect of
proteasome inhibitors on NTZ-mediated inhibition of GLuc secre-
tion following co-treatment with MG132 or ALLN was investigated.
Co-treatment with 0.1–5 μM MG132 (Fig. 4e) or 10 μM ALLN (data
not shown) did not prevent NTZ-mediated inhibition of GLuc
secretion, thus indicating that NTZ-mediated inhibition of GLuc
secretion is unlikely to involve proteasomal degradation.
NTZ inhibits cell proliferation and apoptosis
Cellular mechanisms aimed at counteracting ER stress can result
in the inhibition of cell proliferation or the induction of cell death.
To investigate the effects of NTZ on cell proliferation, cell metabo-
lism and cell division were analysed. Cell metabolism was assayed
using a colorimetric assay that measures absorbance of formazan
produced from reduction of MTS by dehydrogenase enzymes in
metabolically active cells. A 24-h treatment with 10 μM NTZ
inhibited MDBK cell metabolism by  30%, whilst treatment with
higher NTZ concentrations (25 μM and 50 μM) inhibited MDBK cell
metabolism by 70% (Fig. 5a). Inhibition of cell metabolism was also
observed after 6-h treatment with 25 μM and 50 μM (data not
shown). This is in agreement with previously published observa-
tions that NTZ inhibits metabolism of the human colon cancer cell
line Caco2 (Muller et al., 2008a). Cell division was assayed using
CFSE, which is a fluorescent molecule that crosslinks intracellular
proteins upon cleavage by intracellular esterases. The fluorescence
intensity of CFSE halves with each cell division. NTZ treatment
elicited a dose-dependent reduction in cell division of MDBK cells
(Fig. 5b). No cell death was observable by light microscopy follow-
ing 24-h NTZ treatment. Nonetheless, apoptosis was assayed by
flow cytometry using FITC-conjugated Annexin V and propidium
iodide (PI). Early-stage apoptotic cells are Annexin V-positive and
PI-negative, whilst late-stage apoptotic cells are Annexin V-positive
and PI positive. A 24-h NTZ treatment had no significant effect on PI
staining. A small increase in Annexin V staining (MFI¼17.6) of the
total PI-negative population was observed following NTZ treatment,
compared to DMSO treatment (MFI¼10.5) (Fig. 5c).
Treatment with MG132 (0.1–5 μM) resulted in MDBK cell death, as
observed by light microscopy and increased GLuc activity (Fig. 4e).
Thus, the increased GLuc activity was due to GLuc released from dead
cells. However, co-treatment with MG132 and NTZ promoted cell
survival, as determined by light microscopy and GLuc assay (Fig. 4e).
Importantly, 24-h treatment with 5 μM MG132 resulted in a sig-
nificant decrease in forward scatter (i.e. cell size) by flow cytometry
(data not shown), as well as the appearance of two distinct PI-
negative populations: Annexin V-low and Annexin V-high (Fig. 5d).
The Annexin V-high population (i.e. early-stage apoptotic cells)
represented  47% of the total PI-negative population, whilst the
Annexin V-low population had a similar MFI to NTZ treated cells.
MDBK cells co-treated with MG132 and NTZ exhibited similar
forward scatter and Annexin V staining to DMSO-treated cells; thus
NTZ inhibited MG132 induced early-stage apoptosis.
NTZ prevents complex glycan formation
Ca2 þ is required for post-translational modification of newly
synthesised secretory and membrane proteins in the ER (Brostrom
and Brostrom, 2003). Thus, NTZ-mediated depletion of ER Ca2 þ is
likely to perturb protein glycosylation, which is required for correct
protein folding and therefore export from the ER. Thapsigargin-
mediated depletion of ER Ca2 þ stores impairs N-linked glycosylation
of the rotavirus glycoproteins VP7 and NSP4 without inhibiting viral
protein synthesis (Michelangeli et al., 1995), whilst NTZ inhibits
maturation and cell surface expression of influenza virus glycopro-
tein hemagglutinin (Rossignol et al., 2009) and the replication of JEV
at similar concentrations to those used here (Shi et al., 2014).
Interestingly, thapsigargin also inhibits BVDV replication (Fig. 6a).
To determine whether NTZ also affects maturation of BVDV envelope
glycoproteins, MDBK cells were infected with NADL and treated with
NTZ, thapsigargin or the mannosidase inhibitor kifunensine for 24 h.
Secreted virions were collected, concentrated and lysed, then ana-
lysed by Endo H digest and Western blot (Fig. 6b). Kifunensine,
which prevents complex glycan formation, was used as a positive
control for the Endo H digest. Endo H partially digested E2 glyco-
protein from virus produced in DMSO-treated cells, indicating the
presence of a variety of glycan species of which some are complex
(Endo H-resistant) and some are oligomannose (Endo H-sensitive).
Treatment with NTZ increased sensitivity of E2 to Endo H digestion,
indicating that NTZ treatment prevents the formation of complex
glycans on BVDV envelope proteins. Thapsigargin treatment at
0.5 mM did not affect the Endo H sensitivity of E2 to the same extent
as NTZ at 25 mM (Fig. 6b) but increasing the concentration of
thapsigargin to 1.0 mM to demonstrate further sensitivity, led to a
complete ablation of viral biogenesis and as a consequence, a lack of
E2 immuno-detection (see Supplementary Fig 1).
