Download SDS-PAGE and Western blot analysis

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Crimean-Congo hemorrhagic fever replication interplays with regulation mechanisms of
apoptosis
Running title: CCHFV interplays with caspase induced apoptosis
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Helen Karlberg 1,2, Yee-Joo Tan 3,4,* and Ali Mirazimi 1,2,5*
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Public Health Agency of Sweden, SE-171 82 Sweden
Karolinska Institute, Stockholm, SE-171 77, Sweden
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Department of Microbiology, Yong Loo Lin School of Medicine, National University Health
System (NUHS), National University of Singapore, Singapore
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Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and
Research), Singapore.
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National Veterinary Institute, SE- 756 51 Uppsala
Nr of Words Abstract: 223
Nr of Words MS: 4668
Nr of Figures: 6
* Corresponding authors:
E-mails: [email protected] (A. Mirazimi) & [email protected] (Y.-J. Tan)
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Abstract
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Pathogenesis of viral hemorrhagic fevers (VHF) is associated with alteration of vascular
barrier function and hemorrhage. To date, the specific mechanism behind this is unknown.
Programmed cell death and regulation of apoptosis in response to viral infection is an
important factor for host or virus survival but this has not been well-studied in the case of
Crimean-Congo hemorrhagic fever virus (CCHFV).
In this study, we demonstrated that CCHFV infection suppresses cleavage of poly (ADPribose) polymerase (PARP), triggered by staurosporine at early post infection. We also
demonstrated that CCHFV infection suppresses activation of caspase-3 and caspase-9. Most
interestingly, we found that CCHFV N can suppress induction of apoptosis by Bax and inhibit
the release of cytochrome-c from the inner membrane of mitochondria to cytosol. However,
CCHFV infection induces activation of Bid at late post infection, suggesting the activation of
extrinsic apoptotic signaling. Consistently, supernatant from late post-infected cells
stimulated was found to induce PARP cleavage, most probably through the TNF-α dead
receptor pathway. In summary, we found that CCHFV has strategies to interplay with
apoptosis pathways and thereby regulate caspase cascade. We suggest that CCHFV
suppresses caspase activation at early stages of the CCHFV replication cycle, which perhaps
benefits the establishment of infection. Furthermore, we suggest that the host cellular
response at late post infection induces host cellular pro-apoptotic molecules through the death
receptor
pathway.
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Introduction
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Crimean-Congo hemorrhagic fever virus (CCHFV) is a member of the Nairovirus genus of
the family Bunyaviridae. CCHFV is found worldwide (Asia, Africa Middle East, Europe) and
causes severe disease in humans, with a reported mortality rate of up to 30%. CCHFV is
transmitted through tick bites or through contact with viremic blood or tissues from patients
or livestock. Viruses within this family encode for three single-stranded RNA segments with a
negative sense. The large (L) segment encodes the RNA-dependent RNA polymerase, the
medium (M) segment codes for the two mature structural glycoproteins (Gn and Gc) and the
small (S) segment encodes the nucleocapsid protein (N) (Elliott, 1990; Ergonul et al., 2006).
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The pathogenesis of viral hemorrhagic fevers (VHF) is associated with alteration of vascular
barrier function and hemorrhage, most probably due to a combination of factors such as
damage to the epithelium, consumption and degradation of clotting factors, and an overactive
inflammatory response. The reduced capacity or dysfunction of endothelial cells observed in
CCHF cases is believed to be due to virus-mediated host factors such as the pro-inflammatory
response, to the virus infection itself, or a combination of both. Therefore, a hyperactive host
response may be important in progression of the disease. Cell death might contribute to the
increased permeability of the vascular system and bleeding disorders observed among
CCHFV patients. Clinical studies indicate that high viremia is correlated with high cytokine
levels and disease severity (Bray, 2005; Schnittler & Feldmann, 2003; Weber & Mirazimi,
2008).
We have previously demonstrated that CCHF virus can induce cleavage of poly (ADP-ribose)
polymerase (PARP), which is associated with apoptosis and has been cited as one hallmark of
apoptosis and caspase activation at late post infection (Karlberg et al., 2011). Rodrigues et al.
subsequently showed that apoptosis is induced in human hepatoma cells at late stages of
CCHFV infection (Rodrigues et al., 2012). Caspase-dependent apoptosis occurs following the
activation of two main pathways initiated by both internal (intrinsic/mitochondria-dependent
pathway) and external stimuli (extrinsic/death receptor-mediated pathway). Caspases,
cysteine aspartate-specific proteases, are activated and amplify the apoptotic signaling
pathways, leading to cell death. Effector caspases, including caspase-3, -6, and -7, and their
activation are controlled by upstream initiator caspases such as caspase-8 and -9. Extrinsic
and intrinsic pathways may function separately or mediate a crosstalk interconnection through
cleavage of pro-apoptotic Bid, a member of the BCL-2 family of cell death regulators, which
finally ends with cell death (Thornberry, 1998; Zimmermann et al., 2001). Pro-inflammatory
host-derived mediators may contribute to receptor-mediated apoptosis in response to viral
infection. External stimuli activate death receptors that include TNFR, Fas/CD95, and
TRAIL, which function to induce apoptosis. Upon binding of their ligands, these receptors
become activated and initiate activation of caspase-8, which in turn activates Bid.
