Caspase-mediated cleavage of the feline calicivirus capsid protein.
ABSTRACT Feline calicivirus (FCV) is responsible for an acute upper respiratory tract disease in cats. The FCV capsid protein is synthesized as a precursor (76 kDa) that is post-translationally processed into the mature 62 kDa capsid protein by removal of the N-terminal 124 amino acids. Our previous studies have also detected a 40 kDa protein, related to the FCV capsid protein, produced during infection. Here we demonstrate that cleavage of the FCV capsid protein, during infection of cells in culture, was prevented by caspase inhibitors. In addition, caspase-2, -3 and -7 were activated during FCV infection, as shown by pro-form processing, an increase in N-acetyl-Asp-Glu-Val-Asp-7-amido-4-trifluoromethylcoumarin cleavage activity and in situ poly(ADP-ribose) polymerase cleavage. Caspase activation coincided with the induction of apoptosis and capsid cleavage to the 40 kDa fragment. An in vitro cleavage assay, using recombinant human caspases and in vitro-derived FCV capsid protein, revealed that caspase-2, and to a lesser extent caspase-6, cleaved the capsid protein to generate a 40 kDa fragment. Taken together, these results suggest that FCV triggers apoptosis within infected cells and that caspase-induced capsid cleavage occurs concomitantly with apoptosis. The possible role of capsid cleavage in the pathogenesis of FCV infection is discussed.
- SourceAvailable from: David Tulasne[Show abstract] [Hide abstract]
ABSTRACT: Viral infection constitutes an unwanted intrusion that needs to be eradicated by host cells. On one hand, one of the first protective barriers set up to prevent viral replication, spread or persistence involves the induction of apoptotic cell death that aims to limit the availability of the cellular components for viral amplification. On the other hand, while they completely depend on the host molecular machinery, viruses also need to evade the cellular responses that are meant to destroy them. The existence of numerous antiapoptotic products within the viral kingdom proves that apoptosis constitutes a major threat that should better be bypassed. Among the different strategies developed to deal with apoptosis, one is based on what viruses do best: backfiring the cell on itself. Several unrelated viruses have been described to take advantage of apoptosis induction by expressing proteins targeted by caspases, the key effectors of apoptotic cell death. Caspase cleavage of these proteins results in various consequences, from logical apoptosis inhibition to more surprising enhancement or attenuation of viral replication. The present review aims at discussing the characterization and relevance of this post-translational modification that adds a new complexity in the already intricate host-apoptosis-virus triangle.Cell Death & Disease 01/2012; 3:e277. · 6.04 Impact Factor
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ABSTRACT: The infection process by simian virus 40 (SV40) and entry of its genome into nondividing cells are only partly understood. Infection begins by binding to GM1 receptors at the cell surface, cellular entry via caveolar invaginations, and trafficking to the endoplasmic reticulum, where the virus disassembles. To gain a deeper insight into the contribution of host functions to this process, we studied cellular signaling elicited by the infecting virus. Signaling proteins were detected by Western blotting and immunofluorescence staining. The study was assisted by a preliminary proteomic screen. The contribution of signaling proteins to the infection process was evaluated using specific inhibitors. We found that CV-1 cells respond to SV40 infection by activating poly(ADP-ribose) polymerase 1 (PARP-1)-mediated apoptotic signaling, which is arrested by the Akt-1 survival pathway and stress response. A single key regulator orchestrating the three pathways is phospholipase C-gamma (PLCgamma). The counteracting apoptotic and survival pathways are robustly balanced as the infected cells neither undergo apoptosis nor proliferate. Surprisingly, we have found that the apoptotic pathway, including activation of PARP-1 and caspases, is absolutely required for the infection to proceed. Thus, SV40 hijacks the host defense to promote its infection. Activities of PLCgamma and Akt-1 are also required, and their inhibition abrogates the infection. Notably, this signaling network is activated hours before T antigen is expressed. Experiments with recombinant empty capsids, devoid of DNA, indicated that the major capsid protein VP1 alone triggers this early signaling network. The emerging robust signaling network reflects a delicate evolutionary balance between attack and defense in the host-virus relationship.Journal of Virology 04/2010; 84(7):3431-42. · 5.08 Impact Factor