To investigate whether NTZ treatment caused a disruption of
ER–Golgi transport, which could explain the inhibition of complex
glycan formation, MDBK cells were treated with either NTZ or
thapsigargin in the presence of ER α-glucosidase inhibitor NAP-
DNJ. Free oligosaccharides (FOS) were purified and analysed by
normal-phase HPLC (Fig. 6c). Inhibition of ER α-glucosidases
increases the level of cytosolic FOS Glc3Man5GlcNAc1, a marker
of ER-associated degradation (ERAD) (Alonzi et al., 2008), and ER
luminal FOS Glc3Man7GlcNAc2, a marker of a secondary
 O. Ashiru et al. / Virology 462-463 (2014) 135–148 141

Fig. 5. NTZ promotes cell survival. (a) Metabolism, as determined by formazan release using a MTS assay, of MDBK cells treated with 0.1–50 μM NTZ was measured after
24 h, then normalised to the metabolism of DMSO treated MDBK cells. (b) MFI of CFSE-labelled MDBK cells was measured after 24-h treatment with DMSO or NTZ, then
normalised to untreated CFSE-labelled MDBK cells. Note that CFSE fluorescence intensity halves with cell division. (c) Dot plots of MDBK cells stained with Annexin V and
propidium iodide after 24-h DMSO or NTZ treatment. (d) Dot plots of MG132 and/or NTZ treated MDBK cells stained with Annexin V and propidium iodide after 24 h.

degradation pathway for Golgi-retrieved proteins (Alonzi et al.,
2013). Treatment of MDBK cells with either NTZ or thapsigargin, in
the presence of NAP-DNJ, resulted in a concentration-dependent
reduction in ER-localised but not cytosolic FOS (Fig. 6d), indicating
a disruption to ER–Golgi transport. Thapsigargin, which was less
effective in modulating E2 glycosylation at the concentration used,
also affected FOS to a similar extent suggesting that, a global
analysis of disruption of ER–Golgi transport may mask protein-
specific effects on BVDV E2 glycosylation induced by NTZ.
Discussion
NTZ-mediated mobilisation of intracellular Ca2 þ
NTZ is a thiazolide with antiviral properties, as determined
by its effects on a variety of viruses including HCV. In this study,
NTZ was shown to inhibit replication of another member of the
Flaviviridae family. As reported for HCV, NTZ-mediated anti-BVDV
activity also involved PKR phosphorylation. Unexpectedly,
NTZ treatment induced PKR phosphorylation in uninfected MDBK
cells, i.e. in the absence of viral dsRNA. This led to the discovery
that NTZ depletes intracellular Ca2 þ stores – a process that is
known to induce PKR phosphorylation. NTZ depleted ATP-
sensitive intracellular Ca2 þ stores, i.e. the ER and Golgi, but did
not elicit an influx of extracellular Ca2 þ . Thus, it would be
important to ascertain whether NTZ has any effect on store-
operated calcium entry. Interestingly, NTZ is reported to have no
effect cytosolic Ca2 þ levels in erythrocytes, which lack an ER and
Golgi (Arnold et al., 2014). NTZ treatment upregulated BiP expres-
sion and reduced GLuc secretion (hallmarks of ER stress), and also
upregulated TCTP expression. Both TCTP and BiP genes possess
calcium-responsive promoter elements (Lee et al., 2004; Li et al.,
1993). Thus, depletion of ER Ca2 þ stores is likely to be the
142 O. Ashiru et al. / Virology 462-463 (2014) 135–148

Fig. 6. NTZ perturbs N-linked glycosylation of E2. (a) NADL-infected MDBK cells were treated with DMSO or thapsigargin (Tg) for 24 h following virus adsorption. The titre of
progeny virus within each culture supernatant was determined by fluorescent focus assay. (b) NADL-infected MDBK cells were treated with DMSO (lanes 1 and 2), 10 μM NTZ
(lanes 3 and 4), 0.5 μM thapsigargin (lanes 5 and 6) or 10 μM kifunensine (lanes 7 and 8) for 24 h. Secreted virus was lysed and incubated with water (lanes 1, 3, 5, 7) or
EndoH (lanes 2, 4, 6, 8) for 24 h. Lysates were separated on an 8% SDS-PAGE gel and probed for BVDV structural glycoprotein E2 by western blot. The major 65 kD band in the
untreated column (lane 1) represents BVDV E1E2 heterodimer. (c) NADL-infected MDBK cells were treated with a combination of 75 μM NAP-DNJ and either (1) DMSO;
(2) 25 μM NTZ; or (3) 1 μM thapsigargin for 24 h. Free oligosaccharides (FOS) were purified, labelled with 2-anthranilic acid and separated by NP-HPLC. Shown are
representative NP-HPLC profiles of FOS: (i) Glc3Man5GlcNAc1 and (ii) Glc3Man7GlcNAc2. (d) Peak areas of FOS were measured relative to amount of Man4GlcNAc1, which
remained unchanged for all treatments. Error bars indicate standard error.

