School of Biological Sciences, University of Surrey, Guildford, Surrey GU2 5XH, UK1
BBSRC Institute for Animal Health, Pirbright, Woking, Surrey GU24 0NF, UK2
Author for correspondence: Lisa Roberts. Fax +44 1483 300374. e-mail l.roberts{at}surrey.ac.uk
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Abstract |
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The cleavage of eIF4G in entero-/rhinovirus-infected cells has been attributed to the action of the viral 2A protease (Kräusslich 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 (Kräusslich 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 (Kräusslich et al., 1987
). The direct cleavage of eIF4G by recombinant PV 2A was reported recently to be very inefficient (Bovee et al., 1998b
). 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 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 picornavirus-infected 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 Eagles medium (Gibco BRL) and Dulbeccos modified Eagles medium, respectively, supplemented with 10% foetal bovine serum (FBS) at 37 °C, in a humidified atmosphere of 5% CO2 in air. MCF-7 cells were grown under similar conditions in RPMI 1640 medium (Gibco BRL) supplemented with 10% FBS. The 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-ValAlaDL-Aspfluoromethylketone (Z-VAD-FMK; Bachem) or Z-AspGluValAspchloromethylketone (Z-DEVD-CMK; Bachem) were added to a final concentration of 50200 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 °C, cells were harvested in Promega lysis buffer (400 µl) at the indicated times post-infection. Samples were analysed for eIF4GI cleavage by immunoblotting from 7% SDSPAGE minigels (Laemmli, 1970 ). Following transfer, the membranes were incubated with rabbit anti-eIF4G antibodies raised against a C-terminal fragment of eIF4GI (1:2000; kindly donated by S. J. Morley, University of Sussex, UK) followed by peroxidase-labelled 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 protease-induced cleavage of eIF4GI, as detected by Western blot analysis of cell extracts (Fig. 1A). The intact protein (Mr 220000) was lost progressively with the concomitant appearance, within 2 h of infection, of the C-terminal cleavage product, with an apparent Mr of 120000. 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. 1C
), as observed previously (Medina et al., 1993
).
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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 (Jänicke 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 cells. The cells were infected with PV as before and cleavage of eIF4GI was examined. As shown in Fig. 3
, eIF4GI was still cleaved efficiently in PV-infected MCF-7 cells, indicating no significant involvement or requirement for caspase-3 in this event. The rate of cleavage did appear to be slower than that seen in HeLa cells, but we believe that this is due to a less-efficient infection process in this cell type. The eIF4GI cleavage product was indistinguishable in size from that generated in PV-infected HeLa cells (data not shown). Taken together, these data confirm that caspases play no role in the PV 2A- or FMDV L-induced cleavage of eIF4GI.
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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.
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References |
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Received 7 February 2000;
accepted 7 April 2000.