Journal of General Virology (2013), 94, 1636–1646
DOI 10.1099/vir.0.051540-0
Axin expression enhances herpes simplex virus type
1 replication by inhibiting virus-mediated cell death
in L929 cells
Eun-Jin Choi,1 Sewoon Kim,2 Eek-hoon Jho,2 Ki-Joon Song1
and Sun-Ho Kee1
Correspondence
Sun-Ho Kee
1
Laboratory of Cell Biology, Department of Microbiology and Bank for Pathogenic Virus,
College of Medicine, Korea University, Seoul, 136-705, Korea
[email protected]
2
Received 13 January 2013
Accepted 22 March 2013
Herpes simplex virus type 1 (HSV-1) replicates in various cell types and induces early cell death,
which limits viral replication in certain cell types. Axin is a scaffolding protein that regulates Wnt
signalling and participates in various cellular events, including cellular proliferation and cell death.
The effects of axin expression on HSV-1 infection were investigated based on our initial
observation that Wnt3a treatment or axin knockdown reduced HSV-1 replication. L929 cells
expressed the axin protein in a doxycycline-inducible manner (L-axin) and enhanced HSV-1
replication in comparison to control cells (L-EV). HSV-1 infection induced cell death as early as
6 h after infection through the necrotic pathway and required de novo protein synthesis in L929
cells. Subsequent analysis of viral protein expression suggested that axin expression led to
suppression of HSV-1-induced premature cell death, resulting in increased late gene expression.
In analysis of axin deletion mutants, the regulators of the G-protein signalling (RGS) domain were
involved in the axin-mediated enhancement of viral replication and reduction in cell death. These
results suggest that viral replication enhancement might be mediated by the axin RGS domain.
Department of Life Science, University of Seoul, Seoul 130-743, Korea
INTRODUCTION
Herpes simplex virus type 1 (HSV-1) is a large (~152 kb)
fast-replicating DNA virus that infects many cell types
(Whitley & Roizman, 2001). HSV-1 infection causes
various human diseases such as orofacial lesions, and
ocular, brain and disseminated viral diseases (Whitley &
Roizman, 2001). At the cellular level, HSV-1-infected cells
undergo structural and biochemical alterations termed
cytopathic effects, which ultimately lead to cell lysis and
death (Nguyen & Blaho, 2006). In addition to necrotic cell
death, HSV-1 infection triggers the apoptotic cell death
pathway. Apoptosis is first triggered and later blocked in
cells infected with HSV-1 (Aubert et al., 1999; Koyama &
Adachi, 1997; Nguyen & Blaho, 2006), suggesting that
HSV-1 expresses both pro-apoptosis and anti-apoptosis
genes. The triggering of apoptosis seems to occur in the
absence of protein synthesis within 3 h post-infection in
HEp-2 cells (Aubert et al., 1999), but Vero cells require de
novo protein synthesis for HSV-1-dependent apoptosis
(Nguyen et al., 2005). Many lines of evidence have shown
the involvement of mitochondrial membrane perturbances
and caspase-3 activation during HSV-1-mediated apoptosis
(Kraft et al., 2006; Nguyen & Blaho, 2006), whereas HSV-1
mutations such as ICP4 (infected-cell polypeptide 4)
deletion (d120) induce caspase-independent apoptosis
(Galvan et al., 1999). In contrast, anti-apoptosis genes
1636
appear to be expressed 6 h post-infection (Aubert et al.,
1999), and numerous viral gene products such as ICP4,
ICP27, ICP34.5 and US5 kinase have anti-apoptosis effects
(Nguyen & Blaho, 2006). Cellular proteins such as Bcl-2 and
NF-kB seem to be involved during HSV-1-mediated antiapoptosis. In addition, some viral proteins interact with
specific cellular proteins to attenuate apoptosis. For
example, the interaction of HSV-1 U3 kinase with
programmed cell death protein 4 participates in the
attenuation of apoptosis in cells infected with the HSV-1 d
120 mutant (Wang et al., 2011). The balance between proapoptotic and anti-apoptotic effects of HSV-1 gene
expression may determine the rate of infected-cell death. If
the balance in cell death regulation shifts to apoptosis, HSV1 infection may trigger premature death of host cells, which
produces an unfavourable environment for viral replication.
In accordance with this hypothesis, inhibiting apoptosis
using the caspase inhibitor zVAD increases viral persistence
and replication (Wood & Shillitoe, 2011). Among other cell
death-related mechanisms, autophagy seems to inhibit
HSV-1 replication (Pei et al., 2011), and the anti-autophagic
effect of HSV-1 ICP34.5 has been well described (Alexander
et al., 2007; Orvedahl et al., 2007). Additionally, HSV-1
infection induces autophagy in some cells including
fibroblasts and macrophages (English et al., 2009;
McFarlane et al., 2011). As identifying the HSV-1-induced
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Axin facilitates HSV-1 replication
cell death mechanisms appears to be dependent on
differences in cell type (Aubert & Blaho, 2003; English
et al., 2009; Galvan & Roizman, 1998; McFarlane et al., 2011;
Nguyen et al., 2005), cellular factors may be important for
HSV-1-induced cell death and viral replication. Thus, in this
study, the effects of axin expression were investigated in
terms of HSV-1 replication and virus-mediated cell death to
analyse the influence of host factors during HSV-1 infection.
