Download Induction of apoptosis by the Amsacta moorei entomopoxvirus

Journal of General Virology (2013), 94, 1876–1887
DOI 10.1099/vir.0.051888-0
Induction of apoptosis by the Amsacta moorei
entomopoxvirus
Srini Perera,1,2 Peter Krell,2 Zihni Demirbag,3 Remziye Nalçacioğlu3
and Basil Arif1
Correspondence
1
Basil Arif
2
[email protected]
Laboratory for Molecular Virology, Great Lakes Forestry Centre, Sault Ste. Marie, Ontario, Canada
Department of Molecular and Cellular Biology, University of Guelph, Ontario, Canada
3
Department of Biology, Karadeniz Technical University, Trabzon, Turkey
Received 25 January 2013
Accepted 20 April 2013
CF-70-B2 cells derived from the spruce budworm (Choristoneura fumiferana) undergo apoptosis
when infected with Amsacta moorei entomopoxvirus (AMEV), as characterized by membrane
blebbing, formation of apoptotic bodies, TdT-mediated dUTP nick-end labelling (TUNEL) staining,
condensed chromatin and induction of caspase-3/7 activity. The apoptotic response was
reduced when cells were infected with UV-inactivated AMEV, but not when infected in the
presence of the DNA synthesis inhibitor, cytosine b-D-arabinofuranoside. Hence, only pre-DNA
replication events were involved in inducing the antiviral response in CF-70-B2 cells. The virus
eventually overcame the host’s antiviral response and replicated to high progeny virus titres
accompanied by high levels of caspase-3/7 activity. The CF-70-B2 cells were less productive of
progeny virus in comparison to LD-652, a Lymantria dispar cell line routinely used for propagation
of AMEV. At late stages of infection, LD-652 cells also showed characteristics of apoptosis such
as oligosomal DNA fragmentation, TUNEL staining, condensed chromatin and increased
caspase-3/7 activity. Induction of apoptosis in LD-652 cells was dependent on viral DNA
replication and/or late gene expression. A significantly reduced rate of infection was observed in
the presence of general caspase inhibitors Q-VD-OPH and Z-VAD-FMK, indicating caspases
may be involved in productive virus infection.
INTRODUCTION
Apoptosis or programmed cell death is an active process
characterized by various morphological and biochemical
changes eventually leading to cell death. Caspases, a family
of cysteine proteases, play a crucial role in the regulation
and execution of apoptosis. Caspases are present in cells
as inactive zymogens (procaspases) and are activated in
response to apoptotic stimuli. There are two types of
caspases; initiator caspases (caspases-8, -9 and -10) and
effector caspases (caspases-3, -6 and -7). Initiator caspases
are activated through death receptor signalling (extrinsic)
or mitochondrial (intrinsic) pathways, which result in the
activation of effector caspases. The active effector caspases,
through cleavage of specific host proteins, are instrumental
in directing subsequent morphological and biochemical
changes associated with apoptosis. These changes include
reduction of cellular volume, chromatin condensation,
fragmentation of nucleus, phosphatidylserine exposure on
the cell surface, DNA fragmentation, membrane blebbing
and formation of apoptotic bodies. Cells undergoing
apoptosis may exhibit some or all of these features (Fink
& Cookson, 2005; Riedl & Shi, 2004).
Many viruses induce apoptosis, both in vivo and in
cultured cells. In non-permissive hosts, apoptosis is an
1876
effective antiviral mechanism, which can lead directly to
premature cell death limiting viral replication and spread.
The Autographa californica multicapsid nucleopolyhedrosis
virus (AcMNPV) induces apoptosis in Spodoptera littoralis
SL2 cells (Chejanovsky & Gershburg, 1995), Choristoneura
fumiferana CF-203 cells (Palli et al., 1996) and in various
tissues of Spodoptera litura larvae (Zhang et al., 2002). The
IPLB-LD-652Y cells undergo apoptosis in response to
infection by several nucleopolyhedroviruses (NPVs) from
Bombyx mori (BMNPV), Hyphantria cunea (HycuNPV),
Spodoptera exigua (SeMNPV), Orgyia pseudotsugata
(OpMNPV) and S. litura (SpltMNPV) (Ishikawa et al.,
2003). Limited SpltMNPV propagation and infectivity in S.
exigua larvae was attributed to cell death by apoptosis,
which was observed in the haemolymph and fat body
tissues (Feng et al., 2007).