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ABSTRACT: Theiler's murine encephalomyelitis virus (TMEV) results in a persistent central nervous system infection (CNS) and immune-mediated demyelination in mice. TMEV largely persists in macrophages (Ms) in the CNS, and infected Ms in vitro undergo apoptosis, whereas the infection of other rodent cells produces necrosis. We have found that necrosis is the dominant form of cell death in BeAn virus-infected BHK-21 cells but that ~20% of cells undergo apoptosis. Mcl-1 was highly expressed in BHK-21 cells, and protein levels decreased upon infection, consistent with onset of apoptosis. In infected BHK-21 cells in which Mcl-1 expression was knocked down using silencing RNAs there was a 3-fold increase in apoptotic cell death compared to parental cells. The apoptotic program switched on by BeAn virus is similar to that in mouse Ms, with hallmarks of activation of the intrinsic apoptotic pathway in a tumor suppressor protein p53-dependent manner. Infection of stable Mcl-1-knockdown cells led to restricted virus titers and increased physical to infectious particle (PFU) ratios, with additional data suggesting that a late step in the viral life cycle after viral RNA replication, protein synthesis, and polyprotein processing is affected by apoptosis. Together, these results indicate that Mcl-1 acts as a critical prosurvival factor that protects against apoptosis and allows high yields of infectious virus in BHK-21 cells.Journal of Virology 11/2011; 86(4):1922-9. · 5.08 Impact Factor
Caspase-mediated cleavage of the feline
calicivirus capsid protein
Naema Al-Molawi, Victoria A. Beardmore,3 Michael J. Carter,
George E. N. Kass and Lisa O. Roberts
School of Biomedical and Life Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK
Received 19 September 2002
Accepted 13 January 2003
Feline calicivirus (FCV) is responsible for an acute upper respiratory tract disease in cats. The FCV
capsid protein is synthesized as a precursor (76 kDa) that is post-translationally processed into
the mature 62 kDa capsid protein by removal of the N-terminal 124 amino acids. Our previous
studies have also detected a 40 kDa protein, related to the FCV capsid protein, produced during
infection. Here we demonstrate that cleavage of the FCV capsid protein, during infection of cells in
culture, was prevented by caspase inhibitors. In addition, caspase-2, -3 and -7 were activated
during FCV infection, as shown by pro-form processing, an increase in N-acetyl-Asp-Glu-Val-Asp-
7-amido-4-trifluoromethylcoumarin cleavage activity and in situ poly(ADP-ribose) polymerase
cleavage. Caspase activation coincided with the induction of apoptosis and capsid cleavage to the
40 kDa fragment. An in vitro cleavage assay, using recombinant human caspases and in vitro-
derived FCVcapsid protein,revealed that caspase-2,and toa lesser extent caspase-6, cleavedthe
capsid protein to generate a 40 kDa fragment. Taken together, these results suggest that FCV
triggers apoptosis within infected cells and that caspase-induced capsid cleavage occurs
concomitantly with apoptosis. The possible role of capsid cleavage in the pathogenesis of FCV
infection is discussed.
The family Caliciviridae includes several significant patho-
gens of man and animals. Feline calicivirus (FCV) is a major
cause of upper respiratory tract disease in cats. The genome
is a single-stranded positive sense RNA of about 7?5 kb
(Carter et al., 1992a). The genome is polyadenylated and
covalently linked to a 15 kDa viral protein, termed VPg, at
the 59 end and contains three open reading frames (ORF).
ORF1 encodes the non-structural proteins, ORF2 encodes
the capsid protein and ORF3 a small highly basic structural
protein (reviewed in Clarke & Lambden, 1997). ORF2 and
ORF3 are expressed from a single subgenomic mRNA
(Herbert et al., 1996). Calicivirus particles contain a single
capsid protein (58–76 kDa). The capsid of members of the
vesivirus genus, such as FCV and San Miguel sea lion virus
(SMSV), is formed initially as a larger precursor which is
cleaved by the viral protease into the mature capsid protein
(62 kDa in the case of FCV; Neill, 1992; Neill et al., 1991;
Carter et al., 1992b; Sosnovstev et al., 1998). The mature
FCV protein is created by removal of the N-terminal 124
amino acids from the precursor (Carter, 1989). The mature
protein is incorporated into virions.