The intrinsic, or mitochondria, pathway is associated with the release of mitochondria
proteins such as cytochrome-c, which activate downstream caspase activity through activation
of caspase-9 from the intermembrane space into the cytoplasm (Thornberry, 1998;
Zimmermann et al., 2001; Zimmermann & Green, 2001). Intracellular processes can result in
loss of mitochondrial integrity via pro-apototic members of the BCL-2 superfamily members,
for example Bid, Bak and Bax, which act directly on the outer mitochondrial membrane,
facilitating secretion of mitochondria proteins (Zimmermann et al., 2001; Zimmermann &
Green, 2001).
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The aims of this study were to determine whether CCHFV regulates apoptosis pathways and
to investigate the mechanism behind the regulatory properties of CCHFV, which interferes
with the apoptotic signaling chain during the virus replication cycle.
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Results
CCHFV replication suppresses activation of caspase-3 and caspase-9.
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We have previously demonstrated that CCHFV infection induces caspase-3 activation at late
post infection in SW-13 (Karlberg et al., 2011). In order to investigate the events early in the
CCHFV replication cycle, in the present study, mock or CCHFV infected SW-13 cells (MOI
1 at 24 h.p.i.) were treated with different concentrations of STS, a relatively non-selective
protein kinase inhibitor, which is often used as a general method for inducing apoptosis. At 5
hours post treatment, detached cells were harvested and analyzed by Western blot. It was
found that cleavage of PARP was clearly suppressed in the CCHFV-infected cells, compared
with the mock-infected cells, in the 4µM STS treatment (Figure 1A-B). However, at the
highest concentration of STS used, no inhibition of PARP cleavage within infected cells was
observed.
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To investigate whether CCHFV infection regulates caspase-3 activation during STS
treatment, SW13 cells were infected and treated as described above (with 4uM STS) and
analyzed for the presence of caspase-3 cleavage. It was found that the activation of caspase-3
in the CCHFV infected cells was significantly reduced compared with the mock-infected cells
(Figure 2A-B). To further characterize the interaction of CCHFV replication and the
apoptosis pathway, we analyzed these samples for STS-induced cleavage of caspase-9, which
is upstream of caspase-3. The results showed that cleavage of caspase-9 was also suppressed
during the early phase of CCHFV replication cycle in STS-treated infected cells, in contrast to
mock infected cells (Figure 2C-D).
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CCHFV infection suppresses release of cytochrome-c in apoptotic cells
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In order to investigate whether CCHFV infection interferes or interplays with release of
cytochrome-c from inner mitochondria membrane in apoptotic cells caused by STS, mock
infected and CCHFV-infected cells (24 h p.i.) were not treated or treated with STS. Attached
and detached cells were harvested at 5 h post treatment, and the cytosol and mitochondria
fractions were collected separately ((Gogvadze et al., 2003)) and analyzed for estimated
release of cytochrome-c by WB. It was found that release of cytochrome-c into the cytosol
was decreased in infected cells compared with mock-infected cells, for both attached and
detached cells (Figure 3).
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CCHFV N protein suppresses activation of apoptosis by Bax
Release of proteins, such as cytochrome-c, from mitochondria inter-membrane space to
cytosol is controlled by BCL-2 pro-apoptotic members such as Bax. To investigate whether
the CCHFV N protein is involved or contributes to regulation of mitochondria membrane
permabilisation and downstream caspase activation, we established an in vitro system (see
Materials & Methods section). The SW13 cells were transfected with a plasmid coding for
CCHFV ORF N (myc-N), with different concentrations and a plasmid coding for Bax (flagBax), a potent inducer of apoptosis that acts via the mitochondria. Induction of apoptosis in
the presence and absence of CCHFV N was then analyzed by measuring the activation of
caspase-3. The results showed that overexpression of Bax (with a flag epitope at the N
terminus) in SW13 cells induced a high level of apoptosis, as determined by the activation of
caspase-3 and cleavage of endogenous PARP, when compared with mock transfected cells
(Figure 4). However, the level of apoptosis induced by Bax was significantly reduced when
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Bax was co-expressed with different amounts of recombinant N protein. These results suggest
that expression of N in the early phase of CCHFV infection may suppress the apoptotic
processes that are coordinated by Bax or downstream of Bax and cause the CCHFV-infected
cells to become resistant to apoptosis.
