Journal of General Virology (2004), 85, 1125–1130
Short Communication
DOI 10.1099/vir.0.19564-0
Cleavage of eukaryotic initiation factor eIF4G and inhibition of host-cell protein synthesis during feline calicivirus infection Margaret M. Willcocks, Michael J. Carter and Lisa O. Roberts School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK
Correspondence Lisa O. Roberts
[email protected]
Received 8 August 2003 Accepted 16 December 2003
Caliciviruses are small, non-enveloped, positive-stranded RNA viruses that are pathogenic for both animals and man. Although their capsid structure and genomic organization are distinct from picornaviruses, they have similarities to these viruses in their non-structural proteins. Picornaviruses induce a rapid inhibition of host-cell cap-dependent protein synthesis and this is mainly achieved through cleavage of eIF4G and/or dephosphorylation of 4E-BP1. In this study, the effect of calicivirus infection was examined on host-cell protein synthesis in order to determine whether they also induce host shut-off. We report that infection of cells with feline calicivirus (FCV) leads to the inhibition of cellular protein synthesis. This is accompanied by the cleavage of the eukaryotic translation initiation factors eIF4GI and eIF4GII in a manner reminiscent of that induced by picornaviruses. However, the cleavages occur at different sites. The potential mechanisms of these cleavage events and the implications for the translation of calicivirus mRNA are discussed.
The caliciviruses are a family of non-enveloped, positivestranded RNA viruses that infect both animals and man. Feline calicivirus, a member of the Vesivirus genus, is an upper respiratory-tract pathogen of cats and is readily grown in tissue culture cells. Molecular studies have determined the sequence of the complete genome from several strains of this virus (Carter et al., 1992; Oshikamo et al., 1994; Sosnovtsev & Green, 1995) and an infectious cDNA clone has been produced (Sosnovtsev & Green, 1995). Consequently, techniques now exist to dissect both the mechanism of virus replication and the effect of virus infection upon the host-cell. The FCV genome is polyadenylated and has a viral protein (VPg) linked to the 59 end (Herbert et al., 1996); it contains three open-reading frames (ORFs). ORF1 encodes the nonstructural proteins, which are translated from the genomic mRNA. ORF 2 encodes the virus capsid protein and ORF 3 encodes a small basic protein that has been identified as a minor component of the virion (Sosnovtsev & Green, 2000). Both ORF2 and ORF3 are expressed from a subgenomic mRNA and this subgenomic mRNA is also linked to VPg (Herbert et al., 1996). Currently, little is known about the effects of calicivirus infection on the host-cell. It is well established that many picornaviruses [including the entero-/rhinoviruses (e.g. poliovirus, PV) and foot-and-mouth disease virus (FMDV)] induce a dramatic shut-off of host-cell protein synthesis. The entero-/rhinovirus 2A protease and the unrelated 0001-9564 G 2004 SGM
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FMDV L protease are central to the process of picornavirusinduced host protein synthesis shutdown and each induces cleavage of eIF4GI and eIF4GII (Etchison et al., 1982; Etchison & Fout, 1985; Devaney et al., 1988; Gradi et al., 1998; Belsham et al., 2000). It has also been demonstrated that the FMDV 3C protease is capable of inducing the cleavage of eIF4G (Belsham et al., 2000). Due to the similarities of picornavirus proteins to those of caliciviruses, we have studied the effects of FCV infection on host-cell protein synthesis. We demonstrate that FCV infection results in inhibition of host-cell protein synthesis and show that this shut-off is accompanied by the specific cleavage of the initiation factors eIF4GI and eIF4GII. Crandell-Rees feline kidney cells (CRFK) were grown in Eagle’s modified minimal essential medium (Life Technologies) supplemented with 10 % foetal calf serum at 37 uC in an atmosphere containing 5 % CO2. FCV strain F9 was propagated in CRFK cells and the titre determined by plaque assay. CRFK cells (in 35 mm dishes) were infected with FCV (strain F9) at an m.o.i. of 10 p.f.u. per cell or mock infected. At 30 or 60 min intervals the dishes were washed with methionine- and cysteine-free medium and pulse-labelled for 20 min in the same medium with the addition of 10 mCi (370 kBq) [35S] methionine/cysteine [Trans35S-label (ICN); specific activity>1000 Ci mmol21]. At the completion of the pulse, the cells were washed in ice-cold saline and harvested in 400 ml SDS-PAGE sample buffer. Samples were subjected to 10 % SDS-PAGE analysis and autoradiography. Duplicate cultures were lysed in 1M 1125