A smaller form of the capsid protein (approx. 40 kDa by
SDS-PAGE; called p40) has been observed during FCV
infection in cell culture at late times post-infection (Carter
et al., 1989). Similar truncated forms of the capsid protein
have been reported in the late stages of rabbit haemorrhagic
1998). In addition, cleaved soluble forms of the Norwalk
virus (NLV) capsid protein have been associated with
enteric infection in man (Hardy et al., 1995). This cleaved
capsid protein was also present within infected stools.
However, it is not known if these cleaved forms are
generated by similar mechanisms or if they have any role in
Apoptosis is a process of cell death used by organisms
to eliminate superfluous, cancerous or virus-infected cells
(Arends & Wyllie, 1991; Kaufmann & Hengartner, 2001;
Zimmerman et al., 2001). Viruses have been found to either
inhibit or promote this process in host cells, or even to do
both at different stages in their replicative cycles (reviewed
in Everett & McFadden, 1999; Roulston et al., 1999; Alcamı ´
& Koszinowski, 2000; Barber, 2001; Boya et al., 2001). Many
viruses block the apoptotic response to ensureefficient virus
production but some induce apoptosis, resulting in virus
dissemination and protection from an immune response.
Apoptosis progresses through a series of morphological and
biochemical changes: forexample,chromatincondensation,
3Present address: MRC Protein Phosphorylation Unit, Division of Cell
Signalling, School of Life Sciences, University of Dundee, Dundee,
DD1 5EH, UK.
0001-8840 G 2003 SGM
Printed in Great Britain
Journal of General Virology (2003), 84, 1237–1244
nuclear disruption, DNA fragmentation, plasma membrane
blebbing and cell shrinkage. Most, if not all, of thesechanges
are effected by members of a family of cysteine proteases
called caspases. The caspase gene family contains 14 mam-
malian members, of which 11 human enzymes have been
identified. All caspases are expressed as pro-enzymes that
contain an N-terminal pro-domain, a large subunit and a
C-terminal small subunit (Stennicke & Salvesen, 1998).
Phylogenetic and functional analysis has shown that the
caspase gene family can be divided into two subfamilies that
are related to either interleukin-1b-converting enzyme and
play a role in inflammation, or to the mammalian counter-
parts of Ced-3 and are involved in apoptosis. Some of the
latter caspases (e.g. caspase-8, -9 and -10) are characterized
by a long pro-domain and are called initiator caspases
because their pro-domains contain interaction motifs that
allow these caspases to form dynamic complexes with other
proteins and transduce various apoptotic signals into pro-
tease activity. This in turn initiates a caspase cascade that
culminates in the activation of downstream effector or
executioner caspases that are characterized by short pro-
domains. The activated effector caspases-3, -6 and -7 cleave
a number of target proteins, and this is responsible for
the ultimate destruction of the cell (Budihardjo et al.,
1999; Hengartner, 2000; Adrain & Martin, 2001; Bratton &
To date there have been few reports on the molecular effects
of caliciviruses on cells. Apoptosis has been observed in
rabbit liver following infection with RHDV (Alonso et al.,
1998) and in San Miguel sea lion virus infection (J. D. Neill,
personal communication). We set out to study the mechan-
ism of FCV capsid cleavage in tissue culture and were led to
look at potential apoptotic changes in FCV-infected cells.
We report here that, during FCV infection, the activation of
caspases is responsible for proteolysis of the viral capsid
protein as well as cellular apoptosis. In addition, caspase-2
and -6 were shown to cleave the FCV capsid protein to a p40
product in vitro.