To counter the host defence response, many viruses encode
anti-apoptotic proteins, which allow the virus to replicate
to a high titre and establish a successful infection. Insect
viruses inhibit apoptosis by expressing P35 and/or IAP
(inhibitor of apoptosis) proteins, which have been found in
baculoviruses (Clem et al., 2010), entomopoxviruses . (Li
et al., 2005a; Means et al., 2007) and iridoviruses (Ince
et al., 2008). P35 and its homologues inhibit apoptosis by
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Apoptosis in A. moorei entomopoxvirus-infected cells
Members of the family Poxviridae contain large, linear
dsDNA genomes, and infect both vertebrates (Subfamily:
Chordopoxvirinae)
and
invertebrates
(Subfamily:
Entomopoxvirinae) (King et al., 2011). The Amsacta moorei
entomopoxvirus (AMEV) was first isolated from the
lepidopteran red hairy caterpillar (A. moorei) (Roberts &
Granados, 1968). The 232 kb genome of AMEV has been
completely sequenced (Bawden et al., 2000). Several AMEV
genes including FALPE (Alaoui-Ismaili & Richardson,
1996; Alaoui-Ismaili & Richardson, 1998), superoxide
dismutase (Becker et al., 2004), inhibitor of apoptosis (Li
et al., 2005a; Li et al., 2005b), thymidine kinase (Gruidl et
al., 1992; Lytvyn et al., 1992), spheroidin (Hall & Moyer,
1991) and DNA photolyase (Nalcacioglu et al., 2010) have
been well characterized.
AMEV replicates in cultured cells from Estigmene acrea (BTIEAA, BTI-EAH), L. dispar (SCLD-135, IPLB-65z, IPLB-LD652), Heliothis zea (IPLB-1075), Spodoptera frudgiperda
(IPLB-21) and B. mori (BM-N) (Goodwin et al., 1990;
Granados, 1981; Marlow et al., 1993). Of these, IPLB-LD-652
(commonly referred to as LD-652) is the most widely used
cell line for propagation and maintenance of AMEV.
In attempts to find other cell lines permissive to AMEV, we
tested several lines present at the Great Lakes Forestry
Centre. The cell line CF-70-B2 derived from budworm C.
fumiferana (Lepidoptera, Tortricidae) appeared to support
the replication of AMEV. We decided to compare the
replication of the virus in this cell line with that in the
more commonly used LD-652. We observed that AMEV
infection triggered an apoptotic response in both CF-70-B2
and LD-652 cells. Since the virus also replicated productively in both lines, we sought to examine this phenomenon further.
RESULTS
AMEV infection in CF-70-B2 and LD-652 cells
CF-70-B2 was found to support parental AMEV replication
during a preliminary screening of several insect cell lines.
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The kinetics of extracelluar virus production was monitored over a period of 5 days (Fig. 1a). LD-652 cells were
used for assaying virus titres. Extracellular virus production
was detected as early as 1 day p.i. in both cell lines when
(a)
1.00E+09
Titre (TCID50 ml–1)
In some cases, apoptosis is related to viral pathogenesis
rather than host defence; the virus can replicate and produce
infectious progeny despite activation of cellular apoptotic
pathways. In these cases, apoptosis is observed at late
infection stages concurrently with productive infection
(Hinshaw et al., 1994; Ren et al., 2005; Thoulouze et al.,
1997). There is emerging evidence that some of these viruses
actively promote apoptosis for their own survival and spread
(Best & Bloom, 2004; Richard & Tulasne, 2012).
We compared the process of infection between CF-70-B2
cells and the more commonly used LD-652 cells. When
infected at an mo.i. of 10, the LD-652 cells showed
cytopathic effects at 1 day post-infection (p.i.) by rounding
up and detaching from the surface while the same was not
observed until 2 days p.i. in CF-70-B2 cells. At 2 days p.i.,
all the LD-652 cells were clearly infected and many
contained spheroidal occlusion bodies (OBs). Only a few
cells with OBs were observed at 2 days p.i. in CF-70-B2. By
3 days p.i., more cells with OBs were observed in both cell
lines.
1.00E+08
1.00E+07
1.00E+06
LD-652
1.00E+05
CF-70-B2
1.00E+04
0
1
2
3
4
5
Time p.i. (days)
(b)
GFP expression (RFU)
direct interaction with cellular caspases while viral IAPs
appear to act indirectly by inactivating cellular IAP
antagonists such as Hid, Reaper and Grim proteins
(Clem et al., 2010). Recently, a new gene encoding an
apoptosis suppressor was identified in Lymantria dispar
MNPV (LdMNPV) (Yamada et al., 2011).
4000
LD-652
CF-70-B2
3000
2000
1000
0
0
1
2
3
Time p.i. (days)
4
5
Fig. 1. Time-course of AMEV infection in LD-652 and CF-70-B2
cells. (a) Extracellular virus production. The cells were infected with
parental AMEV at an m.o.i. of 10 and virus titres were measured by
TCID50. The results shown are mean±SEM from three independent
experiments, each containing three replicates. (b) Very late gene
expression under the spheroidin promoter. The cells were infected
with a sph”/gfp+ recombinant virus (m.o.i. 10), and gfp fluorescence
was measured over a period of 5 days. The results shown are from
one of two representative experiments, each with five replicates.
Error bars represent±SEM. RFU, Relative fluorescence units.