The most well-known function of axin is negative
regulation of canonical Wnt signalling through formation
of the b-catenin destruction complex. Upon Wnt interaction with its receptor [e.g. frizzled (Fz) or LRP)], bcatenin dissociates from axin and is transported into the
nucleus to transcriptionally upregulate proliferationrelated genes such as myc and cyclin D. In addition, axin
appears to participate in the induction of cell death in
Chinese hamster ovary (CHO) cells (Neo et al., 2000). In
several systems, axin appears to be involved in regulation
rather than direct induction of cell death. Axin overexpression enhances mitotic defect-induced cell death in
L929 cells (Choi et al., 2011). Additionally, axin functions
in UV-induced cell death by phosphorylating p53 at Ser 46,
which is catalysed by HIPK2 (Li et al., 2007). Subsequent
observations showed that Pirh2 competes with HIPK2 to
bind axin and abrogates HIPK2-mediated p53 phosphorylation, which allows cells to survive under low-dose UV
irradiation (Li et al., 2009). Therefore, axin determines cell
fate by controlling p53 activation status. In contrast, some
lines of evidence suggest that axin plays a cytoprotective
role under specific conditions. Reduction of b-catenin
levels showed a neuroprotective effect in neurons and in a
Drosophila model of Huntington’s disease (HD) (Godin
et al., 2010), and axin overexpression increases lifespan in
HD Drosophila (Dupont et al., 2012). Moreover, axin
expression appears to alter mitochondrial function, which
attenuates staurosporine (STS)-induced mitochondriamediated cell death in HeLa cells (Shin et al., 2012). These
observations suggest that axin may play a protective role in
cells under some harmful conditions, although antiproliferative or cell death effects are more frequently observed.
We observed that HSV-1 infected and replicated more
efficiently in axin-expressing L929 cells in comparison to
control cells. This enhanced HSV-1 replication effect was
accompanied by reduced cell death.
RESULTS
Axin expression facilitates HSV-1 replication and
reduces HSV-1-induced cell death
HSV-1-infected cells were treated with Wnt3a-conditioned
medium (Wnt3a-CM) to determine the effects of Wnt
signalling activity on viral replication. Treatment with Wnt3aCM induced an increase in b-catenin levels in L929 cells (Fig.
1a), suggesting the activation of b-catenin-dependent Wnt
signalling. HSV-1 infection reduced b-catenin levels, which
http://vir.sgmjournals.org
was surpassed by Wnt3a-CM treatment (Fig. 1b). Viral
replication, which was monitored by viral capsid protein ICP5
expression in Wnt3a-CM-treated L929 cells, appeared to be
reduced in comparison to that of control untreated cells (Fig.
1b). In the case of the b-catenin knockdown experiment, only
a slight increase of viral replication was observed in L929 cells
(Fig. 1c, d). To evaluate further, knockdown of endogenous
axin, which is a negative regulator of b-catenin-dependent
Wnt signalling, was performed and a reduction of viral
replication was observed in L929 cells (Fig. 1e, f). These results
suggest that HSV-1 replication may be influenced by bcatenin-dependent Wnt signalling activity.
Next, we analysed the effects of axin expression on HSV-1
replication, because axin is an important regulatory protein
in b-catenin-dependent Wnt signalling (Zeng et al., 1997).
For this, axin-expressing L929 cells (L-axin), which express
axin and GFP using a dual promoter system in a
doxycycline-inducible manner, were established. Axin
expression was monitored using the tagged Myc peptide
in L-axin cells (Fig. 2a). Wnt3a-CM treatment increased
the b-catenin level in L-axin cells, but this effect was less
apparent compared to that in L929 cells (Fig. 1b).
Additionally, the efficiency of HSV-1 replication appeared
to be higher in L-axin than in L929 cells regardless of
Wnt3a treatment (Fig. 1b). The increase of HSV-1
replication appeared to be axin-expression-specific since
the increase was more apparent in the presence of
doxycycline (Fig. 2b). A detailed time-dependent analysis
showed that a higher expression of ICP5 was observed
from 16 h post-infection in L-axin cells compared to that
in control L-EV cells (Fig. 2c). The increase in HSV-1
replication in L-axin cells was confirmed by immunofluorescence analysis (Fig. 2d). A quantitative plaqueforming analysis using infected-cell culture supernatant
showed about a fivefold increase in virus release from Laxin cells compared to that from L-EV cells (Fig. 2e, f). In
conclusion, our results suggest that axin expression may
enhance HSV-1 replication, providing supportive evidence
of an inverse relationship between HSV-1 replication and
b-catenin-dependent Wnt signalling.
HSV-1-mediated cell death was analysed. In phase-contrast
microscopic observations, HSV-1 infection-induced cell
death was less clear in L-axin cells than in L-EV cells (Fig.
3a). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that HSV-1-infected LEV cells showed significant cell death in dose- and timedependent manners, but these effects decreased significantly
in L-axin cells (Fig. 3b). Similarly, HSV type 2 (HSV-2)
produced less apparent cell death in L-axin cells than in LEV cells, although this effect was obvious only 48 h after
infection (Fig. 3c). A mixed culture of L-axin and parental
L929 cells was infected with HSV-1 and analysed using timelapse imaging. Because L929 cells did not express GFP, the
GFP-expressing cells were L-axin cells. L929 cells underwent
the cell death process, whereas many GFP-expressing L-axin
cells remained viable throughout the imaging period (Fig.