Virus and cells. Crandell-Rees feline kidney (CRFK) cells were
grown in modified Eagle’s medium (MEM; Gibco-BRL) supple-
mented with 10% foetal bovine serum, non-essential amino acids
(1%), penicillin (100 U ml21) and streptomycin (100 mg ml21) at
37˚C and 5% CO2. FCV F9 strain was propagated in confluent
Morphological analysis of apoptosis. Confluent monolayers
of CRFK cells were grown on microscope slides and infected (or
mock-infected) with FCV F9 at an m.o.i. of 100 p.f.u. per cell. At
various time points post-infection (p.i.), cells were fixed in PBS plus
4% (v/v) formaldehyde, washed and stained with Hoechst 33358
(Calbiochem) in PBS (3 mg ml21). Cells were visualized using a
Zeiss Axiovert 135 fluorescence microscope under UV light.
Western blot analysis of cellular and viral proteins. For ana-
lysis of apoptotic proteins in FCV-infected cells, CRFK monolayers
(in 35 mm dishes) were infected or mock-infected with FCV F9 as
described above. At various times p.i., cells were harvested in 400 ml
buffer C (120 mM NaCl, 50 mM Tris pH 8?0, 0?5% NP40). Super-
natants were assayed for protein concentration (Bio-Rad DC protein
assay) and equal amounts of protein (30 mg) subjected to SDS-
PAGE (10% gels for caspase analysis and 7% for poly(ADP-ribose)
polymerase (PARP) and FCV capsid protein analysis) and immuno-
blotting. Blots were probed with anti-caspase-2 (1:2000, Santa-
Cruz, N19), anti-caspase-3 (1:2000; Calbiochem) and anti-caspase-7
(1:2000; gift from G. M. Cohen, MRC Toxicology Unit, Leicester,
UK) antisera followed by peroxidase-labelled donkey anti-rabbit IgG
(1:2000, Amersham), or with anti-PARP monoclonal antibody
(clone C2-10; 1:10000; Alexis) or anti-FCV capsid antibody (1G9;
1:2000; Carter, 1989) followed by rabbit anti-mouse IgG (1:3000;
Amersham). Detection onto X-ray film was achieved using chem-
iluminescence reagents (Pierce).
For the analysis of PARP cleavage, cell extracts were resuspended
in reducing loading buffer (62?5 mM Tris pH 6?8, 6 M urea,
10% glycerol, 2% SDS. 0?003% bromophenol blue and 5%
2-mercaptoethanol) and sonicated on ice for 20 s prior to separation
by SDS-PAGE in 7% gels (as described in Jones et al., 1999). The
proteins were transferred to Immobilon membranes and detection
was performed as described above.
Effect of caspase inhibitors on FCV capsid cleavage. For
investigation of the role of caspases in FCV capsid protein cleavage,
infections were carried out in the absence or presence of the caspase
inhibitors Z-Val-Ala-DL-Asp-fluoromethyl ketone (Z-VAD-FMK;
Bachem; 100 mM) or Acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-
CHO; Bachem; 100 mM) or the corresponding solvent (dimethyl
sulfoxide). Lysates were subjected to SDS-PAGE (7%) and immuno-
blotting for FCV capsid protein as described above.
Detection of caspase activity. Mock-infected or FCV-infected
CRFK cells were harvested by scraping, washed in buffer A (40 mM
b-glycerophosphate, 50 mM NaCl, 2 mM MgCl2, 5 mM EGTA,
10 mM HEPES, pH 7?0), freeze–thawed three times and centrifuged
(13000 g for 30 min). The supernatants were assayed for DEVDase
activity in the presence of 40 mM Ac-DEVD-AFC using a fluores-
cence microtitre plate reader as previously reported (Jones et al.,
In vitro FCV capsid cleavage assays. The entire FCV F9 capsid
gene has been cloned and expressed previously (Carter et al., 1992b).