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S. Perera and others
infected at an m.o.i. of 10. The highest virus titre was
observed at 2 days p.i. and then declined slightly. The LD652 cells were more productive in terms of virus replication
than CF-70-B2; the extracelluar virus titre was 5-10-fold
lower in CF-70-B2 than in LD-652 at all time points tested
p.i.
AMEV infection in the two cell lines was also tested using a
recombinant virus expressing gfp under the control of the
spheroidin promoter (Fig. 1b). Since spheroidin is a very late
gene, gfp expression from the same locus indicates that the
virus has gone through a full cycle of replication. Both cell
lines showed low levels of autofluorescence at 0 days p.i.
GFP was first observed at 2 days p.i. in both LD-652 and
CF-70-B2 cells. In LD-652, gfp expression peaked at 3 days
p.i. and remained stable throughout the experimental
period while it continued to show a slow but gradual
increase up to 5 days p.i. in CF-70-B2 cells. GFP levels were
considerably higher in LD-652 than in CF-70-B2 from the
time GFP was first detected at 2 days p.i., which confirmed
that the latter is less amenable to infection by AMEV.
Antiviral response (early apoptotic response) in
CF-70-B2 cells
AMEV induces an antiviral response in CF-70-B2 cells.
We observed that CF-70-B2 cells undergo apoptosis as a
result of AMEV infection (Fig. 2). Many cells appear to die
of apoptosis before the virus can complete the infection
cycle and produce OBs, indicating that this is an antiviral
response. The apoptotic response was first evident at 1 day
p.i., with a few blebbing cells. Apoptotic bodies as well as
many cells with blebbing were visible at 2 days p.i. (Fig.
2a). Apoptosis was also tested by TdT-mediated dUTP
nick-end labelling (TUNEL), which is an assay for in situ
detection of DNA strand breaks in apoptotic nuclei, in
combination with DAPI, which stains both apoptotic and
non-apoptotic nuclei. TUNEL-positive (green) nuclei are
seen in Fig. 2(b), confirming that AMEV infection leads
to apoptosis in CF-70-B2 cells. In addition, DAPI staining indicated that nuclei of infected cells were highly
condensed as opposed to those of mock-infected control
cells (Fig. 2b). Next, we quantified the apoptotic response
by measuring the caspase-3/7 activity over a period of
5 days (Fig. 2c). In accordance with morphological
observations, a low level of caspase-3/7 activity was
observed at 1 day p.i. when cell blebbing was first
evident. Caspase-3/7 continued to increase with the
progression of infection leading to high levels of activity
at the end of the observation period (5 days p.i., Fig. 2c).
Viral DNA replication and late gene expression are not
required for the antiviral response in CF-70-B2 cells.
Cytosine b-D-arabinofuranoside (AraC) inhibits AMEV
DNA replication (Winter et al., 1995). In order to
determine if AMEV DNA replication or events thereafter
were needed for the antiviral response in CF-70-B2, we
studied the infection in cells treated with AraC (Fig. 3).
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Mock-infected cells failed to divide in the presence of AraC
indicating that cellular DNA synthesis was suppressed. The
lack of OBs in infected cells suggested that viral DNA
synthesis was also inhibited by AraC. Some mock-infected
cells treated with AraC showed membrane blebbing and
formation of apoptotic bodies, suggesting that the inhibitor
also induces apoptosis. However, the apoptotic response
was more pronounced in AraC-treated infected cells
indicating that virus replication is not required for the
antiviral response in CF-70-B2 cells (Fig. 3a).
The effect of AraC on apoptosis was confirmed by
analysing caspase-3/7 activity in CF-70-B2 cells at 4 days
p.i. (Fig. 3b). In comparison to cells infected in the absence
of AraC, those infected in the presence of the inhibitor
(either 200 or 300 mg ml21) showed a significant (P,0.01)
increase in caspase-3/7 activity. The high levels of caspase3/7 activity in AraC-treated, infected cells indicated that
suppression of viral DNA replication and subsequent
events had no effect on induction of apoptosis in CF-70B2 cells.
The antiviral response is induced by early gene
expression. Data obtained from treatment with AraC
indicated that pre-DNA replication events are needed to
induce apoptosis in CF-70-B2 infected cells. In order to
ascertain that early gene expression was needed to induce
apoptosis, we used UV-inactivated virus inoculum for
infection. Treatment of virus particles with UV radiation
damages the DNA genome and restricts mRNA synthesis,
without compromising virus attachment and cell entry
(Tsung et al., 1996). The virus inoculum was exposed to
5 J cm22 UV radiation before inoculating CF-70-B2 cells
(Fig. 4). As expected, the untreated virus induced membrane
blebbing and activation of caspase-3/7. In contrast, cells
infected with the UV-treated virus remained morphologically similar to mock-infected control cells. Caspase-3/
7 activity in cells infected with the untreated virus was
significantly (P,0.05) higher than that in those infected
with the UV-treated virus. These results indicate that viral
gene expression is essential for induction of apoptosis.