primary signal for NTZ-mediated induction of BiP and TCTP
expression. Indeed, upregulated expression of TCTP and BiP was
observable, by Western blot, from 4 to 6 h post-initiation of NTZ
treatment (data not shown), as has also been shown for thapsi-
gargin (Chen et al., 2000; Xu et al., 1999). TCTP (mRNA and
protein) can only be upregulated by ER stress-inducing agents
that alter intracellular Ca2 þ homoeostasis (Xu et al., 1999). Con-
sequently, the ability of NTZ to induce ER stress and upregulate
TCTP expression further confirms that NTZ raises cytosolic Ca2 þ
levels by depleting (in part) ER Ca2 þ stores. Ionomycin pretreat-
ment experiments indicated that NTZ also depletes Ca2 þ seques-
tered in an acidic compartment. Since lysosomes were ruled out,
the Golgi seemed to be the likely candidate. Ca2 þ plays a role in
regulating intra-Golgi transport (Micaroni, 2012). Interestingly,
elevation of cytosolic Ca2 þ concentration above 100 nM is
reported to inhibit intra-Golgi transport (Porat and Elazar, 2000).
Thus, NTZ-mediated increase of cytosolic Ca2 þ might be expected
to inhibit protein trafficking.
 O. Ashiru et al. / Virology 462-463 (2014) 135–148
NTZ-mediated PKR phosphorylation
Elazar et al. determined, with the aid of a cell-free in vitro
biochemical assay, that NTZ directly enhances viral dsRNA-
mediated PKR autophosphorylation (Elazar et al., 2009). However,
NTZ-mediated PKR phosphorylation within cells could involve
additional or alternative mechanisms, especially since it can be
observed in uninfected cells. In addition to viral dsRNA, PKR is
activated by cellular factors, including PACT. Indeed, PACT is
directly responsible for PKR activation during thapsigargin treat-
ment (Lee et al., 2007). The TCTP mRNA is a cellular mRNA with
extensive secondary structure that is also known to activate PKR
(Bommer et al., 2002). Increased TCTP mRNA transcription and
upregulated TCTP protein expression are induced by depletion of
ER Ca2 þ stores and the resultant increase in cytosolic Ca2 þ ,
respectively (Xu et al., 1999). NTZ-mediated PKR phosphorylation
might involve the upregulation of TCTP mRNA as well as a direct
interaction between PKR and the TCTP mRNA.
In the study by Elazar et al., it was also observed that IFN
enhances NTZ-mediated phosphorylation of PKR and eIF2α (Elazar
et al., 2009). The eif2ak2 gene, which encodes PKR, is an
interferon-stimulated gene (ISG). Thus, IFN-mediated upregulated
PKR gene expression could result in increased PKR protein that
would be available for NTZ-mediated phosphorylation. However,
activation of PKR would ultimately inhibit translation of mRNAs
encoding PKR, other ISGs and IFN – hence this is a self-regulating
system. In concordance, chemical inhibition of PKR phosphoryla-
tion did not inhibit the anti-BVDV activity of NTZ (Data not
shown), and is likely to result in increased IFN and ISG translation.
NTZ and the UPR
Regardless of how NTZ mediates PKR phosphorylation, it is
clear that NTZ-mediated activation of PKR results in the phosphor-
ylation of eIF2α. eIF2α can also be phosphorylated by the kinase
PERK (PKR-like ER kinase), which is one of three inducers of the
UPR. Interestingly, phosphorylation of eIF2α favours translation of
Activating Transcription Factor 4 (ATF4), which drives expression
of pro-survival functions (Harding et al., 2003). Importantly, NTZ
treatment is reported to induce translational activation of ATF4 in
a PKR-dependent manner (Elazar et al., 2009). Clearly, upregula-
tion of ATF4 is a mechanism employed by the cell to adapt to NTZ
treatment. Cells can also adapt to ER stress by inducing
ER-associated degradation and autophagy in order to clear mis-
folded proteins accumulated in the ER. Experiments with MG132
and ALLN suggest that ERAD is not induced by NTZ treatment,
however NTZ has been shown to induce autophagy (Lam et al.,
2012).
Effect of NTZ on cell survival and proliferation
MG132 induces apoptotic cell death through the formation
of reactive oxygen species (Emanuele et al., 2002). NTZ inhibits
MG132-induced apoptosis, thereby indicating that the thiazo-
lide mediates strong pro-survival/anti-apoptotic signals. There
are published data showing that thapsigargin inhibits
cycloheximide-induced apoptosis as a result of plasma mem-
brane depolariation, via depletion of intracellular Ca 2 þ stores
(Chacon-Cruz et al., 1998). Interestingly, NTZ is also reported to
disrupt membrane potential (de Carvalho et al., 2011). NTZ
upregulates TCTP, which is a potent anti-apoptotic protein.
Over-expression of TCTP has been shown to protect
against cycloheximide-induced cell death (Wei et al., 2012).
Furthermore, TCTP is reported to mediate its anti-apoptotic activity
by antagonising the function of pro-apoptotic protein Bax, via inhibi-
tion of Bax dimerisation within the mitochondrial membrane
143
(Susini et al., 2008) and/or inhibition of p53-induced transcriptional
activation of Bax (Chen et al., 2011). Thus, NTZ might protect against
MG132-induced cell apoptosis by disrupting cell membrane potential
and/or by upregulating TCTP protein.