For this study, an initiation codon was inserted immediately adja-
cent to the cleavage site to permit the expression of the mature pro-
tein (62 kDa) without the requirement for proteolytic cleavage. This
section was amplified by PCR
CGAATTCGAGTCATGGCGGAT and Rev 59-TTATGAATTCAATA
TTAGGCC. EcoRI sites were added to enable insertion into EcoRI-
digested pGEM-4Z (Promega) for use in in vitro coupled transcrip-
tion and translation (TNT-Promega) reactions. Plasmids were sequ-
enced to check for correct orientation and to verify the capsid
coding sequence. [35S]Methionine-labelled FCV capsid protein was
produced using the TNT T7 Quick kit (Promega) using 1 mg plas-
mid, as per the manufacturer’s instructions. Samples were cooled to
stop the reaction and recombinant human caspase-1, -2, -3, -6, -7, -8,
-9 and -10 (Calbiochem) were added to initiate FCV capsid proteoly-
sis. The final reaction mixture volume was 15 ml and contained
12?5 ml of TNT sample and 12?5 U of recombinant caspase in
10 mM DTT, 0?1% CHAPS and 5% sucrose. Following incubation
for 1, 2?5 and 5 h at 37˚C, 5 ml samples were removed from the
reaction, mixed with SDS-PAGE loading buffer and were analysed
by SDS-PAGE (10%) and autoradiography.
using the primers For59-
Journal of General Virology 84
N. Al-Molawi and others
Caspase inhibitors prevent cleavage of the FCV
capsid during infection of CRFK cells
In agreement with a previous report (Carter, 1989), the
production of the mature capsid protein during FCV
infection of CRFK cells was followed by the appearance of
an approximately 40 kDa fragment (p40) by 6–8 h p.i., as
detected by Western blot analysis. Modification of other
viral capsid proteins has been reported, for example the
influenza virus nucleocapsid protein is cleaved during
apoptosis, as is the capsid protein of transmissible gastro-
enteritis virus (TGEV) (Zhirnov et al., 1999; Eleouet et al.,
2000). As these capsids were cleaved by caspases, we studied
the possible involvement of this family of proteases in the
FCV capsid cleavage event. In the first approach, CRFK cells
were treated with the pan-caspase inhibitor Z-VAD-FMK
prior to, and during, infection with FCV. In the absence of
caspase inhibitors, cleavage of the 62 kDa protein into a p40
product was observed at about 8 h p.i. (Fig. 1, panel a).
However, addition of the caspase inhibitor Z-VAD-FMK
completely prevented this cleavage. Likewise, addition of
the caspase-3 specific inhibitor Ac-DEVD-CHO also pre-
vented the capsid cleavage (Fig. 1, panel b). These results
suggested that caspases were involved in the FCV capsid
Caspase activation in FCV-infected cells
The involvement of caspases in FCV capsid cleavage impli-
cates an activation of caspases during FCV infection. There-
fore, we examined the activation of a number of caspases by
Western blot analysis. Activation of caspase-3 in FCV-
infected cells was observed from about 4 h p.i., as shown by
the appearance of the p20 fragment, and by 8 h p.i. of p17,
corresponding to the large subunit of the pro-caspase
(Fig. 2). In parallel, a decrease in the levels of the pro-form
(p32) was observed (Fig. 2). We also observed activation
of caspase-7, with the pro-form starting to disappear between
4 and 6 h p.i., although the fragment (p19) representing
the processed large subunit was never detected using the
available antisera (Fig. 2). In the case of caspase-2, the pro-
form was processed from a 48 kDa protein into a 33 kDa
fragment that represents an intermediate cleavage product,
most likely consisting of the large subunit plus pro-domain
(Li et al., 1997; Fig. 2). The activation of caspase-3/-7 was
further confirmed by an increase in caspase-3-like activity
(DEVDase activity) that became detectable by 6 h p.i. (Fig. 3)
and paralleled the pro-caspase processing described above.
The activation of caspases during FCV infection was
Fig. 1. Caspase inhibitors prevent cleavage of the FCV capsid
protein. FCV infections were performed in the absence or pre-
sence of the caspase inhibitor Z-VAD-FMK (100 mM; panel a)
or Ac-DEVD-CHO (100 mM; panel b). Cytoplasmic extracts
were made at the times indicated p.i. and subjected to SDS-
PAGE (7%) and Western blotting. Blots were probed with
monoclonal antibody 1G9 as described in methods. Detection
onto X-ray film was achieved using chemiluminescence. The
capsid cleavage product (p40) is indicated by the arrow.
Fig. 2. Caspase activation in FCV-infected CRFK cells. CRFK
cells were infected with FCV for the times indicated or treated
with 1 mM staurosporine (St) for 20 h or mock-infected (C).