However, as the late events in infection (replication and late
gene expression) have no effect on the apoptotic response
(Fig. 3), it can be concluded that early genes are involved in
inducing apoptosis in CF-70-B2 cells.
Apoptosis in LD-652 cells
AMEV induces apoptosis in LD-652 cells. LD-652 has
been the line of choice to propagate AMEV because almost
every cell becomes infected and the virus replicates to a high
titre. The cells exhibit no visual signs of apoptosis (cell
blebbing or apoptotic bodies) at any time during infection.
As we were conducting a comparative study between CF-70B2 and LD-652, we decided to test for apoptosis in the latter
despite the lack of obvious morphological characteristics.
Interestingly, we found compelling evidence of apoptosis
induction by AMEV at late stages of infection (Fig. 5).
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Journal of General Virology 94
Apoptosis in A. moorei entomopoxvirus-infected cells
(a)
Mock
Infected
(b)
Mock
Light
TUNEL
DAPI
Infected
Light
TUNEL
DAPI
Caspase activity
(c)
30
20
10
0
0
1
2
3
4
Time p.i. (days)
5
Shortly after infection, LD-652 cell DNA became fragmented with an apparent DNA ladder beginning at 3 days p.i.
that became more distinct thereafter (Fig. 5a). TUNEL
staining indicated that a large number of infected cells
underwent apoptosis, at 4 days p.i. (Fig. 5b). In addition,
nuclei of infected cells showed apoptotic morphology as
evidenced by DAPI staining. Control (mock-infected) cells
contained large nuclei with diffused chromatin while the
nuclear chromatin of infected cells was highly condensed
(Fig. 5b). A marked increase in caspase-3/7 activity was also
observed from 3 days p.i., which further confirmed AMEV
induced late apoptosis in LD-652 cells (Fig. 5c).
Viral DNA replication and/or late gene expression are
required to induce apoptosis in LD-652 cells. The DNA
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Fig. 2. Apoptosis in CF-70-B2 cells infected
with AMEV (m.o.i. 10). (a) Light micrographs of
mock-infected and infected cells, at 2 days p.i.
Bars, 20 mm. (b) TUNEL staining (green) and
DAPI counterstaining (blue) of mock-infected
and infected cells at 3 days p.i. The same field
is viewed under normal light and under UV light
with either an FITC or DAPI filter to detect
TUNEL- or DAPI-stained nuclei, respectively.
Bars, 20 mm. (c) Caspase-3/7 activation in
CF-70-B2 cells. Caspase-3/7 activity is normalized to cell viability and expressed as a ratio
of the activity of mock-infected cells at day 0.
The results shown are from one of two
representative experiments, each with three
replicates. Error bars represent±SEM.
synthesis inhibitor AraC prevented cell division of LD-652
(Fig. 6a). No OBs were seen in infected cells in the presence
of 300 mg AraC ml21 (Fig. 6a), whereas a few (one or two)
cells containing OBs were present at a reduced concentration of 200 mg ml21 (not shown). AraC treatment also
induced apoptosis in some mock-infected LD-652 cells as
seen by occasional cell blebbing (not shown).
Fig. 6(b) shows caspase-3/7 activity in LD-652 cells, in the
presence or absence of AraC, at 4 days p.i. Mock-infected
cells showed basal levels of caspase-3/7 activity, which
increased as a result of AraC treatment (200 or 300 mg
ml21). As expected, AMEV infection also resulted in an
increase of caspase-3/7 levels in LD-652 cells in the absence
of the inhibitor. In comparison to cells infected in the
absence of AraC, those infected in the presence of the
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S. Perera and others
Caspases and productive virus infection
(a)
Mock
Infected
Mock (AraC)
Our results show that replication of the viral genome
triggers caspase activation in the highly productive LD-652
cells. To investigate the role of caspases in viral infection,
we infected the cells in the presence of broad spectrum
caspase inhibitors, Q-VD-OPH and Z-VAD-FMK (Fig. 7).
A recombinant virus expressing gfp in the spheroidin locus
was used for infecting the cells at two different m.o.i. (0.1
and 1), and GFP expression was monitored over a period
of 5 days. A detectable increase in GFP was observed at
3 days p.i. and continued to increase up to 5 days p.i. After
3 days p.i., the cells treated with Z-VAD-FMK showed a
significant decrease for both 0.1 m.o.i. (P,0.001) and 1
m.o.i. (3–4 days p.i.: P,0.001; 5 days p.i.: P,0.05) in GFP
expression, compared to control cells (cells infected in the
absence of inhibitors). The inhibitor Q-VD-OPH was less
effective in suppressing GFP expression than Z-VAD-FMK.
Nevertheless, at both 0.1 and 1 m.o.i., GFP expression in
cells treated with Q-VD-OPH was significantly (P,0.05)
lower than the control cells at 3–5 days p.i. Taken together,
our data indicate that caspases may play a role in
productive virus infection.