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Pro-inflammatory cytokines released during CCHFV infection can induce PARP
cleavage
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As demonstrated above, we have evidence that at 24 h p.i., CCHFV has the ability to suppress
caspase-3 activation through inhibition of release of cytochrome-c by interfering with Bax
This finding seems to contradict our previous observation that CCHFV infection induces
apoptosis at late post infection (Karlberg et al., 2011). To investigate whether CCHFV
infection initiates apoptotic signaling pathways through the extrinsic pathway at late post
infection, SW13 were mock or CCHFV-infected (MOI 1). At 24 and 48 h p.i., cells were
harvested and analyzed for activation and cleavage of Bid (truncated Bid, tBid), which is a
pro-apoptotic member of the BCL-2 family, by WB. External stimuli such as proinflammatory factors (TNF-α) activate the extrinsic or death receptor signaling pathways,
which in turn activate caspase-8, leading to activation of Bid. Our results clearly
demonstrated that CCHFV infection at late post infection led to cleavage of Bid to tBid, in
attached and detached cells (Figure 5).
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It has been demonstrated, by us and others (Karlberg et al., 2011; Rodrigues et al., 2012), that
CCHFV infection induces secretion of TNF-α and other pro-inflammatory factors at late post
infection through activation of dendritic cells and macrophages. In the present study we
examined whether supernatant of infected SW13 releases pro-inflammatory factors, such as
TNF-α, during infection, using SW13 cells which were mock infected or CCHFV-infected
(MOI 1) and harvested at 24, 48, and 72 h.p.i. Analysis of supernatants for the presence of
TNF-α showed that infected cells secreted TNF-α at late post infection (data not shown).
However, to confirm whether the induction of cleavage of PARP at late post infection is due
to an extrinsic pathway, all supernatants, mock and CCHFV infected, were UV-inactivated in
order to inactivate the virus (which may be present in the supernatant) and then transferred to
seeded new SW13 cells for 24 hours. The presence of CCHFV N, as an indicator of
inactivation with UV and cleavage of PARP, was determined by Western blot for all lysates
(data not shown). The results showed clearly that activation and cleavage of PARP was
induced in cells where supernatant from infected cells collected at either 48 or 72 h.p.i. was
added (Figure 6A-B). In contrast, no PARP cleavage was observed for cells incubated with
supernatant from mock infected or UV-inactivated virus infected cells.
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In order to characterize the process in more detail, supernatants collected at 48 h.p.i. from
infected cells were mock treated or treated with neutralizing TNF-α antibodies (5ug/mL) for 1
h at 37oC and transferred to seeded SW13. At 24 h post treatment, detached cells were
harvested and analyzed with Western blot. The analysis showed that antibodies against TNF-α
significantly suppressed cleavage of PARP (Figure 6C), indicating that TNF-α secreted from
infected cells is a contributor to induction of the extrinsic apoptotic pathway during the late
phase of infection.
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Discussion
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A characteristic of CCHF and other VHF is loss of epithelium cell function, in particular
function of endothelial cells, which leads to changes in vascular permeability and dysfunction
and imbalanced fluid distribution between the intra- and extra-vascular tissue space, causing
coagulation disorders, hemorrhage, and multi organ failure (Peters & Zaki, 2002).
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Recently, two animal models with defective interferon response, IFNAR- and STAT1- mice,
which are killed by/susceptible to CCHFV infection, have been used to study the pathogenesis
of the disease (Bente et al., 2010; Bereczky et al., 2010). Infected mice developed leukopenia,
thrombocytopenia, and elevated levels of liver enzymes. The highest levels of CCHF vRNA
were detected mainly in liver and spleen. These organs were clearly affected, with visible
symptoms such as necrosis of the liver and massive lymphocyte depletion of the spleen.
Infected mice also had elevated levels of pro-inflammatory cytokine (TNF-α, IL 6, IL10) in
serum/blood samples (Bente et al., 2010; Bereczky et al., 2010). An over-stimulated host
protective response might be one contributing factor to coagulation disturbances, with
hemorrhage and the overall vascular system affected.
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In previous studies, we and others revealed that caspase-dependent apoptotic pathways are
induced in different human cell types late during the CCHFV replication cycle (Fraisier et al.,
2014; Karlberg et al., 2011; Rodrigues et al., 2012). Activation of executor caspase-3 then
leads to cleavage of the viral structural protein, N. We found that caspase activation does not
favor progeny viral production and therefore suggested that it is induced by the host cell as a
protective response, rather than induced by the virus infection (Karlberg et al., 2011). Viruses
have different strategies to interfere with cell death pathways during infection to secure
efficient progeny viral production by expressing viral proteins (encode gene products) or
down-/up-regulate cellular pro- and anti-apoptotic proteins, which interfere with apoptotic
signaling pathways. Rift Valley fever virus, a member of the Bunyaviridae, encodes NSm
protein with anti-apoptotic functions by inhibiting caspase-3 activity and upstream initator
caspases (Won et al., 2007). Another member of the Bunyaviridae, La Crosse virus, induces
increased levels of cellular BCL-2 during infection, both in vivo and in vitro, which prolongs
cell survival and seems to be important in decreasing apoptosis and preventing tissue damage
(Pekosz et al., 1996).