Western blotting as described in methods. Caspase-3: the
32 kDa pro-form (p32) was processed to yield p20/p17 frag-
ments that correspond to the (active) large subunit. Caspase-7:
the 36 kDa pro-form and the inactive p29 intermediate are shown
to be processed during FCV infection. The feline caspase-7
large fragment (p19) was not detected by the antiserum as
shown by its absence following induction of apoptosis with
staurosporine (data not shown). Caspase-2: the 48 kDa pro-
form was processed to a p33 intermediate form that is indica-
tive of activation.
to SDS-PAGE (10%)and
FCV capsid cleavage
furthermore confirmed by investigating the in situ cleavage
of PARP, a protein normally involved in DNA repair
(Lindahl et al., 1995). During apoptosis PARP is cleaved
from a 116 kDa protein into an inactive 85 kDa form
(Kaufmann, 1989; Lazebnik et al., 1994). Cleavage of PARP
was detected in FCV-infected cells from about 4 h p.i.
(Fig. 4) as shown by the appearance of a band correspond-
ing to the 85 kDa form.
Another established target for caspases is ICAD/DFF45 (Liu
et al., 1997; Enari et al., 1998) which becomes inactivated
through proteolytic cleavage, enabling the DNase CAD/
DFF40 to cleave the cellular DNA into oligonucleosomal
length fragments. Cleavage of the cellular DNA during FCV
infection of CRFK cells was detected between 4–8 h p.i.
(data not shown) and this correlated well with the onset of
chromatin condensation observed by microscopy (see
below). However, the DNA fragments could not be resolved
to give the appearance of a sharp ladder in apoptotic CRFK
cells. This was observed even in CRFK cells induced to
undergo apoptosis with a well characterized agent such as
staurosporine (1 mM). We therefore assume that this is an
effect of the cell type used in this study.
FCV triggers chromatin condensation in
infected CRFK cells
The activation of caspases during FCV infection of CRFK
cells suggested that other apoptotic changes might occur
during infection. Indeed, CRFK cells infected with FCV F9
showed additional signs of apoptosis beginning at 6 h p.i.
Apoptotic cells exhibit characteristic morphology including
cell and nuclear shrinkage and condensation of the chro-
matin. Nuclear integrity can be visualized by staining cells
with Hoechst 33358, which penetrates nuclei and binds to
DNA. Typical condensation of chromatin was clearly
evident in most FCV-infected cells by about 9 h p.i.
(Fig. 5, panel b) and was similar to the changes induced
by the apoptosis-inducing agent staurosporine (1 mM for
12 h; data not shown).
In vitro cleavage of the FCV capsid protein by
recombinant human caspases
In an attempt to identify the caspase(s) responsible for FCV
capsid cleavage, we studied the ability of recombinant
Fig. 3. Increase in caspase-3-like activity during FCV infection.
CRFK cells were infected with FCV F9 and at the indicated
time-points the cells were harvested. Cellular extracts were pre-
pared and assayed for Ac-DEVD-AFC cleavage activity as
described in methods.
Fig. 4. FCV infection leads to PARP cleavage. CRFK cells
were infected with FCV F9 for the times indicated. Cell extracts
were subjected to SDS-PAGE (7%) and Western blotting for
the immunodetection of PARP as described in methods.
Cleavage from the 116 kDa native protein to the 85 kDa frag-
ment is shown.
Fig. 5. Induction of apoptosis during FCV infection. CRFK
cells were mock-infected (panel a) or infected with FCV F9
(panel b) for 9 h, fixed with 4% formaldehyde and stained with
Hoechst 33358. The nuclei were visualized by fluorescence
microscopy and photographed. The induction of apoptosis is
shown by the appearance of highly condensed chromatin and
Journal of General Virology 84
N. Al-Molawi and others
human caspases to cleave the capsid protein in an in vitro
assay. [35S]Methionine-labelled capsid protein was synthe-
sized in TNT reactions and incubated with a panel of
recombinant caspases at 37˚C. Samples were removed at
various time points and were subjected to SDS-PAGE and
autoradiography. Cleavage of the 62 kDa form and forma-
tion of p40 was detected only with caspase-2 and to a lesser
extent with caspase-6 (Fig. 6). None of the other caspases
tested here cleaved the capsid protein under the conditions
used in this study. Of note is the lack of accumulation of
p40 despite ongoing cleavage of the native protein at 5 h.