TCTP and BiP are both Ca2 þ -binding proteins (Lievremont et al.,
1997; Sanchez et al., 1997). Based on their intracellular localisation,
it is likely that BiP binds Ca2 þ in the ER as part of a protective
response involved in the restoration of normal ER function, whilst
TCTP binds Ca2 þ in the cytosol to prevent induction of Ca2 þ -
dependent apoptosis pathways. Indeed, Ca2 þ binding is required
for TCTP-mediated protection against Ca2 þ -dependent apoptosis
(Graidist et al., 2007). Thus, it is likely that TCTP and BiP are
upregulated during NTZ treatment as a protective response
designed to buffer Ca2 þ in both the cytoplasm and ER, respec-
tively, thereby preventing apoptosis following NTZ-mediated Ca2 þ
mobilisation and ER stress. It would be interesting to investigate
the effect of NTZ on expression of other Ca2 þ -binding proteins.
In addition to inhibiting apoptosis, NTZ treatment also down-
regulates cell metabolism and cell division. NTZ-mediated ER
stress induces global shutoff of cap-dependent translation (via
phosphorylation of eIF2α); hence, it is likely that NTZ treatment
leads to a block in the synthesis of cellular proteins involved in
running the cell cycle. Interestingly, NTZ-mediated eIF2α phos-
phorylation has been shown to promote translation of ATF4
(Elazar et al., 2009), which drives expression of pro-survival
functions (Harding et al., 2003). Thus, it is likely that NTZ
promotes cell survival by upregulating ATF4, TCTP and BiP. So, it
seems that NTZ holds cells in stasis until Ca2 þ homoeostasis and
cellular functions are restored. Altogether, these data indicate that
24-h NTZ treatment does not induce lethal acute ER stress, but
instead induces chronic sub-lethal ER stress that cells can adapt to
via multiple mechanisms.
Anti-viral consequences of NTZ-mediated Ca2 þ mobilisation
HCV IRES-mediated translation is not suppressed following
eIF2α phosphorylation (Robert et al., 2006). Due to functional
relatedness of HCV and BVDV IRESs, NTZ-mediated phosphoryla-
tion of PKR is unlikely to inhibit BVDV and HCV IRES-mediated
translation. Alternatively, NTZ might inhibit cap-dependent trans-
lation of cellular factor(s) required by Flaviviridae family members.
Indeed, the antiviral effect of NTZ might simply be due to
decreased cellular metabolism. Another possibility is that NTZ
mediates its antiviral effect by upregulating expression of a
cellular protein with anti-viral properties. In support of this, TCTP
has been shown to inhibit replication of white spot syndrome
virus in shrimp (Tonganunt et al., 2008) and Singapore grouper
iridovirus in grouper fish spleen cells (Wei et al., 2012).
HCV inhibits PKR phosphorylation with the aid of the IRES as
well as the viral proteins E2 and NS5A – these are also encoded by
BVDV. However, NTZ treatment is likely to create an environment
in which BVDV and HCV prematurely encounter activated PKR and
the associated intracellular antiviral defence mechanisms, despite
the evasion strategies employed by these viruses. Phosphorylation
of PKR and eIF2α was observable, by Western blot, from 4 to 6 h
post-initiation of NTZ treatment (data not shown). On the other
hand, cytopathic BVDV and HCV trigger phosphorylation of PKR
and eIF2α from 12 h post-infection (Gil et al., 2006; Arnaud et al.,
2010). Non-cytopathic BVDV, which account for the vast majority
of BVDV biotypes circulating in nature, does not induce PKR and
eIF2α phosphorylation throughout the infection (Gil et al., 2006).
Although translation of proteins encoded by species of the
Flaviviridae family is unlikely to be affected by PKR activation,
viral encoded glycoproteins synthesised in an NTZ-treated cell
would be translocated into the lumen of a dysfunctional ER. Hence,
NTZ treatment is likely to result in the accumulation of misfolded
144 O. Ashiru et al. / Virology 462-463 (2014) 135–148
BVDV and HCV glycoproteins. The significantly low progeny BVDV
titre observed after 24-h NTZ treatment could represent the virions
assembled from BVDV proteins “rescued” upon upregulation of ER-
resident molecular chaperones, such as BiP. Thus, NTZ treatment
might ensure that the host immune system is faced with a signifi-
cantly reduced viral burden, and so might result in virus clearance.
NTZ-mediated Ca2 þ mobilisation results in perturbation of
N-linked glycosylation and ER-Golgi trafficking of the BVDV
structural glycoprotein E2. In addition to E2, both BVDV and HCV
encode other glycoproteins (namely the structural proteins E1 and
Erns; the latter being unique to BVDV), which are processed and
folded in the ER. It is likely that NTZ treatment will also inhibit
correct folding and therefore export of these viral glycoproteins
from the ER. Furthermore, NTZ-mediated Ca2 þ mobilisation might
also explain the observation that thiazolides inhibit maturation
and cell surface expression of the influenza virus hemagglutinin
(Rossignol et al., 2009). The ER is also the primary site of genomic
replication and virion assembly for BVDV, HCV and other Flavivir-
idae family members. Interestingly, addition of exogenous CaCl2
inhibited in vitro replication of subgenomic HCV RNA in cytoplas-
mic lysates (Choi et al., 2006). Furthermore, H2O2-mediated
elevation of cytosolic Ca2 þ is reported to inhibit HCV RNA
replication in cells harbouring subgenomic and genomic HCV
RNA by disrupting active HCV replication complexes (Choi et al.,
2006). Note that HCV genome replication is localised to the
membranous web that is formed by modified membranes of the
ER and Golgi. Any effect of NTZ on Flaviviridae RNA replication has
yet to be investigated. Perturbations to ER function and Ca2 þ
homoeostasis would affect many aspects of the life cycle of these
ER-tropic viruses. Ca2 þ plays a key role in several aspects of all
virus life cycle, including virion stability, virus entry, viral DNA
transport, transcription, pathogenesis etc. (Ruiz et al., 2000).