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NaOH and label incorporation determined by quantification of trichloroacetic acid precipitable radiation. FCV infection of CRFK cells resulted in a significant decrease in host-cell protein synthesis; levels of radioactive incorporation declined by about 20 % by 4 h post-infection (p.i.) and were reduced to approximately 50 % of control values by 5 h p.i. This was matched by the reduction in the appearance of nascent, 35S-labelled proteins on the autoradiograph from about 4?5 h p.i. (Fig. 1A, B). The shut-off observed in FCV infection is reminiscent of picornavirus-induced inhibition of host-cell translation. In that case, shut-off is achieved by disruption of the host-cell cap-dependent translation processes by virus proteasemediated cleavage of host translation initiation factors, most markedly cleavage of eIF4GI and its homologue eIF4GII (Krau¨sslich et al., 1987; Devaney et al., 1988, Gradi et al., 1998). We therefore investigated the fate of these proteins in FCV-infected CRFK cells. CRFK cells were either infected with FCV or mock-infected as described above. Cell extracts were subjected to SDS-PAGE analysis (7 % for eIF4GI and eIF4GII and 10 % for other proteins) and immunoblotting. Following transfer, the membranes were incubated with
Fig. 1. Inhibition of host-cell protein synthesis in FCV-infected cells. CRFK cells were infected with FCV strain F9 at an m.o.i. of 10 p.f.u. per cell. At various times p.i., cells were pulse labelled with [35S] methionine/cysteine. (A) Cells were lysed in SDS-PAGE sample buffer and subjected to SDS-PAGE and autoradiography. (B) Cells were lysed in 1 M NaOH and label incorporation was determined by quantification of trichloroacetic acid precipitable radiation. Appearance of viral proteins is indicated. 1126
antisera specific for the C-terminal portion of eIF4GI (amino acids 920–1396; 1 : 2000) or eIF4GII (amino acids 1168–1194; 1 : 1000), eIF4A (1 : 1000) or eIF2a (1 : 1000). Antisera were kindly provided by Dr S. J. Morley, University of Sussex, UK (eIF4GI, eIF4A and eIF2a) or Dr Y. Svitkin, McGill University, Montreal, Canada (eIF4GII). These sera have been previously described (Scorsone et al., 1987; Bushell et al., 2000; Goldstaub et al., 2000). This was followed by peroxidase-labelled donkey anti-rabbit IgG (1 : 2000, Amersham) or anti-mouse IgG in the case of anti-eIF2a. Detection on X-ray film was achieved using chemiluminescence reagents (Pierce). Both eIF4GI and eIF4GII (220 kDa) were cleaved following FCV infection (Fig. 2 A). Cleavage of eIF4GI was first detectable at 4–5 h p.i. By 7 h p.i., approximately 50 % of the eIF4GI was cleaved. Cleavage products of approximately 200, 165, 145 and 137 kDa were detected with the eIF4GI C-terminal antisera (Fig. 2A). Cleavage of eIF4GII was also first detected at 4 h p.i. however, it then proceeded more rapidly such that 90 % of the 220 kDa protein was cleaved by 5 h. One major cleavage product of 155 kDa was detected with antisera against the C terminus of eIF4GII (Fig. 2A). Note, other initiation factors such as eIF2a remain intact throughout the infection (Fig. 2B). The timing and progression of these events correlated with a reduction in total protein synthesis levels in the infected cells of 20 % by 4 h p.i. and 55 % by 6 h p.i. Although in FMDV-infected cells cleavage of the RNA helicase (eIF4A) has also been reported (Belsham et al., 2000), we did not observe any cleavage of eIF4A during FCV infection (data not shown). The eIF4G protein contains binding sites for many of the translation initiation factors, and thus acts to bring these factors and the ribosomal subunits into contact. PVinduced cleavage of eIF4G separates the binding sites for the cap-binding protein (eIF4E) and eIF3, which binds the ribosomal subunit (Lamphear et al., 1993). FCV-infected cell extracts were analysed by 7 % SDS-PAGE alongside PVinfected HeLa cell extracts. HeLa cells