This suggests that p40 may be further cleaved. No distinct
bands of lower molecular mass were detected (data not
shown) suggesting that several additional caspase-2 cleavage
sites exist in p40.
It is widely accepted that viruses can inhibit or activate
the apoptotic process. Induction of apoptosis may serve as
a mechanism for virus dissemination or may be a host
defence mechanism. Conversely, viruses may delay the host
responses until replication is complete. The molecular
mechanisms responsible for cellular damage and disease
induced by caliciviruses have not been widely studied.
However, the induction of apoptosis in RHDV infection is
thought to play a key role in the pathogenesis of disease
(Alonso et al., 1998). Here we demonstrate that FCV also
induces apoptosis in cultured cells and propose that apop-
tosis may be a common feature of calicivirus infections.
FCV infection was shown to induce typical features of
having undergone apoptosis by 12 h. This was shown by the
sation and DNA fragmentation. A novel target for caspases
identified here was the viral capsid protein. A truncated
form of the FCV capsid protein corresponding to p40 has
been observed previously in infected cells (Carter et al.,
1989); however, in this early study the protease responsible
for capsid cleavage remained unknown. Here, we found that
the time-course of capsid cleavage occurred in parallel with
the onset of apoptosis, and two lines of evidence suggest a
inhibitors such as Z-VAD-FMK and Ac-DEVD-CHO pre-
vented this capsid cleavage. Secondly, we report here that
recombinant caspases directly cleaved the capsid protein to
and caspase-6, as other caspases were unable to cleave the
capsid protein under the conditions tested here.
In contrast to most effector and initiator caspases which
played by caspase-2 and caspase-6 is much less extensive.
Caspase-2, initially described as Nedd-2/Ich-1 (Wang et al.,
1994; Kumar et al., 1994), has the unique feature of being
a long prodomain caspase with effector caspase substrate
specificity, and hence may act both as an initiator and
effector caspase. Despite the evidence for participation in
some apoptotic pathways, revealed by the phenotype of
caspase-2 null mice (Bergeron et al., 1998), little is known
about its activation process and its downstream target
polypeptides. Caspase-2 has been suggested to be involved
in the apoptosis of neuronal and other cell types in response
to a range of different apoptotic stimuli that induce cyto-
toxic stress (Troy et al., 2000; Lassus et al., 2002; O’Reilly
et al., 2002; Robertson et al., 2002). However, an emerging
feature is the role of caspase-2 in the cellular response to
viral and bacterial infections. He et al. (2001) recently
showed that apoptosis induction following infection by the
paramyxovirus simian virus 5 requires caspase-2. Likewise,
caspase-2 needs to be expressed for apoptosis to occur in
response to rabies virus infection (Ubol & Kasisith, 2000).
Invasive Salmonella typhimurium was reported to activate
wasalsopartiallydependent on caspase-2 (Jesenbergeretal.,
2000). Besides caspase-2 autocatalysis (Butt et al., 1998), the
only other cellular polypeptide substrates for caspase-2 thus
far known are bid (Guo et al., 2002) and golgin-160
(Mancini et al., 2000). In this study, we found not only that
Fig. 6. In vitro cleavage of the FCV capsid
protein by recombinant caspases. Mature
35S-labelled FCV capsid (62 kDa) was pro-
duced in a TNT reaction. Equal amounts of
capsid protein were incubated at 37˚C in
the absence (2) or presence of recombinant
caspase-1, -2, -3, -6, -7, -8, -9 or -10 as
described in methods. At the indicated time-
points, samples were removed, subjected to
SDS-PAGE and analysed by autoradiogra-
phy. The cleavage product (p40) is indicated
with an arrow.