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In the present study, where SW13 cells were used as a model system, we demonstrated that
apoptotic signaling pathways are regulated early during the CCHFV replication cycle. By
challenging CCHFV-infected cells at 24 h p.i. with STS, we were able to show that activation
both upstream and on executor caspase level (caspase-9 and caspase-3) was suppressed. In
line with these results, we also showed that activation and cleavage of PARP, a marker of
apoptosis, are suppressed.
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Opening of the mitochondrial permeability transition (MPT) pore and loss of membrane
potential leads to release of pro-apoptotic proteins such as cytochrome-c from the
intermembrane space into the cytosol of cells undergoing apoptosis (Zimmermann et al.,
2001). The released cytochrome-c and Apaf-1 form a complex known as the apoptosome,
leading to caspase-9 activation, which further activates downstream effector caspases
(Zimmermann et al., 2001). In the present study, we showed that secretion of cytochrome-c
from the mitochondria into the cytosol was decreased in STS-treated CCHFV-infected cells,
but not in STS-treated mock infected cells. This indicates that the release of mitochondria
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proteins is regulated and caspase activation downstream of mitochondria level, including
caspase-3, is suppressed or delayed during the early phase of CCHFV infection. It has
previously been demonstrated that hepatitis C virus (HCV) encodes proteins with antiapoptotic activity, which interfere with apoptosis signaling pathways by different
mechanisms. HCV NS2 interferes with caspase-dependent induced apoptosis by counteracting
cytochrome-c release from mitochondria, which contributes to viral persistence by interfering
with host cell defense (Erdtmann et al., 2003). In the mitochondrial pathway, pro-apoptotic
members of the BCL-2 super family are associated with the mitochondria and release of
cytochrome-c. Bax is one of the key pro-apoptotic (BCL-2 member) molecules, an inactive
monomer, and is normally found in the cytosol or loosely bound to the mitochondria
membrane. Upon activation by apoptotic stimuli, Bax changes confirmation and forms
oligomers, which are then integrated by formation of membrane pores that facilitate release of
mitochondrial proteins such as cytochrome-c, which leads to downstream caspase activation
(Antonsson et al., 2000; Wolter et al., 1997). By using a recombinant expression system, we
showed that the overexpression of viral structural nucleocapsid (N) could inhibit Bax-induced
apoptosis. These results indicate that the high level of CCHFV N expressed in infected cells
has the ability to protect against apoptotic stimuli by acting at the level of Bax or downstream
of it. Hence N could be one of the major factors controlling cell death in the early phase of
infection. It has recently been demonstrated that rubella virus (RV) capsid protein interferes
with import of Bax into mitochondria and thereby prevents mitochondria membrane
permabilisation and promotes cell survival, and that adenovirus (ADV) induces expression of
anti-apoptotic protein E1B 19K (vBcl-2), which inhibits the TNF-α-mediated death signaling
pathway, including release of mitochondria proteins such as cytochrome-c by directly
interacting with Bax (Ilkow et al., 2011; Sundararajan et al., 2001). It has also been reported
that Hantaan virus (HTNV) nucleocapsid protein modulates apoptosis pathways through NFƙB (Ontiveros et al., 2010).
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As discussed above, we have evidence that at 24 h p.i., CCHFV infection is able to inhibit
caspase-3 activation through regulation of cytochrome-c release by interplaying or interfering
with Bax. This finding can be interpreted as contradicting our previous finding that CCHFV
infection induces apoptosis at late post infection (Karlberg et al., 2011). External stimuli
through the death receptor pathway might initially induce caspase activation, triggered by the
viral infection, and often results in crosstalk between the extrinsic and intrinsic cell death
pathways through the pro-apoptotic member Bid, thereby facilitating induction of the
mitochondrial pathway (Luo et al., 1998; Perez & White, 2000). Transmissible gastroenteritis
virus infection induces both the FasL and mitochondria pathways through interconnection
with Bid and upregulation of Bid (Ontiveros et al., 2010).
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In the present study, we clearly demonstrated that Bid is active at late post infection and also
that supernatant of CCHFV-infected cells contains the pro-inflammatory factors. These
findings together indicate that CCHFV at late post infection most probably induces apoptosis
through induction of pro-inflammatory factors, which in turn activates the extrinsic apoptotic
pathway. This assumption is in line with the data in Figure 6A and B, which show that UVinactivated supernatants collected from CCHFV-infected cells at 48 and 72 h p.i. can induce
cleavage of PARP in fresh cells from as early as 24 h post-treatment. Since all the
supernatants collected were UV-inactivated before being transferred to the fresh cells,
apoptosis could not have been induced by virus replication. Instead, it is most likely caused
by pro-inflammatory mediators released into the medium as a cellular protective response to
infection. This suggests in turn that caspase activation is triggered by external stimuli as a
secondary effect. In the present study, we also demonstrated that neutralizing TNF-α
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antibodies reduced the cleavage of PARP triggered by supernatant from infected SW13 cells
at 48 h p.i. (Fig 6C). However, the neutralization was not complete, most probably because
we could not neutralize all TNF-α or because other molecules are also involved in triggering
the extrinsic pathway.