3d). These cell images were used to directly count viable and
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E.-J. Choi and others
(a)
Wnt3a
– 1/5 1/2 1 (dilution)
b-CTN
24 h
– S B1 B2 B1+2
(c)
siCatenin
48 h
– S B1 B2 B1+2
b-CTN
b-Actin
b-Actin
(b)
HSV-1
Wnt3a
–
–
L929
+ +
– +
–
–
L-axin
+ +
– +
(d)
ICP5
HSV-1 + +
siCatenin S B1+2
ICP5
b-CTN
Myc
b-CTN
b-Actin
b-Actin
(e)
(f)
HSV-1
siAxin
–
–
+
–
+
S
+ +
A1 A2
E5
IP IgG
– – S A1 A2
siAxin
ICP5
G4
b-Actin
HC
Fig. 1. HSV-1 replication is inversely related to Wnt signalling. (a) L929 cells were incubated in media with the indicated
dilutions of Wnt3a-CM or without Wnt3a-CM for 24 h. Activation of Wnt signalling was identified by increased expression of bcatenin (b-CTN). (b) L929 or L-axin cells treated with Wnt3a-CM were infected with HSV-1 (m.o.i.56) for 16 h. Immunoblot
analysis showed that treatment with Wnt3a decreased viral replication in L929 and L-axin cells. (c) L929 cells were transfected
with two kinds of b-catenin siRNA (B1, siCatenin-B1; B2, siCatenin-B2) and scrambled control siRNA (S). At 24 and 48 h after
siRNA transfection, a reduction in b-catenin expression was identified by immunoblot analysis. (d) At 24 h post-transfection of
siRNA, L929 cells were infected with HSV-1 for 16 h and subjected to immunoblot analysis. Knockdown of b-catenin slightly
increased HSV-1 replication in L929 cells. (e) Immunoblot analysis revealed that knockdown of axin decreased HSV-1
replication at 16 h post-infection in L929 cells. (f) A reduction in axin expression by siAxin transfection was confirmed by
immunoprecipitation using the E5 mAb and immunoblot analysis using mAb G4, as previously described (Kim et al., 2009).
Results are representative of three independent experiments. HC, heavy chain of IgG.
dead cells (Fig. 3e). Cell death in L929 cells was observed
from 6 h post-infection, whereas L-axin cells began to die at
15 h post-infection. Taken together, our results suggest that
axin expression reduces or delays HSV-1-induced early cell
death and facilitates viral replication.
The detailed mechanisms of HSV-1-mediated cell death
were elucidated. In live and dead cell staining at 16 h postinfection (Fig. 4a), a greater abundance of dead cells was
observed for L-EV cells in comparison with L-axin cells.
This cell death appeared to occur in a caspase-3independent manner because HSV-1 infection failed to
induce caspase-3 activation or poly(ADP-ribose) polymerase cleavage in both L-EV and L-axin cells (Fig. 4b). For a
detailed analysis of HSV-1-induced cell death, L-EV and Laxin cells were infected with HSV-1, and subjected to flow
cytometric analysis (Fig. 4c). At 16 h post-infection, the
Annexin V-PE and 7-amino-actinomycin D (7-AAD)
positive population was increased from 2.06 to 11.44 %
in L-EV cells, whereas there was no significant increase in
L-axin cells. Annexin V-PE positive and 7-AAD negative
1638
cells were not increased upon HSV-1 infection in both
cells, suggesting that HSV-1 induced non-apoptotic,
necrotic cell death. The proportion of dead cells in both
virus-infected L-EV and L-axin cells appeared to be less
abundant in comparison to the results of the MTT assay
(Fig. 3b). This discrepancy might result from the loss of
shrunken dead cells during stringent washing steps.
Furthermore, treatment with necrostatin-1, which is
known as necroptosis inhibitor (Degterev et al., 2005),
facilitated HSV-1 replication and decreased virus-induced
cell death in L929 cells (Fig. 4d, e). These results suggest
that HSV-1-mediated cell death was induced through the
necrotic pathway and reduction of necrotic cell death
might enhance HSV-1 replication in L-axin cells.
HSV-1-mediated cell death requires viral
replication and de novo protein synthesis
L-EV and L-axin cells were infected with UV-irradiated
HSV-1 to determine whether the axin-mediated reduction
in cell death was caused by engagement of the virus–receptor
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Journal of General Virology 94
Axin facilitates HSV-1 replication
L-EV
(a)
Dox
(b)
L-EV L-axin
– + – +
HSV-1
Dox
– – + +
– + – +
L-axin
– – + +
– + – +
ICP5
Myc
Myc
b-Actin
β-Actin
(d)
(c)
HSV-1
0h
8h
16 h
L-EV
ICP5
24 h
Myc
L-axin
ICP5
Myc
EV Axin EV Axin EV Axin EV Axin
ICP5
0h
Myc
b-Actin
10-3
10-4
10-5
L-EV
L-axin
(f)
10−6× viral titre (p.f.u. ml−1)
(e)
16 h
8
6
4
2
0
L-EV L-axin
Fig. 2. Axin expression enhances replication of HSV-1 in L929 cells. (a) The expression of Myc-tagged axin was analysed by
immunoblot analysis in L929 cells stably expressing doxycycline-inducible axin (L-axin) and control L-EV cells. (b) L-EV and L-axin
cells with or without doxycycline were infected with HSV-1 for 16 h and subjected to immunoblot analysis. In the presence of
doxycycline, L-axin cells showed an increase of HSV-1 replication. (c) L-EV (EV) and L-axin (Axin) cells were infected with HSV-1 for
the indicated times and subjected to immunoblot analysis. A higher expression of ICP5 was observed in L-axin cells than in L-EV cells.
(d) Immunofluorescence analysis showed the increase of viral replication at 16 h post-infection in L-axin cells. Bar, 50 mm. (e) A
plaque-forming assay revealed that productive viral replication occurred in L-axin cells. (f) Plaques observed in (e) were quantified.
Results are representative of three independent experiments and the error bars indicate the SDs. Dox, doxycycline.
complex. Irradiated HSV-1 failed to express the late gene
product ICP5 after infection (Fig. 5a). Upon infection with
irradiated HSV-1, cell death of both L-EV and L-axin cells
decreased significantly in comparison to cell death upon
infection with non-irradiated virus (Fig. 5b). Furthermore,
the protein translation requirement for HSV-1-mediated
cytotoxic effects was analysed using the translation inhibitor
cycloheximide (CHX). The effect of CHX was confirmed by
failure of capsid protein synthesis after HSV-1 infection (Fig.