Perhaps the best-characterised virus from this point of view is
rotavirus – another diarrhoegenic virus. Rotavirus-induced diar-
rhoea is also Ca2 þ -dependent and NTZ has been shown to reduce
the duration of rotavirus disease (Rossignol et al., 2006), and
inhibit rotavirus replication by reducing viroplasm-associated RNA
synthesis (La Frazia et al., 2013).
Attribution of the antiviral effects of NTZ to its ability to deplete
intracellular Ca2 þ stores would be more apparent if the thiazolide
inhibited BVDV replication in a dose dependent manner. Although,
10 μM raised cytosolic Ca2 þ levels, this thiazolide concentration
had no significant effect on BVDV replication. This discrepancy
might be explained by the observation that MDBK and Huh-7 cells
can recover, within 24 h, from ER stress induced by 10 μM NTZ but
not by 25 μM NTZ. Thus, it is possible that although treatment
with 10 μM NTZ depleted ATP-sensitive Ca2 þ stores, this thiazo-
lide concentration was less able to perturb cellular processes and
therefore was unable to induce ER stress and downstream antiviral
consequences. It is important to point out that by targeting cellular
processes, NTZ treatment is likely to significantly reduce the
possibility of viral escape mutants arising. Although NTZ's antiviral
action might also impact on “cellular health”, fortuitously the cell
has several strategies that enable it to survive the “harsh” treat-
ment that rids it of the viral burden. Note that NTZ (Alinias) has
an excellent safety profile in man, where peak plasma concentra-
tions of  37 μM are detected following oral administration of
500 mg NTZ (Stockis et al., 2002). The active metabolite of NTZ in
man is TIZ, following deacetylation, and both compounds are
equally effective inhibitors of protozoan parasites (Muller et al.,
2006) and BVDV biogenesis in MDBK cells (unpublished data).
In Neospora, NTZ and TIZ deplete Ca2 þ stores (Esposito et al., 2007)
and inhibits protein disulphide isomerase (PDI), the ER chaperone
upregulated in response to ER stress (Muller et al., 2008b),
suggesting that common inhibitory mechanisms exist in host cells
for parasitic and viral pathogens.
In conclusion, NTZ inhibits BVDV replication by a post-entry
mechanism that is likely to involve the depletion of ATP-sensitive
Ca2 þ stores, as well as the phosphorylation of PKR and eIF2α.
However, NTZ had no effect on IRES-mediated translation whilst
downregulating cap-dependent translation. Nevertheless, NTZ
treatment resulted in upregulated expression of the Ca2 þ -binding
proteins, BiP (an indicator of ER stress) and TCTP. TCTP is a protein
(encoded by a highly structured mRNA that can activate PKR) that
might play a key role in the anti-viral and pro-survival properties
of NTZ. Importantly, Ca2 þ mobilisation is the primary signal
elicited by NTZ, and might underpin many of the anti-viral
mechanisms attributed to NTZ to date.
Materials and methods
Reagents
The following compounds were supplied as follows: Nitazox-
anide (NTZ; Romark Laboratories), 1,2-bis(2-aminophenoxy)ethane-
N,N,N0 ,N0 -tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-
AM; Invitrogen), Thapsigargin (Calbiochem), Ionomycin (free acid;
Enzo Life Sciences), Carbonyl cyanide 3-chlorophenylhydrazone
(CCCP; Sigma-Aldrich), Gly-Phe β-naphthylamide (GPN; Santa Cruz
Biotechnology), Adenosine 50 -triphosphate disodium salt (ATP;
Enzo Life Sciences), Brefeldin A (BFA; Sigma-Aldrich), MG132
(Sigma-Aldrich) and kifunensine (Sigma-Aldrich).
ATP was dissolved in deionised water. All other compounds
were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) or
anhydrous DMSO (Invitrogen). All compounds were reconstituted
as 1000  stocks, in light of the fact that DMSO concentrations of
0.2% or higher increase intracellular Ca2 þ levels (Morley and
Whitfield, 1993). Cells were treated with these compounds or
0.1% vehicle/diluent for various time points ranging from 10 min to
24 h.