Hepatitis C virus induces apoptosis by an indirect immunologically mediated mechanism and
viral core protein enhances this process through TNF signaling pathways (Zhu et al., 1998).
Cytokines and chemokines regulate the host immune response, but when present in high
concentrations might contribute to toxic effects. Dysregulation of the cytokine and chemokine
response is anticipated to cause capillary fragility, leading to leakage of erythrocytes and
plasma through the vascular endothelium and resulting in dangerous hypotension, which is a
prominent feature of VHF (Geisbert & Jahrling, 2004). Previous studies have shown that key
molecules in CCHF progression seem to be cytokines such as IL 6, IL 8, and TNF-alpha. In
an animal model study, mice lacking STAT-1 had elevated levels of IL-6, IL-10, and TNFalpha concentrations, confirming findings in some severe human cases (Bente et al., 2010;
Ergonul et al., 2006; Papa et al., 2006).
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In summary, this study showed that CCHFV has strategies to interplay with apoptotic
molecular signaling pathways and thereby regulatory properties. It also showed that CCHFV
N possesses the function of suppressing mitochondria permeability through Bax, which in
turn has an influence on downstream executor caspase activity. This delays or suppresses
induced caspase activation during the early phase of the CCHFV replication cycle. This event
perhaps works to the benefit of the virus, helping or allowing it to establish infection. In later
phases of CCHFV infection, we suggest that the host cellular response may induce the death
receptor pathway, leading to activation of Bid. However, this hypothesis should be followed
up in future experiments.
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Methods
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Cells, antibodies, chemicals and virus
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SW13 cells (human adrenal cortex adeno carcinoma cells) were maintained in Leibovitz’s
medium (L15) supplemented with 2% fetal bovine serum and antibiotics (10 U/mL penicillin
and 10 µg/mL streptomycin). The antibodies used in this study included a rabbit polyclonal
anti-CCHFV nucleocapsid antibody (Andersson et al., 2004). Anti-myc monoclonal (Santa
Cruz Biotechnology, Santa Cruz, CA), anti-actin monoclonal, anti-flag polyclonal (Sigma, St.
Louis, MO), anti-PARP polyclonal, anti-caspase 3, 9 monoclonal (Cell Signaling Technology,
Beverly, MA), anti-cytochrome c and anti-TNF-α antibodies, recombinant TNF-α (rTNF-α)
(BD Pharmingen), and staurosporine (STS) (Cell Signaling Technology) were used according
to the manufacturer’s instructions. Horseradish peroxidase (HRP)-conjugated antibodies
(Pierce, Rockford, IL or Bio-Rad, Hercules, CA) were used, again according to the
manufacturer’s instructions. The Nigerian CCHFV Ibar10200-strain, originally isolated in
Nigeria, was used in the experiments and all handling of live virus was performed in a BSL-4
facility.
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UV-inactivation
Harvested cell supernatant was centrifuged at 13000 rpm for 10 minutes and irradiated at 254
nm (UVG-54) in order to inactivate viral particles.
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In vitro infection model (challenging SW13 cells)
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SW13 cells were seeded in 6-well plates and then infected with CCHFV (MOI=1). After 24
hours post infection (h.p.i.), cells were challenged with 4, 8, 16, or 32 µM STS to induce
apoptosis. The cells were then further incubated for 5 hours, harvested (detached and attached
cells separately), and analyzed with Western blot analysis for activation of caspase-3,
caspase-9 and induced cleavage of PARP.
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SW13 cells were infected with CCHFV, mock infected or treated with UV-inactivated virus.
At different times post infection (24, 48 and 72 h p.i.), supernatants were harvested and UVinactivated. These supernatants were then transferred to new SW13 cells seeded in 6-well
plates. At 24 h p.i., the cells was collected and analyzed for cleavage of PARP in the absence
of replicating virus.
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Cytosolic and mitochondrial cytochrome-c analysis
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In order to analyze the release of cytochrome-c from mitochondria, SW13 cells were seeded
in 6-well plates and either infected with CCHFV (MOI=1) or not infected. At 24 h p.i., the
cells were incubated with apoptotic stimuli (0.1 µM STS) overnight 37°C, and attached and
detached cells were harvested separately, centrifuged for 5 min at 5000 rpm, washed with icecold PBS, and centrifuged a second time. The pellets were resuspended in a solution
containing 0.15 M KCl, 5mM Tris, 1mM MgCl2, and 0.01% digitonin, incubated on ice for 20
min, and thereafter centrifuged at 10 min at 13000 rpm. Cytosol and mitochondria fractions
(Gogvadze et al., 2003) of mock and infected cells, detached and attached, were harvested
and analyzed using Western blot with anti-cytochrome-c antibodies.