5c). CHX treatment also reduced HSV-1-mediated cell
death in both L-EV and L-axin cells (Fig. 5d). Because the
reduction in cell death was greater in L-EV cells, cell viability
became similar in both L-EV and L-axin cells, suggesting
that HSV-1-mediated cell death may require newly synthesized viral proteins after viral entry into L929 cells and that
axin expression may suppress this HSV-1-mediated cell
death. Next, expression of various viral proteins was
investigated to identify which replication steps were
influenced by cell death (Fig. 5e). For this, immunoblot
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analysis was performed using antibodies detecting an
immediate-early (ICP4), early (ICP8) or late (ICP5) protein
(Bryant et al., 2012; Grondin & DeLuca, 2000). Whereas the
expression patterns of ICP4 and ICP8 were similar between
L-EV and L-axin cells after HSV-1 infection, ICP5
expression level was higher in L-axin cells than in L-EV
cells. These results suggest that HSV-1 induces cell death in
an early phase of infection, which may reduce late gene
expression. Therefore, axin-mediated inhibition of cell death
may influence late gene expression more significantly than
immediate-early or early gene expression.
Regulators of G-protein signalling (RGS) domain
of the axin protein participate in facilitating HSV-1
replication
To analyse the axin protein domain responsible for
enhancing HSV-1 replication, various axin deletion mutants
were constructed, and L929 cells expressing these mutant
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E.-J. Choi and others
(a)
L-axin
(b)
m.o.i.=13
Cell viability (%)
L-EV
Ctrl
120
100
80
60
40
20
0
0
24 h
m.o.i.=6
L-EV
L-Axin
9
16 24
120
100
80
60
40
20
0
m.o.i.=3
L-EV
L-Axin
0
9
Time (h)
16 24
120
100
80
60
40
20
0
L-EV
L-axin
0
9
16 24
(d)
04:00
12:00
14:42
24:00
13:24
(c)
L-EV
L-axin
14:12
L929: GFP (–)
L-axin: GFP (+)
Ctrl
(e)
120
48 h
Cell viability (%)
24 h
L929
L-axin
100
80
60
40
20
0
4 5 6 7 8 9 101112131415161718192021222324
Time (h)
Fig. 3. Axin expression reduces HSV-1-mediated cell death. (a) L-EV and L-axin cells before infection (Ctrl) and at 24 h postinfection with HSV-1 were observed through a phase-contrast microscope. A clearer morphology of cell death was observed in
L-EV than L-axin cells. Bar, 20 mm. (b) L-EV and L-axin cells were infected with HSV-1 at different m.o.i. levels for the indicated
times and subjected to MTT assay. L-axin cells showed higher viability than L-EV cells in time- and dose-dependent manners.
The error bars indicate the SDs. (c) In a phase-contrast microscopy observation, HSV-2-induced cell death appeared more
clearly in L-EV than L-axin cells. Bar, 20 mm. (d) Mixed L929 and L-axin cells were infected with HSV-1 and subjected to timelapse imaging analysis. HSV-1 infection led to cell death in GFP-negative parental L929 cells, but this death was delayed in
GFP-positive L-axin cells. Dead cells were determined using morphological characteristics such as loss of refractivity, flattening
of the cells and loss of cell membrane integrity. Arrowheads indicate L-axin cells and arrows indicate dead cells. Bar, 20 mm. (e)
From time-lapse images, 50 cells were counted directly and presented in the graph. Results are representative of three
independent experiments.
axin genes were established (Fig. 6a). All cells expressed
mutant axin and GFP simultaneously in a doxycyclineinducible manner. An initial analysis of infected-cell
cytotoxicity showed that all deletion mutant-expressing
cells, except those with the RGS domain deletion (L-DR)
mutant, showed cytoprotective effects upon HSV-1 infection (Fig. 6b). Subsequent immunoblot analysis of ICP5
expression from infected cells revealed that infected L-DR
cells showed an apparent reduction in ICP5 expression (Fig.
6c, d), suggesting suppression of viral replication. Although
deletion of the glycogen synthase kinase 3 (GSK3b)-binding
domain (L-DG) also produced some cell death and reduced
1640
viral replication compared to those of L-axin cells, these
effects appeared less clear than those in L-EV and L-DR cells
(Fig. 6b–d). These results raised the possibility that the axin
RGS domain may influence HSV-1 infection through a
distinct mechanism in addition to regulating b-catenin
stability through destruction of the b-catenin complex.