Infected (AraC)
(b)
Caspase activity
25
20
Mock
Infected
15
10
DISCUSSION
5
0
0
200
AraC (µg ml–1)
300
Fig. 3. The effect of the DNA synthesis inhibitor cytosine AraC on
CF-70-B2 cells, at 4 days p.i. An m.o.i. of 10 was used for
infection. (a) Light micrographs showing mock-infected (control)
and infected cells in the presence or absence of 300 mg AraC
ml”1. Bars, 20 mm. (b) Caspase-3/7 activation in CF-70-B2 cells
treated with AraC. Caspase-3/7 activity (normalized to viability) is
expressed as a ratio of the mock-infected control. Results shown
are from one of three representative experiments, each containing
three replicates. Error bars represent±SEM.
inhibitor (200 or 300 mg ml21) showed a significant
(P,0.05) reduction in caspase-3/7 activity. Hence, suppression of DNA synthesis by AraC treatment inhibits
caspase-3/7 activity in infected cells. These results suggest
that late events in infection (viral DNA synthesis and/or
late gene expression) are involved in inducing apoptosis in
LD-652 cells.
In the presence of AraC, the cells infected with AMEV
showed less caspase-3/7 activity than mock-infected cells
(Fig. 6b). The lower caspase-3/7 activity in infected cells
could be attributed to the expression of anti-apoptotic
proteins P33 and IAP (Li et al., 2005a; Means et al.,
2007).
1880
During the course of virus infection, many cells limit
infection from spreading to the rest of the organism by
undergoing apoptosis. For most RNA viruses, apoptosis
does not appear to be a limiting factor for infection; some
even utilize it as a means of disseminating progeny virions
while evading inflammatory and immune responses of the
host (Koyama et al., 1998; Teodoro & Branton, 1997). In
many DNA viruses, apoptosis is an antiviral mechanism
where the host cell sacrifices itself to restrict the replication
and spread of virus to neighbouring cells (reviewed by
Clem et al., 2010). There are exceptions, however, where
the virus induces apoptosis in host cells with no detectable
effect on the replication of its DNA genome or production
of infectious progeny. Here, we present evidence to place
AMEV in this category. We show that while AMEV can
induce an antiviral response in some cell lines, an apoptotic
response is initiated late in the replication cycle in
permissive cells, without apparent effect on productive
virus replication.
The antiviral response
We investigated AMEV-induced apoptosis in two lepidopteran cell lines. Of these two, LD-652 is the most
commonly used cell line for propagation of AMEV. The
second cell line, CF-70-B2, has not been previously tested
for AMEV infection but it clearly supports viral replication,
albeit not to the same extent as LD-652 cells. We observed
that infection with AMEV induces an early apoptotic
response in CF-70-B2 as evidenced by high levels of caspase
activity, TUNEL staining, chromatin condensation and
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Journal of General Virology 94
Apoptosis in A. moorei entomopoxvirus-infected cells
(a)
Mock
Infected
Infected (UV)
(b)
Caspase activity
20
15
10
5
0
Mock
Infected
Infected
(UV)
Fig. 4. The effect of UV irradiation of AMEV inoculum, on infected CF-70-B2 cells, at 3 days p.i. An m.o.i. of 10 was used for
infection. (a) Light micrographs, Bars, 20 mm. (b) Caspase-3/7 activation in mock-infected cells and cells infected with AMEV or
UV-irradiated (5 J cm”2) AMEV. Caspase-3/7 activity (normalized to viability) is expressed as a ratio of the mock-infected
control. Results shown are from one of three representative experiments, each containing three replicates. Error bars
represent±SEM.
severe blebbing, leading to apoptotic cell death. Oligosomal
DNA fragmentation was not observed in CF-70-B2 cells
(data not shown). The antiviral response was first evident
at early stages of infection. However, a subset of CF-70-B2
cells succumbs to infection and produces extracellular virus
and OBs, though at lower levels compared with the more
productive LD-652.
Activation of the apoptotic cascade can occur at any stage
of the virus life cycle, depending on the specific interactions between the virus and a particular host. Some viruses
such as bovine herpesvirus 1 (BHV-1), avian leukosis–
sarcoma virus and reovirus induce apoptosis at the time of
attachment and some (e.g. Sindbis virus, mouse hepatitis
virus) do so during fusion and entry (Brojatsch et al., 2000;
Brojatsch et al., 1996; Chitnis et al., 2008; Connolly et al.,
2001; Hanon et al., 1998; Jan et al., 2000; Liu & Zhang,
2007; Ramsey-Ewing & Moss, 1998). Yet others require a
post-entry step (e.g. frog virus 3) such as uncoating (e.g.