Cells
The adherent cell lines Madin Darby Bovine Kidney (MDBK),
Huh-7 (human hepatoma) and 293T (SV40 transformed human
embryonic kidney) were cultured in media and maintained at
37 1C in humidified 5% CO2, unless otherwise stated. MDBK cells
were cultured in RPMI 1640 media supplemented with 10% heat
inactivated, BVDV-free Foetal Calf Serum (FCS; Invitrogen), 2 mM
L-glutamine, 20 U/ml penicillin and 50 μg/ml streptomycin. During
BVDV infections, 10% horse serum (Sigma-Aldrich) was used
instead of FCS. Huh-7 and 293T cells were cultured in high glucose
(4.5 g/L) Dulbecco's modified eagle media (DMEM) supplemented
with 10% heat inactivated FCS, 2 mM L-glutamine, 20 U/ml peni-
cillin, 50 μg/ml streptomycin and 1  Minimal Essential Medium
(MEM) non-essential amino acids (Invitrogen). All cell culture
reagents were obtained from PAA, unless otherwise stated.
BVDV
The BVDV strains Pe515 (non-cytopathic) and NADL (cyto-
pathic) were kind gifts from Dr. Bryan Charleston (The Pirbright
Institute, UK) and Professor Nicole Zitzmann (Oxford Glycobiology
Institute, University of Oxford, UK). In order to propagate BVDV,
MDBK cells were infected at a multiplicity of infection (MOI) of
0.01 in horse serum-containing media at 37 1C in humidified 5%
CO2. Three days post-infection, virus-containing supernatants
were harvested, centrifuged at 2000 rpm for 10 min to remove
cellular debris, aliquoted and stored at  70 1C. Virus titres were
determined by Fluorescent Focus Assay.
 O. Ashiru et al. / Virology 462-463 (2014) 135–148
In order to investigate the antiviral effects of NTZ, confluent
MDBK cells (1  106 per well) in 6-well plates were washed with
PBS then infected with BVDV at an MOI of 1. Following an hour
incubation at 37 1C in humidified 5% CO2 (to allow virus adsorp-
tion), cells were washed and incubated with 2 ml cell culture
media (supplemented with horse serum) containing DMSO or NTZ
for 24 h at 37 1C in humidified 5% CO2. DMSO- or NTZ-treated
uninfected cells were set up in parallel as a control. Following the
24-h treatment, cells were washed in PBS then harvested in 500 μl
of Laemmli buffer and syringed to reduce viscosity, prior to
analysis by Western blot. The virus-containing supernatants were
centrifuged for 10 min at 2000 rpm (in order to remove cell
debris), and the progeny virion titre for each supernatant was
determined by the Fluorescence Focus Assay. Infection efficiency
was assessed by immunofluorescence microscope analysis of
untreated BVDV-infected MDBK cells.
Antibodies
The primary antibodies used were mouse anti-pestivirus NS2-3
(WB103/105; Veterinary Laboratories Agency), mouse anti-BVDV-
1 E2 (WB214; Veterinary Laboratories Agency), rabbit anti-
phosphorylated PKR (Thr 451; Santa Cruz Biotechnology), rabbit
anti-PKR (D-20; Santa Cruz Biotechnology), rabbit anti-
phosphorylated eIF2α (Ser51; cell Signaling Technology), rabbit
anti-eIF2α (FL-315; Santa Cruz Biotechnology), mouse anti-BiP (BD
Transduction Laboratories), rabbit anti-TCTP (abcam), rabbit anti-
GAPDH (Sigma-Aldrich) and mouse anti-Gaussia Luciferase (Nano-
Light Technology). Secondary antibodies used were FITC conju-
gated sheep anti-mouse (Sigma), HRP conjugated goat anti-mouse
(Dako) and HRP conjugated goat anti-rabbit (Dako).
Immunofluorescence microscopy
Cells were fixed for 30 min in 4% formaldehyde (methanol-free;
Polysciences), permeabilised for 10 min in 0.5% Triton-X-100
(Sigma-Aldrich), then blocked for 30 min in 10% FCS/0.5% Tween
20 (Sigma-Aldrich), which was designated IF blocking solution.
This was followed by hour-long incubations with primary anti-
body then species-specific fluorescence-conjugated secondary,
diluted in IF blocking solution. Unless otherwise stated, all
reagents were diluted in PBS (Sigma-Aldrich) and incubations
were carried out at room temperature. PBS washes were carried
out between each step. Nuclei were stained (and photobleaching
was prevented) by the addition of Vectashield mounting medium
with DAPI (Vector Laboratories). Fluorescence was visualised using
a Nikon Eclipse TE2000-U inverted microscope.
Fluorescent focus assay
BVDV titres were determined by Fluorescent Focus Assays
(FFAs). Briefly, 10-fold dilutions of virus-containing supernatants
were carried out using horse serum-containing cell culture media.
Confluent MDBK cells (5  104 per well) in 96-well plates were
infected using 50 μl of neat or diluted supernatant, in triplicate.
Following an hour incubation at 37 1C in humidified 5% CO2, the
virus inocula were removed. After washing with PBS, 100 μl of
horse serum containing media was added to the infected cells.
Cells were incubated at 37 1C in humidified 5% CO2 for 24 h, then
analysed by immunofluorescence microscopy. Virus titre was
determined by counting fluorescent foci and defined as Fluores-
cent Focus Units per millilitre (FFU/ml).