Together with the DIX domain, the RGS domain is well
conserved between axin and its homologue axin2 (Behrens
et al., 1998). Because the RGS domain appears to be related
to axin-mediated enhancement of viral replication and
suppression of cytotoxicity, we speculated that axin2 may
have a similar effect to axin. To evaluate the effect of axin2 on
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Journal of General Virology 94
Axin facilitates HSV-1 replication
Mock
EV Ax
(b)
(a)
Dead
Merged
Live
PH
HSV-1
EV Ax
STS
EV
ICP5
Active
caspase-3
L-EV
PARP-1
L-axin
Myc
b-Actin
(c)
Mock
0.41
(d)
HSV-1
2.06
0.99
HSV-1
Nec-1
– + + + +
– – 10 20 50 (μM)
11.44
b-Actin
96.78
0.75
83.65
3.92
7-AAD
(e)
2.94
2.12
2.77
2.06
94.51
0.43
93.07
2.10
L-axin
Cell viability (%)
Annexin
ICP5
L-EV
120
100
80
60
40
20
0
HSV-1
Nec-1
– + + + +
– – 10 20 50
(μM)
Fig. 4. HSV-1-induced cell death occurred through a necrotic pathway in L929 cells. (a) HSV-1-infected L-EV and L-axin cells
were subjected to live and dead staining at 16 h post-infection. Dead cells with red signals were increased in HSV-1-infected
L-EV cells. PH, phase contrast images. (b) HSV-1 infection for 16 h did not induce caspase-3 activation or poly(ADP-ribose)
polymerase-1 cleavage in L-EV (EV) and L-axin (Ax) cells. STS-treated L-EV cells were used as positive controls. (c) At 16 h
post-infection with HSV-1, L-EV and L-axin cells were stained using Annexin V-PE and 7-AAD, and then subjected to flow
cytometry. Necrotic cell death (Annexin V-PE and 7-AAD positive) was detected in HSV-1-infected L-EV cells, but not in L-axin
cells. (d, e) L-EV cells were treated with the indicated dose of necrostatin-1 for 1 h prior to infection. After infection with HSV-1,
cells were cultivated in fresh media containing necrostatin-1 for 16 h and then subjected to immunoblot analysis (d) or MTT
assay (e). Treatment with necrostatin-1 increased HSV-1 replication and cell viability in HSV-1-infected L-EV cells. Results are
representative of three independent experiments and the error bars indicate the SDs.
HSV-1 replication, L929 cells expressing axin2 were established
(L-axin2). L-axin2 cells infected with HSV-1 showed increased
ICP5 expression compared to that in L-EV cells (Fig. 7a),
although this increase in ICP5 expression appeared to be less
efficient than that in L-axin cells. Subsequent phase-contrast
microscopic observations revealed that L-axin2 cells were
resistant to the cytotoxic effects of HSV-1 compared to those
of L-EV cells (Fig. 7b). This result was confirmed through the
quantification of cell viability, which was performed by direct
counting of viable and dead cells using the phase-contrast
images from Fig. 7b (Fig. 7c). Taken together, the effect of axin
on HSV-1 replication might be mediated, at least in part, by a
function related to the axin RGS domain.
DISCUSSION
Axin is a concentration-limiting factor regulating bcatenin-dependent Wnt signalling activity (Salahshor &
http://vir.sgmjournals.org
Woodgett, 2005), and the increase in axin level results in
antiproliferative effects in various cells (Huang et al., 2009;
Salahshor & Woodgett, 2005). In this study, we showed
that Wnt3a treatment and axin knockdown reduced HSV-1
replication (Fig. 1), suggesting an inverse relationship
between Wnt signalling activity and viral replication. In the
case of b-catenin knockdown, only a slight increase of
HSV-1 replication was observed (Fig. 1d). This result led us
to speculate that there are additional mechanisms in
addition to the regulation of b-catenin-dependent Wnt
signalling. Given that the axin mutant with deletion of the
GSK3b-binding domain (L-DG) showed relatively higher
HSV-1 replication than L-EV and L-DR (Fig. 6), axin
affects HSV-1 replication through RGS domain-related
functions, as well as by regulation of b-catenin-dependent
Wnt signalling. Therefore, the effect of b-catenin knockdown on HSV-1 replication may be less efficient in
comparison to that of axin expression. Although axin
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1641
E.-J. Choi and others
L-EV
HSV-1 – + +
UV – – +
ICP5
L-axin
– + +
– – +
Mock
HSV-1
HSV-1+UV
(b)
Cell viability (%)
(a)
Myc
b-Actin
120
100
80
60
40
20
0
Myc
b-Actin
L-axin
– + +
– – +
Cell viability (%)
L-EV
HSV-1 – + +
CHX – – +
ICP5
L-EV
HSV-1
L-axin
– 1 4 8 16 – 1 4 8 16 (h)
ICP4
L-EV
(d)
(c)
(e)
L-axin
Mock
HSV-1
HSV-1+CHX
120
100
80
60
40
20
0
ICP8
ICP5
Myc
b-Actin
L-EV
L-axin
Fig. 5. HSV-1-mediated cell death requires viral replication and de novo protein synthesis. (a, b) L-EV and L-axin cells were
infected with UV-irradiated HSV-1 for 16 h and subjected to immunoblot analysis (a) or MTT assay (b). UVC irradiation blocked
viral replication and UV-irradiated HSV-1 did not efficiently induce cell death. The error bars indicate the SDs. (c, d) L-EV and Laxin cells were infected with HSV-1 in the presence of CHX for 16 h and subjected to immunoblot analysis (c) or MTT assay (d).
Inhibition of viral protein synthesis by CHX did not efficiently induce HSV-1-mediated cell death. The error bars indicate the SDs.
(e) L-EV and L-axin cells were infected with HSV-1 for the indicated times and then subjected to immunoblot analysis using
anti-ICP4, anti-ICP8 and anti-ICP5 antibodies. In both cell types, the expression pattern of ICP4 and ICP8 was similar, whereas
ICP5 expression was higher in L-axin cells than in L-EV cells. Results are representative of three independent experiments.
participates in regulation of the cell death mechanism
(Choi et al., 2011; Li et al., 2007, 2009; Neo et al., 2000),
recent evidence has revealed a cytoprotective role for axin
under specific conditions. Axin overexpression confers
protection against mutant huntingtin toxicity in the
Drosophila model (Dupont et al., 2012). In addition,
axin expression attenuates STS-induced mitochondrialmediated cell death in HeLa cells (Shin et al., 2012). Axin
also shows a protective effect against Salmonella invasiveness in intestinal epithelial cells (Zhang et al., 2012). In that
report, the DIX domain but not the RGS domain was
required for axin to inhibit Salmonella invasion. In our
results, enhancement of HSV-1 replication required the
RGS domain, suggesting that different intrinsic properties
of axin may be involved in these two events. Axin interacts
with numerous cellular proteins as a scaffolding protein
(Salahshor & Woodgett, 2005), and a small amount of axin
is sufficient in normal cells (Lee et al., 2003).
