Journal
of General Virology (2000), 81, 1703–1707. Printed in Great Britain
..........................................................................................................................................................................................................
SHORT COMMUNICATION
Caspases are not involved in the cleavage of translation
initiation factor eIF4GI during picornavirus infection
Lisa O. Roberts,1 Angela J. Boxall,1 Louisa J. Lewis,1 Graham J. Belsham2 and George E. N. Kass1
1
2
School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, UK
BBSRC Institute for Animal Health, Pirbright, Woking, Surrey GU24 0NF, UK
Infection of cells by many picornaviruses results in
the rapid inhibition of cellular protein synthesis due
to cleavage of the translation initiation factor eIF4G.
The poliovirus (PV) 2A and foot-and-mouth disease
virus (FMDV) L proteases are each sufficient to
mediate this cleavage, but the cleavage mechanism
may be indirect, involving an unidentified cellular
protease(s). eIF4G is also targetted for cleavage by
caspase-3 during apoptosis. Here, it is shown that
caspase inhibitors do not inhibit the cleavage of
eIF4GI during PV or FMDV infection. Similarly, in
transient-expression studies, the cleavage of
eIF4GI induced by PV 2A or FMDV L was unaffected
by these inhibitors. Furthermore, the cleavage of
eIF4GI was observed in PV-infected MCF-7 cells
lacking caspase-3. These data, and the fact that
induction of apoptosis yields different eIF4GI cleavage fragments, indicate that caspases do not have a
major role in the cleavage of eIF4GI during PV or
FMDV infection.
Infection of cells by many picornaviruses (enteroviruses,
rhinoviruses and aphthoviruses) leads to the rapid inhibition of
cellular (cap-dependent) translation. This is accompanied by
cleavage of the eukaryotic translation initiation factor 4G
(eIF4G) (Etchison et al., 1982 ; Etchison & Fout, 1985 ; Devaney
et al., 1988). There are two species of eIF4G, termed eIF4GI and
eIF4GII, which appear to be functionally equivalent but are
only about 50 % identical (Gradi et al., 1998). eIF4G is a
component of the cap-binding complex (eIF4F), together with
the cap-binding protein eIF4E and the RNA helicase eIF4A.
Cleavage of eIF4G results in disruption of the eIF4F complex
and, hence, inhibition of cap-dependent translation. Picornavirus RNAs have in their 5h untranslated regions a complex
structure known as an internal ribosome entry site (IRES)
element, which directs a cap-independent mechanism of
translation (reviewed in Jackson et al., 1994 ; Belsham &
Author for correspondence : Lisa Roberts.
Fax j44 1483 300374. e-mail l.roberts!surrey.ac.uk
0001-6963 # 2000 SGM
Sonenberg, 1996). IRES-directed initiation of protein synthesis
is maintained following the cleavage of eIF4G (Ohlmann et al.,
1996 ; Borman et al., 1997 ; Gradi et al., 1998 ; Roberts et al.,
1998).
The cleavage of eIF4G in entero-\rhinovirus-infected cells
has been attributed to the action of the viral 2A protease
(Kra$ usslich et al., 1987) and, in foot-and-mouth disease virus
(FMDV)-infected cells, to the L protease (Devaney et al., 1988).
These two proteases are unrelated and are members of different
families of proteases ; 2A belongs to the trypsin-like family of
serine proteases whereas L belongs to the papain-like family of
cysteine proteases (Ryan & Flint, 1997). The sites at which
these proteases cleave eIF4GI in vitro lie just seven amino acids
apart (Kirchweger et al., 1994 ; Lamphear et al., 1995). FMDV L
cleaves eIF4GI at residues 635\636 and poliovirus (PV) 2A
cleaves at residues 642\643 (numbering taken from Imataka et
al., 1998).
It is currently unclear whether the 2A and L proteases
cleave eIF4G directly in vivo, or whether the cleavage process
is mediated by a cellular protease that requires prior activation
by 2A or L. This uncertainty stems from the observation that
eIF4G is cleaved in PV- and FMDV-infected cells without any
virus replication (e.g. in guanidine-treated cells) and when the
levels of 2A and L are undetectable (Bonneau & Sonenberg,
1987 ; Belsham et al., 2000). PV 2A alone is responsible for
inducing the cleavage of eIF4G (Kra$ usslich et al., 1987 ; Lloyd
et al., 1988). However, fractionation of PV-infected HeLa cell
extracts resulted in the separation of eIF4G-specific cleavage
activity from PV 2A (Lloyd et al., 1986 ; Bovee et al., 1998 a). In
addition, an anti-2A antiserum that blocks PV 2A processing
activity did not prevent eIF4G cleavage in an in vitro system
(Kra$ usslich et al., 1987). The direct cleavage of eIF4G by
recombinant PV 2A was reported recently to be very inefficient
(Bovee et al., 1998 b). However, it has been demonstrated that
addition of eIF4E to eIF4GI enhances the susceptibility of
eIF4GI to cleavage by PV 2A (Haghighat & Sonenberg, 1997).