were infected with PV1 (Mahoney) as described in Roberts et al. (2000). The eIF4GI cleavage products generated in FCV infection are larger than the main product detected in PV-infected cells (~120 kDa), suggesting that cleavage occurred closer to the eIF4E-binding site in FCV infection (Fig. 2C). To determine whether the eIF4E-binding site was retained on the eIF4GI C-terminal cleavage products, cap-Sepharose binding experiments were carried out on FCV-infected cell extracts. CRFK cells (in 60 mm dishes) were infected with FCV as before and harvested at 7 h p.i. in 100 ml buffer A (20 mM HEPES, pH 7?5; 100 mM KCl; 2 mM MgCl2) plus protease inhibitors (Roche; used at the manufacturer’s recommended concentration) and 1 % Nonidet P40. Cells were incubated on ice for 5 min and the lysate clarified in a microcentrifuge for 5 min at 4 uC. 7-Methyl GTP Sepharose [30 ml (Pharmacia); pre-equilibrated in buffer A] was added to 50 ml cell lysate plus 50 ml buffer A and rotated for 2 h Journal of General Virology 85
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Fig. 2. Cleavage of eIF4GI and eIF4GII in FCV-infected cells. (A) CRFK cells were infected with FCV as outlined in Fig. 1 and, at various times p.i., lysed in SDS-PAGE sample buffer. Extracts from time 0, 5 and 7 h p.i. were subjected to SDSPAGE (7 %) and immunoblotting. Blots were probed with antisera to the C terminus of eIF4GI or eIF4GII. (B) CRFK cells were mock infected (M) or infected (I) with FCV for 5 and 7 h and cell lysates subjected to SDS-PAGE (10 %) and immunoblotting. Blots were probed with antisera to eIF2a, as described in the text. (C) HeLa cells were infected with PV1 (Mahoney) and cell extracts made at 4 h p.i. PV- and FCV-infected cell extracts were subjected to SDS-PAGE (7 %) and immunoblotting with eIF4GI antisera. (D) CRFK cells were mock infected (M) or infected (I) with FCV for 7 h. Cell extracts were incubated with cap-Sepharose as described in the text and the eIF4GI cleavage products retained were detected by immunoblotting The different eIF4GI cleavage products are indicated.
at 4 uC to allow binding to occur. The 7-methyl GTP Sepharose-bound proteins were recovered by centrifugation at 8000 r.p.m. for 1 min and unbound proteins were removed by washing twice in buffer A. The Sepharosebound proteins were resuspended in 50 ml SDS-PAGE sample buffer and subjected to 7 % SDS-PAGE analysis and immunoblotting. In Fig. 2(D), the 200, 165, 145 and 137 kDa products of eIF4GI were all retained by the cap-Sepharose, suggesting that all contain the eIF4E-binding site. However, a smaller 70 kDa product detected using an N-terminal antiserum was not retained by the cap-Sepharose, suggesting both that the eIF4E-binding site was not contained in this part of the protein and that proteins were not being retained as a result of non-specific binding to – or incomplete washing of – the cap-Sepharose (data not shown). Cleavage of eIF4GI and eIF4GII has also been demonstrated in cells undergoing apoptosis (Clemens et al., 1998; Marissen & Lloyd, 1998; Morley et al., 1998) and it is known that caspase-3 efficiently induces this cleavage (Marissen & Lloyd, 1998; Bushell et al., 1999). During apoptosis eIF4GI cleavage products of 150 and 76 kDa are observed in Jurkat and BJAB cells (Clemens et al., 1998; Morley et al., 1998). To examine any role of caspases in the FCV-induced cleavage of eIF4G, we performed http://vir.sgmjournals.org
infections in the presence of the general caspase inhibitor z-Val-Ala-DL-Asp-fluoromethylketone (z-VAD-FMK; Bachem) (Roberts et al., 2000). Cells were infected in the absence or presence of z-VAD-FMK at a final concentration of 100 mM (45 min prior to and during the infection). Cell extracts were subjected to SDS-PAGE analysis and immunoblotting as described above. Although this inhibitor slowed down the onset of eIF4GI or eIF4GII cleavage (observed at 7 h p.i. versus 5 h p.i.) it did not prevent cleavage (Fig. 3A). To confirm that the eIF4G cleavage products seen in CRFK cells infected with FCV were different to those observed in CRFK cells undergoing apoptosis, CRFK cells were treated overnight with 1 mM or 10 mM staurosporine to induce apoptosis (Roberts et al., 2000). Overnight incubation with staurosporine was required in order to observe apoptotic changes in the CRFK cells. Cell extracts were prepared as described above and subjected to 6 % SDS-PAGE, alongside FCV-infected CRFK cell extracts (Fig. 3B). Western blot analysis for eIF4GI revealed the characteristic p150 and p76 eIF4GI cleavage products in the extracts from cells undergoing apoptosis. These are clearly different to the cleavage products seen during FCV infection of CRFK cells. These results suggest that caspases do not have any role in cleavage of eIF4G in FCV infection. The results presented here demonstrate that FCV infection of cells in culture leads to shut-off of host-cell translation. 1127