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Transient transfections and CaspACE fluorometric assay
Transient transfections of SW13 cells were performed using Lipofectamine 2000 reagent
(Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Expression plasmids for
N with N-terminal myc-tag and Bax with N-terminal flag-tag were previously constructed
(Karlberg et al., 2011; Mohd-Ismail et al., 2009).Approximately 24 h after transfection, the
cells were harvested by scraping them into the medium, spun down in a bench-top centrifuge,
and washed twice with cold PBS. The cell pellets were then resuspended in RIPA buffer (50
mM Tris (pH 8.0), 150 mM NaCl, 0.5% NP40, 0.5% deoxycholic acid, 0.005% SDS, and 1
mM phenylmethylsulfonyl fluoride) and subjected to freeze-thawing five times before being
centrifuged at 13000 rpm to remove cellular debris. The cell lysate was then used for Western
blot analysis and the activation of caspase-3 was quantified using the CaspACE fluorometric
assay system from Promega Corporation (Madison, WI).
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SDS-PAGE and Western blot analysis
Samples were resuspended in reducing sample buffer (10mM Tris-HCl, 0.5% SDS, 10%
glycerol; 2% -mercaptpethenol, bromophenol blue), boiled for 5 minutes, and separated by
SDS-PAGE using pre-cast gels from Life technologies (Europe). Electrophoresis was carried
out at 200V and proteins were transferred to nitrocellulose membranes using a transfer buffer
containing 25 mM Tris, 192mM glycine, and 20% methanol at 100 V for 1 hour. Membranes
were blocked in 5% non-fat dry milk overnight at 4C. After washing in PBS containing
0.01% Tween (PBST), the membranes were incubated with primary antibody for 1 hour at
room temperature or overnight at 4C. The membranes were then washed with PBST before
addition of secondary antibody. After incubation at room temperature for 1 hour, the
membranes were washed in PBST. Proteins were detected with ECL Plus Western Blotting
Detection Reagents (Amersham Pharmacia, Buckinghamshire, UK) according to the
manufacturer’s instructions. The intensity of bands were analysed within the linear range of
the detector. Percent of cleaved product is analyzed compared with total density of product
(Cleaved and uncleaved) in each well. Mean of three independent experiments are analyzed
(see statistical analysis).
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Measurement of pro-inflammatory mediators
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Supernatant from infected SW13 cells (MOI 1) were collected at 24, 48, and 72 h p.i.,
centrifuged at 13000 rpm 10 min, and assayed for different cytokines and chemokines
according to the manufacturer’s instructions (Qiagen Mix-N-Match Multi-Analyte ElisaArray
kit).
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Neutralizing assay
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SW13 were infected with CCHFV, mock infected, or treated with UV-inactivated virus.
Supernatants were harvested 48 h p.i., centrifuged at 13000 rpm for 10 min, and UVinactivated. The supernatants and rTNF-α suspension (2ng/mL) were pre-incubated separately
with or without neutralizing antibody against TNF-α (5 μg/mL) for 1 h at 37C and
subsequently transferred to seeded SW13 and incubated for 24 hours. All material was
analyzed with Western blot for PARP activation.
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Densitometric and Statistical analysis
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Densitometric analysis was done on the western blot results using quantity software to
determine the intensity of each band. Each band (cleavage products and uncleaved product)
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were correlated to the total amount of each proteins PARP, Caspase3, etc), which then be
used for statistical analysis. An unpaired student’s t-test was applied to evaluate the statistical
significance of differences measured from the data sets, with P<0.05 considered statistically
significant.
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Acknowledgement
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The CCH Fever Network supported by the European Commission under the Health
Cooperation Work Program of the 7th Framework Program (no. 260427).
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REFERENCES
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413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
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446
447
448
449
450
451
452
453
Andersson, I., Simon, M., Lundkvist, A., Nilsson, M., Holmstrom, A., Elgh, F. & Mirazimi, A. (2004).
Role of actin filaments in targeting of Crimean Congo hemorrhagic fever virus nucleocapsid
protein to perinuclear regions of mammalian cells. Journal of medical virology 72, 83-93.
Antonsson, B., Montessuit, S., Lauper, S., Eskes, R. & Martinou, J. C. (2000). Bax oligomerization is
required for channel-forming activity in liposomes and to trigger cytochrome c release from
mitochondria. The Biochemical journal 345 Pt 2, 271-278.
Bente, D. A., Alimonti, J. B., Shieh, W. J., Camus, G., Stroher, U., Zaki, S. & Jones, S. M. (2010).
Pathogenesis and immune response of Crimean-Congo hemorrhagic fever virus in a STAT-1
knockout mouse model. Journal of virology 84, 11089-11100.
Bereczky, S., Lindegren, G., Karlberg, H., Akerstrom, S., Klingstrom, J. & Mirazimi, A. (2010).
Crimean Congo haemorrhagic fever virus infection is lethal for adult 1 type I interferon
receptor knock-out mice. The Journal of general virology.
Bray, M. (2005). Pathogenesis of viral hemorrhagic fever. Current opinion in immunology 17, 399403.
Elliott, R. M. (1990). Molecular biology of the Bunyaviridae. The Journal of general virology 71 ( Pt 3),
501-522.