Taken together, these data leave open the possibility of the
involvement of an unidentified cellular protease(s) in the
cleavage of eIF4G induced by PV 2A.
It has been demonstrated recently that eIF4G is also
targetted for cleavage in cells undergoing apoptosis (Clemens
et al., 1998 ; Marissen & Lloyd, 1998 ; Morley et al., 1998). The
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L. O. Roberts and others
Fig. 1. Caspase inhibitors have no effect on PV- or FMDV-induced cleavage of eIF4GI. Picornavirus infections were performed
in the absence or presence of caspase inhibitors. Cytoplasmic extracts were made at the times indicated post-infection (p.i.)
and subjected to SDS–PAGE and Western blotting. Blots were probed with anti-eIF4G antisera as described in the text and
detection onto X-ray film was achieved by using chemiluminescence. (A)–(B) PV infection of HeLa cells in the presence or
absence of Z-VAD-FMK (200 µM) (A) or Z-DEVD-CMK (200 µM) (B). (C) FMDV infection of BHK cells in the absence or
presence of Z-VAD-FMK (200 µM). The C-terminal cleavage fragment of eIF4GI is referred to as Ct.
protease responsible for the cleavage of eIF4G during
apoptosis was identified as caspase-3 (a member of the caspase
family of Asp-specific cysteine proteases) in cells and in vitro
(Marissen & Lloyd, 1998 ; Bushell et al., 1999). Recombinant
human caspase-3 was shown to cleave purified eIF4G within
the eIF4F complex (Bushell et al., 1999). It has been shown
recently that caspase inhibitors prevent PV-induced apoptosis
in HeLa cells (Agol et al., 1998), implying a role for caspases in
cell death during picornavirus infection. In the light of these
findings, we set out to determine whether caspases were
involved in the mechanism of eIF4GI cleavage in picornavirusinfected cells (mediated by either PV 2A or FMDV L protease).
We now report that caspases play no major role in the
mechanism of picornavirus-induced cleavage of eIF4GI.
HeLa cells and BHK cells were grown in modified Eagle’s
medium (Gibco BRL) and Dulbecco’s modified Eagle’s medium,
respectively, supplemented with 10 % foetal bovine serum
(FBS) at 37 mC, in a humidified atmosphere of 5 % CO in air.
#
MCF-7 cells were grown under similar conditions in RPMI
1640 medium (Gibco BRL) supplemented with 10 % FBS. The
BHAE
cells were plated into 35 mm dishes 20 h prior to infection. PV
type 1 (Mahoney, PV1) was added to HeLa or MCF-7 cells at
approximately 10 p.f.u. per cell in their respective medium
containing 2 % FBS. After 1 h incubation with rocking, virus
was removed and the cells were washed and incubated with
fresh medium containing 10 % FBS. Similarly, BHK cells
were infected with FMDV (type O) at approximately 10 p.f.u.
per cell using the same protocol. Mock-infected cells
were treated similarly. Where indicated, the caspase inhibitors Z-Val–Ala–-Asp–fluoromethylketone (Z-VAD-FMK ;
Bachem) or Z-Asp–Glu–Val–Asp–chloromethylketone (ZDEVD-CMK ; Bachem) were added to a final concentration of
50–200 µM to the cells in medium containing 2 % FBS for
45 min prior to virus infection. To ensure the continued
presence of the inhibitors throughout the experiment, the
medium added to the cells after the virus had been removed
was also supplemented, where appropriate, with the inhibitor
at the concentrations indicated. After incubation at 37 mC, cells
were harvested in Promega lysis buffer (400 µl) at the indicated
times post-infection. Samples were analysed for eIF4GI
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Cleavage of eIF4G in picornavirus infection
Fig. 2. FMDV L and PV 2A proteases induce the cleavage of eIF4GI within cells in the presence of Z-VAD-FMK. (A) HeLa cells
were treated with Z-VAD-FMK (200 µM) prior to (and during) infection with PV as detailed in Fig. 1 or prior to (and during)
treatment with TRAIL (250 ng/ml) or staurosporine (Stauro) (1 µM) for 4 h as indicated. Extracts were analysed as described
in Fig. 1. The full-length and Ct fragments of eIF4GI induced during PV infection are indicated as before. The eIF4GI fragments
generated after TRAIL or staurosporine treatment are indicated with asterisks. (B) The plasmids encoding FMDV L (pLb) or PV
2A protease (pA∆258 and pA∆802) were transfected into vTF7-3-infected BHK cells as described in the text. Cells were
incubated in the absence or presence of Z-VAD-FMK (200 µM) as indicated. After 20 h, cell extracts were prepared and
analysed as in Fig. 1. Note that in BHK cells, a species of intermediate mobility between eIF4GI and Ct is observed, which is
lost when the full-length protein is completely cleaved ; presumably this is a non-specific breakdown product of eIF4GI (see also
Fig. 1 C). A minor background band migrating just ahead of Ct is also observed.
cleavage by immunoblotting from 7 % SDS–PAGE minigels
(Laemmli, 1970). Following transfer, the membranes were
incubated with rabbit anti-eIF4G antibodies raised against a Cterminal fragment of eIF4GI (1 : 2000 ; kindly donated by S. J.