avian reovirus S1133) to elicit an apoptotic response
(Chinchar et al., 2003; Labrada et al., 2002). In some cases,
apoptosis induction is dependent on de novo viral protein
synthesis (e.g. vaccinia virus), virus replication and/or
post-replication events (e.g. Semliki Forest virus,
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AcMNPV) of the life cycle (Humlova et al., 2002; Schultz
& Friesen, 2009; Urban et al., 2008). In this study, AMEV
infection of CF-70-B2 cells resulted in apoptotic cell death;
this was suppressed by UV irradiation of the inoculum
prior to infection, but not by treatment of cells with AraC,
a DNA synthesis inhibitor. These results suggest that the
antiviral response triggered by AMEV in CF-70-B2 cells is
mediated by early gene products. A similar observation was
reported for the chordopoxvirus vaccinia virus (VACV)
where apoptotic cell death was dependent on early gene
expression in the murine macrophage line J774.G8
(Humlova et al., 2002). However, in Chinese hamster
ovary (CHO) cells, a post-binding step associated with cell
entry by VACV was sufficient to induce apoptosis
(Ramsey-Ewing & Moss, 1998).
Apoptosis and productive infection
Many RNA viruses induce apoptosis in permissive cells
without detrimental effects on virus replication (Bitzer
et al., 1999; Du et al., 2004; Ren et al., 2005). In this study,
we observed increased caspase levels, oligosomal DNA
fragmentation, condensed chromatin and TUNEL-positive
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S. Perera and others
(a)
M
0
3
4
5
6
(c)
Caspase activity
6
1000
500
200
4
2
0
100
0
1
2
3
Time p.i. (days)
4
5
(b )
Mock
Light
TUNEL
DAPI
Infected
Light
TUNEL
DAPI
Merged (light/TUNEL)
Fig. 5. Apoptosis in LD-652 cells infected (m.o.i. 10) with AMEV. (a) DNA fragmentation in infected cells at 0, 3–6 days p.i. M,
molecular mass markers (bp). (b) TUNEL staining (green) and DAPI counterstaining (blue) of mock-infected and infected cells
at 4 days p.i. The same field is viewed under normal light; and under UV light with either an FITC or DAPI filter to detect TUNELor DAPI-stained nuclei, respectively. Note the TUNEL-positive (green) nuclei in infected cells containing OBs, on the merged
image. Bars, 20 mm. (c) Caspase-3/7 activation in infected cells. Caspase-3/7 activity (normalized to viability) is expressed as a
ratio of the mock-infected control at day 0. The results shown are from one of two representative experiments, each with three
replicates. Error bars represent±SEM.
nuclei in infected LD-652 cells. The apoptotic characteristics in LD-652 were observed at late stages of infection,
together with high virus titres, suggesting a process other
than an antiviral response. Productive infection associated
with apoptosis has also been reported in a few DNA
viruses. In the red sea bream iridovirus (RSIV), virusinduced DNA fragmentation was seen in parallel with viral
DNA replication (Imajoh et al., 2004). The varicella-zoster
virus (VZV) induced apoptosis in human foreskin
fibroblasts but not in human sensory neurons, although
both were susceptible to VZV infection (Hood et al., 2003).
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Mosquito densoviruses usually establish persistent infections in mosquito cells with no obvious cytopathic effects.
One exception is the Hemagogus equinus densovirus
(HeDNV) which induces apoptosis accompanied by
vigorous production of progeny in the mosquito cell line
C6/36 (Paterson et al., 2005).
Unlike the host defence response in CF-70-B2 where a
number of cells die before infection is established,
apoptosis in LD-652 cells was exclusively dependent on
virus replication and/or late gene expression. Many other
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Apoptosis in A. moorei entomopoxvirus-infected cells
0.1 m.o.i.
(a)
5.00E+04
Infected
GFP expression (RFU)
Mock
Infected
Infected (Q-VD-OPH)
Infected (Z-VAD-FMK)
4.00E+04
3.00E+04
2.00E+04
1.00E+04
0
Mock (AraC)
0
Infected (AraC)
1
2
3
Time p.i. (days)
4
5
4
5
1 m.o.i.
GFP expression (RFU)
5.00E+04
Caspase activity
(b)
4
Mock
Infected
Infected
Infected (Q-VD-OPH)
Infected (Z-VAD-FMK)
4.00E+04
3.00E+04
2.00E+04
1.00E+04
0
0
3
1
2
3
Time p.i. (days)
2
1
0
0
200
AraC (µg ml–1)
300
Fig. 6. The effect of the DNA synthesis inhibitor cytosine AraC on
LD-652 cells, at 4 days p.i. (a) Light micrographs showing mockinfected and infected (m.o.i. 10) cells, in the presence (300 mg
ml”1) or absence of AraC. Bars, 20 mm. (b) Caspase-3/7 activity in
AraC-treated LD-652 cells. Caspase-3/7 activity (normalized to
viability) is expressed as a ratio of the mock-infected control.