145
Western blot
Lysates were reduced and denatured by addition of β-merc-
aptoethanol (Sigma-Aldrich) and heating at 90 1C for 5 min,
respectively. Proteins were separated on 8% or 10% SDS-PAGE gels,
and transferred unto Immobilon-P membranes (Millipore). PVDF
membranes were blocked overnight at 4 1C in 5% BSA or milk/0.1%
Tween 20/PBS (Sigma-Aldrich), which was designated WB block-
ing solution. This was followed by hour-long incubations with
primary antibody then species-specific HRP-conjugated secondary,
diluted in WB blocking solution. Unless otherwise stated, all
incubations were carried out at room temperature on a rocking
platform. Washes using 0.1% Tween 20/PBS were carried out
between each step. Protein bands were visualised by chemilumi-
nescence using ECL Western Blotting Substrate or SuperSignal
West Pico Chemiluminescent Substate (Pierce Biotechnology) and
Amersham Hyperfilm MP (GE Healthcare). Where necessary
membranes were reprobed for a different antigen. Firstly, mem-
branes were incubated in stripping buffer [1 M Tris–HCl (pH 6.8),
10% SDS, 0.8% β-mercaptoethanol] (Sigma-Aldrich) for 30 min to
1 h at 80 1C. Following washes in 0.1% Tween 20/PBS, stripped
membranes were incubated in WB blocking solution overnight at
4 1C, then probed for proteins of interest.
Endo H digest
Confluent MDBK cells in 25 cm2 flasks were infected with BVDV
at an MOI of 1. After virus adsorption, cells were incubated in 5 ml
media containing DMSO, NTZ, thapsigargin or kifunensine for 24 h
at 37 1C in humidified 5% CO2. Virus-containing supernatants were
centrifuged for 10 min at 2000 rpm to remove cell debris, then
ultracentrifuged for 2 h at 20,000 rpm at 4 1C. Viral pellets were
resuspended in 500 μl PBS, then sonicated using a Branson
Sonifier S-250A (50% duty cycle, 20 s) to lyse virions. Lysates were
incubated with Endoglycosidase H (Endo H; NEB) or water at 37 1C
for 24 h. Non-reduced proteins were run on an 8% SDS-PAGE gel
then analysed by western blot.
Translation reporter assay
Triplicate wells of MDBK cells (4  104 per well) in white
96-well plates were transfected with psiCHECK2-HCV IRES
(Wang et al., 2011), which was a kind gift from Dr. Michael Lai
(Academia Sinica, Taiwan). psiCHECK2-HCV IRES is a bicistronic
plasmid carrying firefly luciferase (FLuc) and renilla luciferase
(RLuc) genes driven by HCV IRES- and cap-dependent translation,
respectively. At 20 h post-transfection, cells were treated with
DMSO or NTZ for 24 h. FLuc and RLuc activities were determined
using the Dual-Glos Luciferase Assay System (Promega) and a
SpectraMax M5 plate reader (Molecular Devices). For each well,
the ratio of FLuc to RLuc activity was calculated.
Measurement of cytosolic Ca2 þ
All fluorometric determinations of cytosolic Ca2 þ were carried
out at room temperature using Hank's Balanced Salt Solution
(HBSS; Invitrogen) with or without 1.3 mM CaCl2. MDBK or Huh-
7 cells (4  104 per well) in μClear black 96-well plates (Greiner)
were loaded with Fura-2 acetoxymethyl (AM; Invitrogen) and
pluronic acid (Invitrogen) for 30 min in the dark. The cells were
washed twice with HBSS, then incubated in 200 μl HBSS for a
further 30 min to allow complete de-esterification of intracellular
Fura-2 AM esters. Where necessary, the de-esterification step was
carried out in the presence of 10 μM BAPTA-AM or DMSO. Baseline
emissions of intracellular Fura-2 were measured for 5 min, at
1-min intervals using a SpectraMax M5 plate reader (Molecular
146 O. Ashiru et al. / Virology 462-463 (2014) 135–148
Devices). Fura-2 was alternatively excited at wavelengths of
340 nm and 380 nm, and emissions at 510 nm were measured.
Compounds of interest (including vehicle/diluent) were diluted
1:25 in HBSS in order to reduce cell damage from DMSO-
containing compounds. Each diluted compound (5 μl) was added
to Fura-2 loaded cells in triplicate, such that a final 1:1000 dilution
of each compound stock was achieved. Following compound
addition, Fura-2 emissions were measured for 10 min. Where
necessary, this was followed by the addition of a second com-
pound and a further 10-min measurement of Fura-2 emissions. For
each well and time point, the ratio of 340/380 nm emission was
calculated then normalised to the average baseline 340/380 ratio.
Note that no compartmentalisation of Fura-2 was observed, as
determined by the IonOptix Fluorescence Measurement System
(data not shown).
Generation of Gaussia luciferase expressing cell lines
To generate lentivirus bicistronically expressing humanised
Gaussia luciferase (GLuc) and cerulean fluorescent protein (CFP),
293T cells were cotransfected with pCSCW-GLuc-IRES-CFP (a kind
gift from Dr. B.A. Tannous, Harvard Medical School, USA) (Badr
et al., 2007), gag-pol expression plasmid pCMV8.91 and Vesicular
Stomatitis Virus G envelope expression plasmid pMD-G (both
were kind gifts from Professor P.J. Lehner, University of Cambridge,
UK), using X-tremeGENE 9 DNA Transfection Reagent (Roche). At
48 h post-transfection, lentivirus-containing supernatant was har-
vested, centrifuged at 2000 rpm for 10 min to remove cellular
debris, and used to transduce MDBK and Huh-7 cells.