Morley, University of Sussex, UK) followed by peroxidaselabelled donkey anti-rabbit IgG (1 : 2500 ; Amersham). Detection onto X-ray film was achieved by using chemiluminescence reagents (Pierce).
PV infection of HeLa cells resulted in the rapid 2A proteaseinduced cleavage of eIF4GI, as detected by Western blot
analysis of cell extracts (Fig. 1 A). The intact protein (Mr
220 000) was lost progressively with the concomitant appearance, within 2 h of infection, of the C-terminal cleavage
product, with an apparent Mr of 120 000. By 4 h, complete loss
of the intact protein was apparent. The cleavage of eIF4GI
occurred before any noticeable morphological changes of the
cells, such as rounding up, blebbing or detachment from the
dish (data not shown). Similarly, FMDV infection of BHK cells
resulted in rapid L protease-induced cleavage of eIF4GI to
generate similar-sized cleavage products of eIF4GI (Fig. 1 C), as
observed previously (Medina et al., 1993).
The presence of the broad-spectrum tripeptide caspase
inhibitor Z-VAD-FMK added at 50 and 200 µM had no effect
on the extent or the kinetics of eIF4GI cleavage in PV-infected
cells (Fig. 1 A). Likewise, Z-DEVD-CMK (200 µM), a modified
tetrapeptide with higher specificity for members of the caspase3 subfamily of caspases, did not modify eIF4GI cleavage in PVinfected cells (Fig. 1 B). Similarly, the rapid cleavage of eIF4GI
in FMDV-infected BHK cells was unaffected by the presence of
Z-VAD-FMK (Fig. 1 C). The concentrations of inhibitor used
have been shown previously to result in efficient blocking of
caspase activity (Chow et al., 1995 ; Jones et al., 1998). This was
confirmed by showing (Fig. 2 A) that Z-VAD-FMK (200 µM)
completely prevented cleavage of eIF4GI in HeLa cells induced
by staurosporine (1 µM) or recombinant human TRAIL
(250 ng\ml, Apo-2 ligand) (MacFarlane et al., 1997 ; a gift from
A. E. Willis, University of Leicester, UK). The cleavage
products of eIF4GI induced by these treatments had approximate Mr of 150 000 and 80 000 and hence migrated very
differently when resolved by SDS–PAGE from those fragments generated by virus infection. These results suggested
that caspases do not play any detectable role in picornavirusinduced eIF4GI cleavage.
In order to examine the properties of the viral proteases
individually, PV 2A and FMDV Lb were expressed transiently
within BHK cells. Plasmids pA∆802 and pLb (encoding PV 2A
and FMDV Lb, respectively, under the control of a T7
promoter) have been described previously (Kaminski et al.,
1990 ; Medina et al., 1993). These plasmids (2 µg) were
transfected into BHK cells (35 mm dishes) using Lipofectin
(8 µl ; Life Technologies) and Optimem (Gibco-BRL) after
infection for 1 h with the recombinant vaccinia virus vTF7-3,
which expresses T7 RNA polymerase (Fuerst et al., 1986).
After incubation for 20 h in the presence or absence of ZVAD-FMK (200 µM), cell extracts were prepared and samples
were analysed for eIF4GI cleavage as outlined previously.
Western blot analysis of cell extracts showed evidence of
eIF4GI cleavage induced by both proteases to the characteristic
cleavage product corresponding to a fragment of Mr 120 000
(Fig. 2 B). Cleavage of eIF4GI occurred independently of the
presence of the caspase inhibitor Z-VAD-FMK (200 µM) (Fig.
2 B), further supporting the observation described above that
caspases are not involved in picornavirus protease-induced
cleavage of eIF4GI.
Finally, we examined the role of caspase-3 in PV-induced
cleavage of eIF4GI by utilizing MCF-7 cells, which do not
express caspase-3 due to a deletion mutation within the
procaspase-3 gene (Ja$ nicke et al., 1998). If caspase-3 plays a
major role in PV-induced cleavage of eIF4GI, then no cleavage
of this protein would be expected within PV-infected MCF-7
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L. O. Roberts and others
References
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In conclusion, it is clear that the inhibition of protein
synthesis during picornavirus infection, resulting from eIF4GI
cleavage, does not involve Z-VAD-FMK-sensitive proteins. If
the model of a cellular protease being required for eIF4GI
cleavage is correct, then another protein(s) must be implicated.
Further work will be required to elucidate the nature of this
protease and to identify the mechanism by which this occurs.
We thank Dr M. J. Carter (University of Surrey) for encouragement
and support. L. J. L. gratefully acknowledges the award of a Wellcome
Trust Vacation Scholarship. We also thank Steven Archibald (IAH) for
preparation of the figures.
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Received 7 February 2000 ; Accepted 7 April 2000
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