Results shown are from one of three representative experiments,
each with three replicates. Error bars represent±SEM.
viruses exhibit replication-dependent apoptosis (Acrani
et al., 2010; Chen et al., 2010; Du et al., 2004; Kassis et al.,
2004; Krejbich-Trotot et al., 2011; Ren et al., 2005; Schultz
& Friesen, 2009; Urban et al., 2008). In AcMNPV, events
associated with viral DNA replication and inhibition of
host protein synthesis could trigger the host apoptotic
response (Schultz & Friesen, 2009). In another DNA virus,
the minute virus of canines (MVC), a moderate level of
genome replication in permissive cells induces a cellular
DNA damage response, which contributes to enhanced
virus replication and cell death, suggesting an intricate
relationship between viral replication and host death
responses (Luo et al., 2011).
http://vir.sgmjournals.org
Fig. 7. The effect of the broad-spectrum caspase inhibitors Q-VDOPH and Z-VAD-FMK on AMEV infection. Cells were infected with
an sph”/gfp+ recombinant virus at 0.1 or 1 m.o.i., and GFP
expression was measured over a period of 5 days. Data shown are
from one of two representative experiments, each with five replicates.
Error bars represent±SEM. RFU, Relative fluorescence units.
In VACV, the presence of phosphatidylserine on the
surface of mature virions was essential for virus entry,
which led Mercer & Helenius (2008) to conclude that
VACV uses apoptotic mimicry to enter host cells. They also
observed that many cells at the edge of viral plaques
displayed exposed phosphatidylserine which led them to
suggest that VACV-infected cells undergo apoptosis at late
stages of infection. However, the significance of late stage
apoptosis in chordopoxvirus infection is yet to be
investigated.
Role of caspases in virus infection
Caspases are essential for propagation of some viruses
through their involvement in replication, maturation or
virus release from infected cells (reviewed by Best & Bloom,
2004; Richard & Tulasne, 2012). In maedi-visna virus
(MVV, a lentivirus), caspase inhibitors delayed MVVinduced cell lysis and subsequent virus release (Duval et al.,
2002). In enterovirus 70 (EV70) and human astrovirus
(HAstV) caspase inhibitors reduced the release of virus
progeny without affecting intracellular virus production
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S. Perera and others
(Chen et al., 2006; Mendez et al., 2004). Conversely,
caspases appear to inhibit total virus yield while enhancing
their release in BHV-1 (Devireddy & Jones, 1999) and in
adenovirus-infected cells (Chiou & White, 1998). In
Chikungunya virus (CHIKV) and S. frugiperda ascovirus
(SfAV), caspase activation promotes the formation of
apoptotic bodies which are used for virus dissemination
(Bideshi et al., 2005; Krejbich-Trotot et al., 2011).
In addition to regulating virus release from infected cells,
caspases are also involved in virus replication and
maturation. In Aleutian mink disease virus (AMDV),
caspases play opposing roles in infection. They are involved
in cleaving the non-structural protein NS1, resulting in
virus replication. In contrast, caspase cleavage of the capsid
protein leads to limited production of mature virions.
Hence, caspases play a vital role in balancing virus
production to ensure persistent infection (Best et al.,
2002, 2003; Cheng et al., 2010). In HAstV, caspases were
reported to be involved in virus maturation through their
role in proteolytic processing of the structural protein
VP90 to the mature capsid protein VP70. In HAstV,
caspases were also involved in virus release through a nonlytic mechanism (Banos-Lara & Méndez, 2010; Mendez et
al., 2004). Caspases play an active role in influenza virus
propagation by facilitating the export of viral ribonucleoprotein complexes formed in the nucleus to the cytoplasm
(Wurzer et al., 2003). Interestingly, in baculoviruses,
caspases have a detrimental effect on virus particles. It
was shown that activation of caspases during infection of a
p35 mutant AcMNPV would lead to the production of
progeny virions with defects in stability and infectivity
(Bryant & Clem, 2009). Evidently caspases play a number
of roles in response to virus infection and are not confined
to limiting virus replication and propagation.
In this study, when LD-652 cells were treated with general
caspase inhibitors Q-VD-OPH or Z-VAD-FMK, virus
infection declined significantly. This suggests that caspases
may be involved in productive virus infection. However,
the reduction in infection was more pronounced in the
presence of Z-VAD-FMK. It has been shown that Z-VADFMK is less effective in inhibiting caspase activity than the
more potent inhibitor, Q-VD-OPH (Chauvier et al., 2007).
Hence, the drastic reduction in infection in the presence of
Z-VAD-FMK could not be attributed exclusively to caspase
inhibition. Z-VAD-FMK has also been shown to inhibit
other cysteine proteases such as cathepsins, papain and
legumain (Chauvier et al., 2007; Rozman-Pungercar et al.,
2003; Schotte et al., 1999). Therefore, it is possible that in
addition to caspases, these other cysteine proteases are also
contributing to AMEV-induced apoptosis and cell death.