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replication (as seen with poliovirus in Roberts et al., 2000). As FCV induces apoptosis in CRFK cells (Al-Molawi et al., 2003; Sosnovtsev et al., 2003), the addition of a caspase inhibitor may generally slow down the whole replication process. In addition, the eIF4G cleavage products observed in FCV infection of CRFK cells are different in size to those produced in apoptosis of these cells. Furthermore, the cap-Sepharose experiment showed that the eIF4GI cleavage products generated in FCV infection were retained on the cap column. However, it has been demonstrated that the p150 and p76 eIF4GI cleavage products generated during apoptosis are not retained on cap-Sepharose (Clemens et al., 1998). We therefore believe that caspases do not play a major role in the cleavage of eIF4G associated with host-cell shut-off in FCV infection and that the cleavage reported here is not a result of apoptosis of infected cells. The mechanism of FCV-induced eIF4G cleavage (e.g. which viral protein is responsible) is currently under investigation.
Fig. 3. Caspases do not play a major role in the cleavage of eIF4G in FCV infection. (A) CRFK cells were mock infected (M) or infected with FCV in the absence (-) or presence (+) of 100 mM of the caspase inhibitor z-VAD-FMK. At 0, 5 and 7 h p.i., cell extracts were made and analysed for cleavage of eIF4GI and eIF4GII as described in Fig. 1. (B) CRFK cells were treated overnight with 10 mM or 100 mM of the apoptosisinducing agent staurosporine (St.). Cell extracts were prepared as described in Fig. 1 and were subjected to SDS-PAGE (6 %) and immunoblotting alongside FCV-infected cell extracts (FCV). Blots were probed with antisera to eIF4GI as described in Fig. 1. The p150 and p76 eIF4GI cleavage products produced in apoptosis are indicated on the right (black arrows) and the different size cleavage products generated during FCV infection are indicated on the left (white arrows).
This shut-off occurs concomitantly with the cleavage of eIF4GI and eIF4GII. Although caspase inhibitors reduced the rate of this cleavage, they did not prevent it. We believe the reduction in rate is a result of a general reduction in the rate of virus 1128
The eIF4G protein acts as a ‘bridge’ in the eIF4F capbinding complex to facilitate the assembly of the translation initiation complex. The cap-binding protein (eIF4E) binds to eIF4GI between amino acid positions 570–582, thus associating the 59 end of the mRNA with this protein. At the same time the ribosomal 40S subunit is bound to eIF3, which itself then binds to eIF4G at about amino acid 970 (Morino et al., 2000). This effectively brings both the mRNA 59 terminus and the ribosome into close proximity and facilitates initiation of protein synthesis. Poliovirusinduced eIF4GI cleavage occurs at amino acid 642 (Lamphear et al., 1995) and separates the binding site for eIF4E from that for eIF3 and thus cap-dependent initiation of translation is efficiently inhibited. In FCV infection, cleavage of eIF4G occurs closer to the N terminus of the protein (the smallest C-terminal cleavage product seen is 137 kDa compared with 120 kDa in poliovirus infection of HeLa cells) and, as shown by the cap-Sepharose binding results, the eIF4E binding site remains on this cleavage product. These observations raise some interesting questions because the exact mechanism of translational initiation in FCV infection is not understood. Early experiments indicated that a covalently linked protein, VPg, was essential for RNA infectivity in vesicular exanthema of swine virus, a calicivirus related to FCV (Burroughs & Brown, 1978). It was later shown that addition of cap analogues to translation reactions failed to inhibit translation, and also that FCV mRNA could be translated at salt concentrations at which cellular messages were not recognized (Herbert et al., 1996b). All these data suggest that FCV mRNA translation proceeds through an eIF4F-independent mechanism. In recent times an infectious cDNA clone has been produced (Sosnovtsev & Green, 1995) and it was reported by these authors that synthetic RNA transcripts (that lack a 59terminal VPg) were not translated by the cell and that such molecules are not infectious; however, the addition of an artificial cap structure to these synthetic transcripts can Journal of General Virology 85
