School of Biomedical and Molecular Sciences, University of Surrey, Guildford, Surrey GU2 7XH, UK
Correspondence
Lisa O. Roberts
l.roberts{at}surrey.ac.uk
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ABSTRACT |
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MAIN TEXT |
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The FCV genome is polyadenylated and has a viral protein (VPg) linked to the 5' end (Herbert et al., 1996); it contains three open-reading frames (ORFs). ORF1 encodes the non-structural 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 FMDV L protease are central to the process of picornavirus-induced 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 °C 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 µCi (370 kBq) [35S] methionine/cysteine [Trans35S-label (ICN); specific activity>1000 Ci mmol1]. At the completion of the pulse, the cells were washed in ice-cold saline and harvested in 400 µl SDS-PAGE sample buffer. Samples were subjected to 10 % SDS-PAGE analysis and autoradiography. Duplicate cultures were lysed in 1M 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).
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Both eIF4GI and eIF4GII (220 kDa) were cleaved following FCV infection (Fig. 2 A). Cleavage of eIF4GI was first detectable at 45 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 eIF2
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).
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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 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 µM (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. 3
A). 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 µM or 10 µM 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.
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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 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.
The eIF4G protein acts as a bridge in the eIF4F cap-binding complex to facilitate the assembly of the translation initiation complex. The cap-binding protein (eIF4E) binds to eIF4GI between amino acid positions 570582, thus associating the 5' 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 5' terminus and the ribosome into close proximity and facilitates initiation of protein synthesis. Poliovirus-induced 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 5'-terminal 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 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 eIF2
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.
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ACKNOWLEDGEMENTS |
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Received 8 August 2003;
accepted 16 December 2003.