In vitro studies have shown that cathepsins can induce
apoptosis by activating the pro-apoptotic protein Bid and
by degrading anti-apoptotic Bcl-2 proteins, leading to
downstream activation of caspases (Droga-Mazovec et al.,
2008; Stoka et al., 2001). Cathepsin activation followed by
apoptosis has also been reported for at least two viruses:
the herpes simplex virus type 1 and murine norovirus
1884
(Furman et al., 2009; Peri et al., 2011). Lysosomal
cathepsins are also involved in autophagy, a highly
conserved cellular degradation pathway, which plays a
major role in innate and adaptive immunity against viral
infections (Chiramel et al., 2013; Kudchodkar & Levine,
2009; Müller et al., 2012). The difference between the
effect of Q-VD-OPH and Z-VAD-FMK on AMEV
infection suggests that autophagy may play a role in
AMEV infection. It will be of interest to investigate if
cathepsins are involved in AMEV infection, either through
autophagy, or by contributing to caspase activation and
apoptosis.
Concluding remarks
This is the first study to show that AMEV replicates
productively in CF-70-B2 cells and delineates a comprehensive study on the mechanism of cell death by
entomopoxvirus infection. We show that early gene
products of AMEV can induce an antiviral response in
CF-70-B2 cells, which limits virus replication and yield.
More importantly, our findings suggest that virus replication/late gene expression triggers apoptosis in permissive
LD-652 cells, and that caspase activation does not appear
to limit infection. It is tempting to suggest that in the case
of AMEV, caspases may play a role in productive infection.
METHODS
Cells and virus. The LD-652 (IPLB-LD-652) and CF-70-B2 (FPMI-
CF-70-B2) cells were maintained at 28 uC. LD-652 cells were
propagated in a medium consisting of 45 % GM (Sigma-Aldrich),
45 % Excell 420 (SAFC Biosciences) and 10 % FBS (PAA, The Tissue
Culture Company). The CF-70-B2 cell line was maintained in 90 %
GM supplemented with 10 % FBS.
The parental AMEV stock used in this study was kindly provided by
Drs Marie Becker and Richard Moyer (University of Florida,
Gainesville, FL, USA). The recombinant AMEV expressing gfp was
constructed in our laboratory (Perera et al., 2010). The gene encoding
GFP was inserted in the sph locus under the sph promoter.
Cells were infected with AMEV at an m.o.i. of 10 p.f.u. per cell, unless
otherwise stated. For infection, cells were seeded in tissue culture
plates at 16105 cells cm22. After allowing the cells to attach for at
least 1 h at room temperature, the culture medium was removed and
virus inoculum was added. The plates were incubated at 28 uC with
gentle rocking to allow for virus adsorption for 2 h. At the end of the
incubation period, the inoculum was replaced with culture medium
and the plates were incubated at 28 uC.
UV irradiation of virus. An aliquot of AMEV in an uncovered, clear
plastic tissue culture plate was exposed to a total dose of 5 J cm22 UV
radiation in a Stratalinker UV Cross-linker (Stratagene).
Assays for AMEV infection
TCID50 assay. LD-652 cells were seeded in tissue culture plates and
infected at an m.o.i. of 10 as described above. To assay extracellular
virus production, the cells and media were collected at various times
p.i. and centrifuged at 1000 g for 5 min. The virus titres in the
supernatant were determined by TCID50 analysis as described by Reed
& Muench (1938).
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Journal of General Virology 94
Apoptosis in A. moorei entomopoxvirus-infected cells
GFP expression. Cells were plated in 96-well tissue culture plates
and infected at an m.o.i. of 10, 1 or 0.1 as described above, and
infected with a recombinant AMEV expressing gfp in the spheroidin
locus. GFP fluorescence in the wells was measured directly in a plate
reader equipped with 485Ex/520Em filters.
Assays for apoptosis
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Caspase and viability assays. Cells were infected at an m.o.i. of 10
and harvested at desired time points. Caspase-3/7 activity and cell
viability were measured using the Caspase-Glo 3/7 Assay (Promega)
and Cell Titre-Blue Cell Viability Assay (Promega) according to
manufacturer’s instructions. Each assay was done in triplicate.
Caspase activity was normalized to cell viability and presented as a
ratio of the mock-infected control cells.
Fragmentation of DNA. Cells were seeded on 25 cm2 flasks and
infected as described above. Cells were collected by centrifugation at
1000 g for 5 min and DNA was extracted according to published
protocols (Sambrook et al., 1989). Ten micrograms of DNA was
analysed on a 1.5 % agarose gel.
TUNEL assay. Cells were infected and collected by centrifugation as
described above. The TUNEL assay was carried out using the
DeadEnd Fluorometric TUNEL system (Promega). Fixing and
staining of cells were done according to the Promega technical
bulletin (TB235) provided with the TUNEL assay kit. Cells were
counterstained with DAPI (VECTASHIELD Hardset Mounting
Medium with DAPI) and visualized in a Nikon Eclipse TS100
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FMK (MP Biomedicals) and Q-VD-OPH (MP Biomedicals) were
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ACKNOWLEDGEMENTS
This research was supported by a Genomics grant from the Canadian
Forest Service.
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