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bring about their translation and thus such modified molecules are infectious. Subsequent rounds of RNA replication combine new RNA with VPg as in a normal infection. Consequently, any cap-independent mechanism for initiation of FCV translation must differ fundamentally from that of PV and may imply a central role for VPg. It should be noted that the calicivirus VPg is around 15 kDa and quite unlike the small 2?5 kDa peptide of picornaviruses, so it may have a completely different function. The role of VPg in calicivirus infection is not understood but it might, for instance, act like a cap structure in recruiting the ribosome to the viral mRNA. In this case, eIF4E may in fact be required for viral mRNA translation. As we have observed, cleavage of eIF4GI and II during FCV infection results in the production of fragments that retain the eIF4E-binding site. Indeed, we have also demonstrated that FCV VPg binds to eIF4E (unpublished data). In addition, it has recently been demonstrated that Norwalk virus VPg binds to eIF3 (Daughenbaugh et al., 2003). The same authors also reported that VPg binds to eIF4E and eIF2a in pull-down assays. Taken together, the data suggest that calicivirus VPg may act as a cap substitute to bind several initiation factors, although the exact mechanism of how ribosomes are recruited to calicivirus mRNA remains to be elucidated. How sequestration of initiation factors by VPg may contribute to the inhibition of host-cell protein synthesis reported here also remains to be determined.
to induction of apoptosis in human lymphoma cell lines. Oncogene 17, 2921–2931. Daughenbaugh, K. F., Fraser, C. S., Hershey, J. W. B. & Hardy, M. E. (2003). The genome-linked protein VPg of the Norwalk virus
binds eIF3, suggesting its role in translation initiation complex recruitment. EMBO J 22, 2852–2859. Devaney, M. A., Vakharia, V. N., Lloyd, R. E., Ehrenfeld, E. & Grubman, M. J. (1988). Leader protein of foot-and-mouth-disease
virus is required for cleavage of the p220 component of the capbinding protein complex. J Virol 62, 4407–4409. Etchison, D. & Fout, S. (1985). Human rhinovirus 14 infection of HeLa cells results in the proteolytic cleavage of the p220 cap-binding complex subunit and inactivates globin mRNA translation in vitro. J Virol 54, 634–638. Etchison, D., Milburn, S. C., Edery, I., Sonenberg, N. & Hershey, J. W. (1982). Inhibition of HeLa cell protein synthesis following
poliovirus infection correlates with the proteolysis of a 220,000dalton polypeptide associated with eucaryotic initiation factor 3 and a cap binding protein complex. J Biol Chem 257, 14806–14810. Goldstaub, D., Gradi, A., Bercovitch, Z., Grosmann, Z., Nophar, Y., Luria, S., Sonenberg, N. & Kahana, C. (2000). Poliovirus 2A protease
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Proteolysis of human eukaryotic translation initiation factor eIF4GII, but not eIF4GI, coincides with the shutoff of host protein synthesis after poliovirus infection. Proc Natl Acad Sci U S A 95, 11089–11094. Herbert, T. P., Brierley, I. & Brown, T. D. K. (1996). Identification of a protein linked to the genomic and subgenomic mRNAs of feline calicivirus and its role in translation. J Gen Virol 78, 1033–1040. Krau¨sslich, H.-G., Nicklin, M. J. H., Toyoda, H., Etchison, D. & Wimmer, E. (1987). Poliovirus proteinase 2A induces cleavage of
Acknowledgements We thank Dr Graham Belsham (BBSRC Institute for Animal Health) for helpful discussions. We thank Mrs Marion Chadd for technical assistance and Mr Alessandro Natoni for help with the apoptosis experiments. We also gratefully acknowledge support from The Wellcome Trust.
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