HSV-1 infection leads cells to undergo structural and
biochemical alterations, which result in necrotic cell death
involving cell lysis (Nguyen & Blaho, 2006; Roizman,
1974). Additionally, HSV-1 can trigger apoptosis at
multiple steps of the infection through expression of proapoptotic and anti-apoptotic molecules (Nguyen & Blaho,
2006). During the early stage of infection, HSV-1 infection
induces apoptotic early death, whereas apoptosis is blocked
by viral proteins in the late stage, and necrotic cell death
1642
ensues. Our results show HSV-1 infection-induced cell
death occurred as early as 6 h post-infection in L929 cells,
but this early cell death was apparently reduced in L-axin
cells (Fig. 3d, e). However, apoptotic signs related to HSV1-induced cell death in control L-EV cells were difficult to
detect. Instead, HSV-1 might induce necrotic cell death
because infected L-EV cells showed perturbation of the cell
membrane, which was assessed by 7-AAD staining (Fig.
4c). Moreover HSV-1-induced cell death was inhibited by
treatment with necrostatin-1 in L929 cells (Fig. 4e). These
results suggest that HSV-1 infection induced necrotic cell
death in L929 cells. Previous observations showed that
L929 cells are vulnerable to necrotic cell death under
specific conditions. For example, zVAD, which is a caspase3 inhibitor that generally shows low cytotoxicity in most
cell lines (Van Noorden, 2001), produces robust necrotic
cell death and autophagy in L929 cells (Chen et al., 2011;
Wu et al., 2011). Different from other cells, treatment of
L929 cells with TNF causes necrotic cell death and
autophagy (Vercammen et al., 1998a; Ye et al., 2011),
and this cell death was exaggerated by treating the L929
cells with zDEVD or zVAD (Vercammen et al., 1998b).
Considering this unique property of L929 cells in response
to cell death, HSV-1 infection preferentially induced
premature necrotic cell death in L929 cells, which might
cause limited viral replication. In addition, HSV-1-induced
cell death seemed to require de novo protein synthesis (Fig.
5c, d), and axin expression did not affect ICP4 and ICP8
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Journal of General Virology 94
Axin facilitates HSV-1 replication
(a)
(b)
Myc tag
127
406
508
ΔP
ΔG
ΔP
Axin
711
(c)
(d)
Mock
ΔM
ΔR
DIX
354
377
ΔG
EV
PP2Ac
226
228
ΔM
GSK b-CTN
MEKK1
HSV-1
EV Axin ΔR ΔG EV Axin ΔR ΔG
Mock
HSV-1
EV Axin ΔP ΔM EV Axin ΔP ΔM
ICP5
Myc
b-Actin
ICP5 protein level (fold change)
RGS
Mock
ΔR
N-term
HSV-1
FL
Splicing site
4
3
2
1
0
EV
ΔR ΔM ΔG ΔP Axin
Fig. 6. Deletion of the RGS domain abolishes the effects of axin on HSV-1 infection in L929 cells. (a) Schematic diagram of
deletion mutant constructs of the axin gene. Numbers indicates the amino acid numbers according to the axin sequence
(GenBank accession no. NP_001153070.1). DR, DM, DG and DP indicate deletion of RGS, mitogen-activated protein kinase
kinase (MEKK), glycogen synthase kinase (GSK) and protein phosphatase 2A (PP2A) axin domains, respectively. FL, full-length
axin. (b) L929 cells expressing various axin deletion mutants were infected with HSV-1 for 16 h and subjected to phasecontrast microscopy observations. Dead cells were determined using morphological characteristics such as loss of refractivity,
flattening of the cells and loss of cell membrane integrity. Bar, 20 mm. (c) L929 cells expressing various axin deletion mutants
were infected with HSV-1 for 16 h and subjected to immunoblot analysis. Compared to the other axin deletion mutants and fulllength axin, deletion of the RGS domain resulted in a lower level of ICP5 expression. (d) ICP5 protein expression relative to bactin of each axin mutant-expressing cell line was measured from (c) using densitometry, and a quantitative comparison was
performed. Results are representative of three independent experiments. EV, L-EV calls; axin, L-axin cells.
expression (Fig. 5e). These results suggest that immediateearly or early proteins might induce premature necrotic cell
death in L929 cells, which might cause restriction of late
gene product expression and viral replication. In L-axin
cells, suppressing HSV-1-induced cell death may produce a
more favourable situation for viral replication.