Erdtmann, L., Franck, N., Lerat, H., Le Seyec, J., Gilot, D., Cannie, I., Gripon, P., Hibner, U. & GuguenGuillouzo, C. (2003). The hepatitis C virus NS2 protein is an inhibitor of CIDE-B-induced
apoptosis. J Biol Chem 278, 18256-18264.
Ergonul, O., Tuncbilek, S., Baykam, N., Celikbas, A. & Dokuzoguz, B. (2006). Evaluation of serum
levels of interleukin (IL)-6, IL-10, and tumor necrosis factor-alpha in patients with CrimeanCongo hemorrhagic fever. The Journal of infectious diseases 193, 941-944.
Fraisier, C., Rodrigues, R., Vu Hai, V., Belghazi, M., Bourdon, S., Paranhos-Baccala, G., Camoin, L.,
Almeras, L. & Peyrefitte, C. N. (2014). Hepatocyte pathway alterations in response to in vitro
Crimean Congo hemorrhagic fever virus infection. Virus research 179, 187-203.
Geisbert, T. W. & Jahrling, P. B. (2004). Exotic emerging viral diseases: progress and challenges.
Nature medicine 10, S110-121.
Gogvadze, V., Orrenius, S. & Zhivotovsky, B. (2003). Analysis of mitochondrial dysfunction during
cell death. Current protocols in cell biology / editorial board, Juan S Bonifacino [et al]
Chapter 18, Unit 18 15.
Ilkow, C. S., Goping, I. S. & Hobman, T. C. (2011). The Rubella virus capsid is an anti-apoptotic
protein that attenuates the pore-forming ability of Bax. PLoS pathogens 7, e1001291.
Karlberg, H., Tan, Y. J. & Mirazimi, A. (2011). Induction of caspase activation and cleavage of the
viral nucleocapsid protein in different cell types during Crimean-Congo hemorrhagic fever
virus infection. J Biol Chem 286, 3227-3234.
Luo, X., Budihardjo, I., Zou, H., Slaughter, C. & Wang, X. (1998). Bid, a Bcl2 interacting protein,
mediates cytochrome c release from mitochondria in response to activation of cell surface
death receptors. Cell 94, 481-490.
Mohd-Ismail, N. K., Deng, L., Sukumaran, S. K., Yu, V. C., Hotta, H. & Tan, Y. J. (2009). The hepatitis
C virus core protein contains a BH3 domain that regulates apoptosis through specific
interaction with human Mcl-1. Journal of virology 83, 9993-10006.
Ontiveros, S. J., Li, Q. & Jonsson, C. B. (2010). Modulation of apoptosis and immune signaling
pathways by the Hantaan virus nucleocapsid protein. Virology 401, 165-178.
Papa, A., Bino, S., Velo, E., Harxhi, A., Kota, M. & Antoniadis, A. (2006). Cytokine levels in CrimeanCongo hemorrhagic fever. Journal of clinical virology : the official publication of the Pan
American Society for Clinical Virology 36, 272-276.
14
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
Pekosz, A., Phillips, J., Pleasure, D., Merry, D. & Gonzalez-Scarano, F. (1996). Induction of apoptosis
by La Crosse virus infection and role of neuronal differentiation and human bcl-2 expression
in its prevention. Journal of virology 70, 5329-5335.
Perez, D. & White, E. (2000). TNF-alpha signals apoptosis through a bid-dependent conformational
change in Bax that is inhibited by E1B 19K. Molecular cell 6, 53-63.
Peters, C. J. & Zaki, S. R. (2002). Role of the endothelium in viral hemorrhagic fevers. Critical care
medicine 30, S268-273.
Rodrigues, R., Paranhos-Baccala, G., Vernet, G. & Peyrefitte, C. N. (2012). Crimean-Congo
hemorrhagic fever virus-infected hepatocytes induce ER-stress and apoptosis crosstalk. PloS
one 7, e29712.
Schnittler, H. J. & Feldmann, H. (2003). Viral hemorrhagic fever--a vascular disease? Thrombosis and
haemostasis 89, 967-972.
Sundararajan, R., Cuconati, A., Nelson, D. & White, E. (2001). Tumor necrosis factor-alpha induces
Bax-Bak interaction and apoptosis, which is inhibited by adenovirus E1B 19K. J Biol Chem 276,
45120-45127.
Thornberry, N. A. (1998). Caspases: key mediators of apoptosis. Chemistry & biology 5, R97-103.
Weber, F. & Mirazimi, A. (2008). Interferon and cytokine responses to Crimean Congo hemorrhagic
fever virus; an emerging and neglected viral zonoosis. Cytokine Growth Factor Rev 19, 395404.
Wolter, K. G., Hsu, Y. T., Smith, C. L., Nechushtan, A., Xi, X. G. & Youle, R. J. (1997). Movement of
Bax from the cytosol to mitochondria during apoptosis. J Cell Biol 139, 1281-1292.