Gaussia luciferase assay
In 24-well plates, MDBK or Huh-7 cells stably expressing GLuc
and CFP (1  105 per well) were incubated in 500 μl of media
containing DMSO, NTZ or BFA. At various time points after
compound treatments were initiated, 15 μl aliquots of conditioned
media were collected into μClear white 96-well plates (Greiner)
then assayed for GLuc assay. Briefly, 50 μl of 20 μM coelenterazine
(NanoLight Technology) was added to the aliquots of conditioned
media, and GLuc activity was measured using a SpectraMax M5
plate reader (Molecular Devices) in relative light units (RLU).
Where necessary, cells were harvested in 200 μl of Laemmli buffer
(per well) after 24-h compound treatments, then analysed for
GLuc protein by Western blot. Note that NTZ has no inhibitory
effect on GLuc activity (data not shown).
Apoptosis assay
In 6-well plates, MDBK cells (1  105 per well) were treated
with DMSO, NTZ and/or MG132 for 24 h. Cells (present in the
culture supernatants, and detached by EDTA) were harvested,
centrifuged then washed twice with PBS. Apoptosis was detected
using the Annexin V Apoptosis Detection Kit FITC (eBiosciences),
then analysed by flow cytometry (FACSCalibur; BD Biosciences).
MTS assay
Triplicate wells of MDBK cells (4  104 per well) in 96-well
plates were incubated in 200 μl media containing different NTZ
concentrations or DMSO, for 24 h at 37 1C in humidified 5% CO2.
In parallel, triplicate wells containing only diluted NTZ (i.e. no
cells) were set up as blanks, due to the yellow appearance of NTZ.
A 1:20 mixture of phenazine methosulfate (PMS; 1 mg/ml; Pro-
mega) and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-
phenyl)-2-(4-sulfophenyl)-2 H-tetrazolium (MTS; 2 mg/ml;
Promega) was added (40 μl) to all wells. MTS is bioreduced by
cells into a formazan product that is soluble in tissue culture
medium. Following a 3-h incubation at 37 1C in humidified 5% CO2,
the absorbance of formazan at 490 nm was measured using a
UVmax plate reader (Molecular Devices). The corrected A490 values
for NTZ-treated cells were normalised to the A490 value for DMSO-
treated cells. A sigmoidal dose–response (variable slope) curve
was generated using GraphPad Prism software.
CFSE assay
MDBK cells (1  106 per well) in 6-well plates were incubated
with 10 μM CFSE (Carboxyfluorescein succinimidyl ester;
eBioscience) for 15 min at 37 1C in humidified 5% CO2, then
incubated in media for a further 30 min. To measure the CFSE
level of undivided cells (designated “Day 0”), untreated CFSE-
labelled MDBK cells were detached using trypsin, fixed in 4%
formaldehyde for 15 min at room temperature, then analysed by
flow cytometry (FACSCalibur; BD Biosciences). The remaining
CFSE-labelled cells were washed in PBS, incubated with media
containing DMSO or NTZ for 24 h at 37 1C in humidified 5% CO2,
then analysed by flow cytometry. The geometric mean of the
fluorescent intensity (MFI) was determined for each treatment,
and normalised to the MFI of “Day 0” cells.
FOS analysis
MDBK cells (1  106 per well) in 6-well plates were incubated
in media containing the α-glucosidase inhibitor NAP-DNJ
(Rawlings et al., 2009) with DMSO, NTZ or thapsigargin for 24 h
at 37 1C in humidified 5% CO2. The cells were dounce-
homogenised then free oligosaccharides (FOS) were extracted
and purified as previously described (Alonzi et al., 2008; Neville
et al., 2004). Briefly, FOS were labelled with 2-anthranilic acid and
purified by chromatography using Discovery Speed Amide-2
columns. Labelled FOS were further purified by chromatography
using Concanavalin A-Sepharose 4B columns (100 ml packed
resin), then separated by normal phase-HPLC (Waters) using a
4.6  250 mm2 TSKgel Amide 80 column (Anachem) as des-
cribed previously (Alonzi et al., 2009).
Statistical analysis
All statistical analyses were performed using GraphPad Prism
software. A two-tailed unpaired Student's t-test using 95% con-
fidence intervals was used to assess statistical significance
(p o0.05). Where n ¼p r0.05, nn ¼p r0.01 and nnn ¼p r0.001
relative to control. Unless otherwise indicated, each value reported
is the mean and standard deviation of three or more independent
experiments.
Acknowledgments
We would like to thank Professor Anant Parekh (University of
Oxford, UK) for his support and scientific advice, as well as for
careful review of the manuscript. We also thank Dr. Juris Galva-
novskis and Professor Frances Ashcroft (University of Oxford, UK)
for their technical advice, and use of their IonOptix Fluorescence
Measurement System. Professor Raymond Dwek is thanked for
helpful discussion. This work was supported by the Oxford
Glycobiology Institute, UK and Romark Laboratories, USA.
Appendix A. Supplementary material
Supplementary data associated with this article can be found in
the online version at http://dx.doi.org/10.1016/j.virol.2014.05.015.
 O. Ashiru et al. / Virology 462-463 (2014) 135–148
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