In analysis of deletion mutants of axin, both the RGS and
GSK3b-binding domain of axin may participate in axinmediated modulation of HSV-1 replication and cell death
(Fig. 6). Both domains were involved in b-catenindependent Wnt signalling through binding to adenomatosis polyposis coli (APC) and GSK3b (Itoh et al., 1998;
Kishida et al., 1998). Quantification of the results suggests
that the RGS domain may have additional functions to
axin-mediated regulation of b-catenin-dependent Wnt
signalling activity because deletion of the GSK3b-binding
domain showed only partial reversion of axin effects on
viral replication and cell death (Fig. 6). This speculation
may be supported by the results that b-catenin knockdown
affects viral replication only slightly (Fig. 1d). In the
literature, APC and the alpha subunit of the trimeric
G-protein (Gao) have been well described as RGS-binding
http://vir.sgmjournals.org
proteins. In addition to regulation of b-catenin-dependent
Wnt signalling, APC participates in numerous cellular
events, such as cell migration, polarization, microtubule
stability and cell death (Hanson & Miller, 2005; McCartney
& Näthke, 2008). In terms of cell death, APC plays
complicated roles. The expression of full-length APC in a
colon carcinoma cell line induces apoptosis (Morin et al.,
1996), and conversely loss of Drosophila APC induces
apoptosis of retinal neurons (Ahmed et al., 1998). In some
cases, APC-mediated regulation of cell cycle progress
appears to be independent of b-catenin-mediated signals
(Ishidate et al., 2000). More recently, it was described that
Gao directly acts on the axin RGS domain, which
neutralizes the axin inhibitory function for Wnt signalling
(Egger-Adam & Katanaev, 2010). Gao may participate in
overactivation of Wnt/Fz signalling, and the interaction of
Gao with Rab4 and Rab5 may be involved in Gaomediated Wnt signalling activation (Egger-Adam &
Katanaev, 2008; Koval et al., 2011). In addition to Gao
binding ability, both the Rab5 and axin proteins show
neuroprotective effects in the HD Drosophila model
(Dupont et al., 2012; Ravikumar et al., 2008). These
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1643
E.-J. Choi and others
which had a deletion of a specific domain, was transfected into L929
cells and cultured in DMEM containing G418. Final clones expressing
the appropriate axin mutant were selected. Each mutant axin was
expressed by the doxycycline-inducible system. Each axin deletion
mutant contained an RGS domain deletion (amino acid numbers
127–226; DR), a mitogen-activated protein kinase kinase 1 deletion
(228–354; DM), a GSK3b deletion (377–406; DG), or a protein
phosphatase 2A deletion (508–711; DP). STS, necrostatin-1 and CHX
were purchased from Sigma. The following antibodies were used: bactin, ICP5, Myc (Santa Cruz Biotechnology), ICP4, ICP8 (Abcam),
b-catenin (BD Pharmingen), active caspase-3 (Cell Signaling
Technology).
HSV-1
Ctrl
EV Ax Ax2 EV Ax Ax2
(a)
ICP5
Myc
b-Actin
L-EV
(b)
L-axin
L-axin2
Ctrl
Virus and drug treatment. HSV-1 strain HF (ATCC VR-260) and
(c)
Cell viability (%)
16 h
120
100
80
60
40
20
0
Ctrl
HSV-1
L-EV L-axin L-axin2
Fig. 7. Axin2 expression enhances HSV-1 replication and reduces
HSV-1-mediated cell death. L-EV (EV), L-axin (Ax) and L-axin2
(Ax2) cells were infected with HSV-1 and subjected to immunoblot
analysis using the indicated antibodies (a) or phase-contrast
microscopy observations (b) before infection (Ctrl) or at 16 h postinfection. Bar, 20 mm. (c) Viable and dead cells were directly
counted from phase-contrast images that were taken at 16 h postinfection with HSV-1 and presented as a graph. Approximately
150 cells were counted for each cell line in triplicate. The error
bars indicate the SDs.
HSV-2 strain G (ATCC VR-734) were propagated in Vero cells using
Eagle’s minimum essential medium (EMEM) containing 2 % FBS.
Inactivated HSV-1 was prepared by UVC irradiation (12 000 mJ)
using an XL-1500 UV cross-linker (Spectronics). L929 cells were
seeded and treated with doxycycline (1 mg ml21) for 24 h and then
infected with virus. Cells were inoculated with virus stock (m.o.i.56)
in serum-free media, and culture media were exchanged with fresh
EMEM containing 2 % FBS and doxycycline after 2 h for an
additional 16 h, unless indicated. Cells were pretreated with CHX
(10 mg ml21) or necrostatin-1 for 1 h and were maintained in these
drugs during the infection. The Wnt3a-CM was produced from L929
cells, as described previously (Shin et al., 2012). Briefly, control and
Wnt3a-expressing L929 cells were grown for 24 h, and the culture
medium was exchanged with fresh medium. After 24 h incubation,
the medium was collected and used as control-conditioned medium
(control-CM) or Wnt3a-CM. These conditioned media were used for
activating the b-catenin-dependent Wnt signalling cascade in L-EV
and L-axin cells.
Plaque assay. The supernatant obtained from HSV-1-infected L-EV
or L-axin cells was diluted in tenfold steps and inoculated into
confluent Vero cells. After 2 h incubation, the viruses were removed
and the first overlay medium (EMEM, 1 % agarose) was added to
each well. After 4 days, a second overlay medium (EMEM, 1 %
agarose, 5 % neutral red) was added and incubated for 1 day. The
plaques were counted after fixation in 4 % formaldehyde and
detachment of the agarose.
Transfection. Cells were grown to 40–60 % confluency and
observations lead to the speculation that axin, APC, Gao
and Rab proteins may interrelate to regulate some
common cellular events including cell death.
Our results indicate that axin expression suppresses HSV1-induced premature cell death, which results in facilitated
viral replication. These axin effects may be related to
suppression of Wnt signalling activity but the intrinsic
functions of axin mediated by the RGS domain are also
involved in these axin effects on HSV-1 infection.
transfected with siRNA using Lipofectamine 2000 reagent
(Invitrogen) according to the manufacturer’s instructions. The
sequences of two siRNAs specific to mouse axin and to mouse bcatenin were 59-GGCAGAGAGCUCAGGUAUG-39 (siAxin-A1), 59TGCCAAGAAGGCTGATGCG-39 (siAxin-A2), 59-AAGGCUUUUCCCAGUCCUUCA-39 (siCatenin-B1) and 59-AAGAUGAUGGUGUGCCAAGUG-39 (siCatenin-B2). siAxin or siCatenin was transfected
into L929 cells and cultivated for 24 h. Then, the transfected cells
were infected with HSV and subjected to immunoblot analysis at 16 h
post-infection. The reduction in axin expression was monitored by
immunoprecipitation using the E5 and G4 axin-specific monoclonal
antibodies (Kim et al., 2009).