Won, S., Ikegami, T., Peters, C. J. & Makino, S. (2007). NSm protein of Rift Valley fever virus
suppresses virus-induced apoptosis. Journal of virology 81, 13335-13345.
Zhu, N., Khoshnan, A., Schneider, R., Matsumoto, M., Dennert, G., Ware, C. & Lai, M. M. (1998).
Hepatitis C virus core protein binds to the cytoplasmic domain of tumor necrosis factor (TNF)
receptor 1 and enhances TNF-induced apoptosis. Journal of virology 72, 3691-3697.
Zimmermann, K. C., Bonzon, C. & Green, D. R. (2001). The machinery of programmed cell death.
Pharmacol Ther 92, 57-70.
Zimmermann, K. C. & Green, D. R. (2001). How cells die: apoptosis pathways. J Allergy Clin Immunol
108, S99-103.
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Figure legends
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Figure 1. CCHFV infection inhibits PARP cleavage induced by STS. SW13 cells were mock or
CCHFV infected (MOI= 1). At 24 h p.i., cells were treated with 4-32 uM STS. After 5 hours post
treatment, detached cells were harvested and analyzed with Western blot. (A) Percent of cleaved
PARP compared with total density of PARP. Mean of three independent experiments. Error bars =
S.D. Significance level: p<0.001 (***), p<0.01 (**). NS: Non-Significance. (B) Cleavage of PARP in
treated cells with 4 or 32 µM STS in one representative gel, as analyzed in Figure 1A.
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Figure 2. CCHFV infection inhibits activation of caspase-3 and caspase-9 induced by STS. SW13
cells were mock or CCHFV infected (MOI=1). At 24 h p.i., detached cells were treated with 4 uM
STS. After 5 hours post treatment, detached cells were harvested and analyzed with Western blot. (A)
Percent of cleaved caspase-3 relative to total density of caspase-3. Mean of three independent
experiments. Error bars = S.D. (B) Cleavage of caspase-3 in one representative gel, as analyzed in
Figure 2A. (C) Percent of cleaved caspase-9 relative to total amount of caspase-9. Mean of three
independent experiments. Error bars = S.D. (D) Cleavage of caspase-9 in one representative gel, as
analyzed in Figure 2C. Significance level: p<0.0005
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Figure 3. Release of cytochrome-c is suppressed in STS-treated CCHF virus-infected cells. SW13
cells were mock or CCHFV infected (MOI=1). At 24 h p.i., cells were treated with 4 STS uM for 5
hours. Attached and detached cells was separated and fractionated into cytosolic and mitochondrial
fractions. Release of cytochrome-c from mitochondria to the cytosol following CCHFV infection was
analyzed by Western blot with anti-cytochrome c antibody. (A) Percent of cytochrome-c released to
the cytosol. Mean of three independent experiments. Error bars = S.D. Significance level: p<0.0005
(***). (B) A representative gel, as analyzed in Figure 2A. Mitochondrial fraction (a and c) and
cytosolic fraction (b and d).
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Figure 4. CCHFV N protein suppresses activation of apoptosis by Bax. (A) A CaspACE fluorometric
assay system from Promega Corporation (Madison, WI) was used to measure the activation of
caspase-3 in SW13 cells that were mock transfected or transfected with CCHFV N only, or Bax in the
absence or presence of CCHFV N. DNAs used in each of the transfections are indicated in
micrograms. In each transfection, the total amount of DNA was normalized to 1.8 µg with the addition
of empty vector if necessary. All experiments were performed in triplicate, and mean with standard
deviations are plotted. (B) Western blot analysis also was performed to determine the cleavage of
endogenous PARP (top). Similarly, the expression levels of the different proteins were determined
using anti-flag or anti-myc antibodies (middle). The amounts of total cell lysates loaded were verified
by measuring the levels of endogenous actin (bottom). Significance level: p<0.05 (*).
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Figure 6. CCHFV infection induces apoptosis through induction of pro-inflammatory response in
cells. SW13 cells were CCHFV (a), UV-treated CCHFV (b) or Mock infected (c) (MOI=1). At 24, 48,
and 72 h p.i., the supernatant was collected and treated with UV. The UV-treated supernatant was then
transferred to new freshly seeded SW13. After 24 h, cells were harvested and analyzed by Western
blot. (A) Percent of cleaved PARP compared with the total amount of PARP. Mean of three
independent experiments. (B) Cleavage of PARP in one representative gel, as analyzed in Figure 6A.
(C) SW13 cells were incubated with rTNF-α or supernatant from 48 h p.i. infected cells, with or
without anti TNF-α. After 24 h, cells were harvested and analyzed by Western blot. Percent of cleaved
PARP compared with the total density of PARP. Mean of three independent experiments. Significance
levels: p < 0.0005 (***), p < 0.005 (**), p < 0.05 (*)
Figure 5. CCHFV infection induces activation of Bid. SW13 cells were mock or CCHFV infected
(MOI=1). At 24 and 48 h p.i., attached and detached cells were harvested and analyzed for presence of
activation of Bid (tBid).
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