FCV capsid cleavage
FCV infection resulted in the activation of caspase-2 but
also that caspase-2 was by far the most active at cleaving
FCV capsid protein to generate the p40 fragment, and
thereby identified FCV capsid as a novel protein substrate
Caspase-6 plays a major role in nuclear condensation and
apoptotic body formation as a result of its ability to cleave
nuclear lamin A (Lazebnik et al., 1995; Takahashi et al.,
1996; Ruchaud et al., 2002). During apoptosis, lamins A
and C are exclusively degraded by caspase-6 (Orth et al.,
1996; Takahashi et al., 1996), and although other polypep-
tide substrates for caspase-6 have been reported, such as
cytokeratin-18 (Caulin et al., 1997), focal adhesion kinase
(Gervais et al., 1998), NuMA (Hirata et al., 1998), topo-
isomerase I (Samejima et al., 1999) and vimentin (Byun
et al., 2001), these are also cleaved by other caspases,
especially caspase-3. Here, we have identified another
polypeptide, the FCV capsid, that is cleaved by caspase-6.
However, unlike lamins A and C, its cleavage is shared with
caspase-2. In both cases the same fragment (p40) appears to
have been generated; however, whether both caspases target
the same cleavage site needs further investigation. Recently,
it was reported that the viral capsid protein of TGEV is
cleaved by caspase-6 and caspase-7 during apoptosis
following infection of HRT18 cells (Eleouet et al., 2000).
We were unable to demonstrate caspase-6 activation in
FCV-infected cells because none of the commercially
available antibodies tested in this study recognized the
feline form. However, its activation in FCVinfection is most
known to process caspase-6 in turn (Srinivasula et al., 1996;
Slee et al., 2001).
The monoclonal antibody used in this study to detect p40
has been previously mapped to recognize an epitope
consisting of Gly303-Glu-Leu-Ile-Pro-Ala-Gly309within the
FCV capsid protein (Carter, 1989). Another monoclonal
antibodydirected towards the C-terminal end of the protein
and recognizing Pro443-Ile-Phe-Tyr-Lys447does not react
with p40. Consequently, the caspase cleavage site must be
located between Asp320and Asp424. This portion of the
cleavage sites, including Asp-Thr-Ala-Asp331QIle. Cleavage
at this site would produce a fragment of the size of p40.
However, in the absence of any knowledge about possible
additional cleavage at the N terminus of the capsid protein
required for the generation of p40, further work will be
necessary to identify the exact site(s) cleaved by caspase-2
The impact of cleavage of the FCV capsid on virus repli-
cation and cell survival are presently unclear. Cleavage of
viral structural proteins by caspases has been reported for
other viruses, namely TGEV and influenza virus. In the case
of the coronavirus TGEV the capsid protein is cleaved by
caspase-6 and -7 (Eleouet et al., 2000). The authors suggest
that this cleavage may affect particle formation, although
protein of influenza virus is also targeted by caspases
(Zhirnov et al., 1999) and the authors believe that this may
affect virus assembly. It is not known whether the cleaved
capsid interferes with assembly and therefore represents a
cellular defence mechanism, or if the cleaved product has a
role in pathogenesis. For example, cleaved molecules presu-
mably would not be assembled and may actually interfere
with the assembly process, or at least lead to reduced yields
of virus from infected cells. Conversely, cleaved capsid
proteins may elicit non-neutralizing antibodies during
infection and so may in fact be beneficial to pathogenesis
of the virus (as suggested by Hardy et al., 1995). Ofpotential
importance is the recent finding that the human adeno-
associated virus capsid protein, although not cytotoxic to
the cell by itself, significantly enhances apoptosis induced
by the chemotherapeutic drug cisplatin (Duverger et al.,
2002). This apoptosis-modulatory role of the viral capsid
may have critical implications on the development of the
disease (Sasaki et al., 2002).
In conclusion, we report here that FCV infection triggered
an apoptotic response in the host cell that was mediated by
caspases. Caspases were implicated in the cleavage of the
viral capsid protein in infected cells, and studies in vitro
with recombinant caspases identified caspase-2 and to a
lesser extent, caspase-6 as being able to cleave the FCV
We thank Marion Chadd for technical assistance. N.A.M. gratefully
acknowledges sponsorship from the State of Qatar Government.
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