MTT assay. The MTT assay was performed using the CellTiter 96
METHODS
Cells, chemicals and antibodies. L929 cells were grown in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10 % (v/v) FBS (Cambrex). L929 cells expressed ectopic axin (L-axin),
axin2 (L-axin2) or axin deletion mutants together with GFP
simultaneously in a dual doxycycline-inducible manner (Jeon et al.,
2007; Kim et al., 2009). Control cells were established by transfection
with an empty vector with GFP (L-EV). Each mutant axin gene,
1644
non-radioactive cell proliferation assay kit (Promega) according to
the manufacturer’s instructions. Coloured products were measured at
an absorbance of 570 nm using a microplate reader (Spectramax;
Molecular Devices). All experiments were performed in triplicate.
Live imaging. L929 and L-axin cells were mixed and plated on
35 mm dishes. At 4 h post-infection with HSV-1, live images were
taken at 6 min intervals for 20 h using an Observer D1 phase-contrast
microscope equipped with a charge coupled device camera (Carl
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Journal of General Virology 94
Axin facilitates HSV-1 replication
Zeiss). Digital images were processed using AxioVision software (Carl
Zeiss).
Chen, S.-Y, Chiu, L.-Y., Maa, M.-C., Wang, J.-S., Chien, C.-L. & Lin,
W.-W. (2011). zVAD-induced autophagic cell death requires c-Src-
Immunofluorescence assay. The immunofluorescence assay was
dependent ERK and JNK activation and reactive oxygen species
generation. Autophagy 7, 217–228.
performed as described previously (Jeon et al., 2007; Kim et al., 2009).
Briefly, cells were grown on coverslips and infected with HSV-1. After
washing with PBS (pH 7.4), the cells were pretreated with 0.4 %
Triton X-100 and 0.4 % paraformaldehyde in PBS to remove soluble
proteins, and then fixed. Following permeabilization, the cells were
incubated with primary and secondary antibodies. Stained cells were
analysed by fluorescence microscopy (Axioscope; Carl Zeiss). The live
and dead assay was performed using LIVE/DEAD viability/cytotoxicity kit (Molecular Probes) according to the manufacturer’s
instructions.
Immunoblot analysis. The immunoblot analysis was performed as
Choi, E.-J., Kim, S.-M., Song, K.-J., Lee, J.-M. & Kee, S.-H. (2011).
Axin1 expression facilitates cell death induced by aurora kinase
inhibition through PARP activation. J Cell Biochem 112, 2392–2402.
Degterev, A., Huang, Z., Boyce, M., Li, Y., Jagtap, P., Mizushima, N.,
Cuny, G. D., Mitchison, T. J., Moskowitz, M. A. & Yuan, J. (2005).
Chemical inhibitor of nonapoptotic cell death with therapeutic
potential for ischemic brain injury. Nat Chem Biol 1, 112–119.
Dupont, P., Besson, M.-T., Devaux, J. & Liévens, J.-C. (2012).
Reducing canonical Wingless/Wnt signaling pathway confers protection against mutant Huntingtin toxicity in Drosophila. Neurobiology
Dis 47, 237–247.
described previously (Jeon et al., 2007; Kim et al., 2009). Briefly, cells
were cultured in 100 mm dishes and infected with HSV-1. Following
preparation of the cell lysates, SDS-PAGE was performed, and the
proteins were transferred to PVDF membranes. After reacting the
membranes with primary and secondary antibodies, target proteins
were visualized using enhanced chemiluminescence (PerkinElmer).
Egger-Adam, D. & Katanaev, V. L. (2008). Trimeric G protein-
Flow cytometry. FACS analysis was conducted as previously
English, L., Chemali, M., Duron, J., Rondeau, C., Laplante, A.,
Gingras, D., Alexander, D., Leib, D., Norbury, C. & other authors
(2009). Autophagy enhances the presentation of endogenous viral
described (Choi et al., 2011) and the Annexin V-PE apoptosis
detection kit (BD Pharmingen) was used according to the
manufacturer’s instructions. Briefly, cells infected with HSV-1 were
collected and resuspended in binding buffer (10 mM HEPES,
140 mM NaCl, 2.5 mM CaCl2). After addition of Annexin V-PE or
7-AAD, cells were incubated in the dark for 15 min at room
temperature. Stained cells were subjected to flow cytometry using a
FACSCantoII flow cytometer (BD Biosciences) with WinMDI 2.9
(Joseph Trotter, Scripps Research Institute, La Jolla, CA, USA)
software.
dependent signaling by Frizzled receptors in animal development.
Front Biosci 13, 4740–4755.
Egger-Adam, D. & Katanaev, V. L. (2010). The trimeric G protein Go
inflicts a double impact on axin in the Wnt/frizzled signaling
pathway. Dev Dyn 239, 168–183.
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Galvan, V. & Roizman, B. (1998). Herpes simplex virus 1 induces and
blocks apoptosis at multiple steps during infection and protects cells
from exogenous inducers in a cell-type-dependent manner. Proc Natl
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ACKNOWLEDGEMENTS
Godin, J. D., Poizat, G., Hickey, M. A., Maschat, F. & Humbert, S.
(2010). Mutant huntingtin-impaired degradation of b-catenin causes
This research was supported by the Basic Science Research Program
through the National Research Foundation of Korea (NRF) funded by
the Ministry of Education, Science and Technology (2010-0022963).
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