Institute for Animal Health, Pirbright, Woking, Surrey GU24 0NF, UK
Correspondence
Graham J. Belsham
graham.belsham{at}bbsrc.ac.uk
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the partial eIF4GI cDNA sequences reported in this paper are AJ746218 (ovine), AJ746219 (bovine), AJ746220 (hamster), AJ746221 (equine), AJ746222 (murine), AJ746223 (porcine) and AJ746224 (rabbit).
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INTRODUCTION |
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The initiation of translation of picornavirus RNA is achieved by a cap-independent mechanism and is directed by a large, complex RNA structure, the internal ribosome entry site (IRES), that is located within the long 5'-untranslated region (UTR) of the viral RNA [reviewed by Belsham & Jackson (2000)]. The FMDV 5'-UTR is >1300 nt in length and the IRES (about 435 nt) is located at the 3' end of this region. Initiation of protein synthesis occurs at two sites that are 84 nt apart, resulting in the synthesis of two forms of the leader (L) protein, termed Lab and Lb (Sangar et al., 1987
; Belsham, 1992
) at the N-terminus of the polyprotein. Both forms of the L protein are active proteases (Medina et al., 1993
) that cleave the L/P1 junction within the viral polyprotein and also induce cleavage of eIF4GI and eIF4GII (Devaney et al., 1988
; Medina et al., 1993
; Gradi et al., 2004
). Cleavage sites within eIF4GI and eIF4GII that are generated in vitro by the FMDV Lb protease (Lbpro) and the entero- and rhinovirus 2A proteases (2Apro) have been identified (Lamphear et al., 1993
; Kirchweger et al., 1994
; Gradi et al., 2003
, 2004
). For eIF4GI, the cleavage sites for Lbpro and the enterovirus 2Apro are on the C-terminal side of residues G674 and R681, respectively [renumbered according to the system of Byrd et al. (2002)
for the longest form, eIF4GIa, which comprises 1600 aa]. Cleavage of eIF4G separates the N-terminal domain, which interacts with the cap-binding protein eIF4E, away from the C-terminal region that interacts with eIF4A and eIF3. The C-terminal fragment is sufficient to support FMDV IRES-directed translation initiation. The FMDV IRES interacts directly with eIF4G (López de Quinto & Martínez-Salas, 2000
; Stassinopoulos & Belsham, 2001
) and the binding site has been mapped to the JK domains of this IRES. Similarly, the equivalent region of the encephalomyocarditis virus (EMCV) IRES also binds to eIF4G and individual bases that are important in this interaction have been identified (Kolupaeva et al., 1998
, 2003
; Clark et al., 2003
).
FMDV infection of cells results in very rapid inhibition of host-cell protein synthesis (e.g. Belsham et al., 2000; Gradi et al., 2004
) and this correlates with loss of intact eIF4G. The eIF4GI and eIF4GII proteins are cleaved with similar kinetics in FMDV-infected cells (Gradi et al., 2004
). Cleavage of eIF4GI induced by FMDV Lpro can be observed even in the absence of virus replication, when virus protein expression is very low (Belsham et al., 2000
). It has been noted that the expression of the second trans-acting FMDV protease, 3C (termed 3Cpro), within baby hamster kidney (BHK) cells can also induce cleavage of eIF4GI (Belsham et al., 2000
). This may explain the cleavage of eIF4GI, which has been detected in BHK cells infected with a mutant form of FMDV that lacks the Lbpro (Piccone et al., 1995
; Belsham et al., 2000
). The cleavage site generated by 3Cpro was shown to be on the C-terminal side of that generated by Lpro (Belsham et al., 2000
). FMDV 3Cpro is also responsible for cleaving eIF4AI, one of two species of eIF4A that are present within the cytoplasm of the cell. The cleavage site in eIF4AI has been identified and cleavage results in loss of activity (Li et al., 2001
); however, eIF4AII is not modified by FMDV 3Cpro.
FMDV Lpro and 3Cpro are unrelated to each other. Lpro is related to the papain-like family of cysteine proteases (Roberts & Belsham, 1995; Guarné et al., 1998
), whereas FMDV 3Cpro and enterovirus 2Apro are related to the serine proteases (Allaire et al., 1994
; Ryan & Flint, 1997
). It has been shown that proteases expressed by human immunodeficiency virus type 1 (HIV-1) and HIV-2 can also cleave eIF4GI and the cleavage sites have been identified (Ohlmann et al., 2002
). Furthermore, cleavage of eIF4GI occurs in feline calicivirus-infected cells (Willcocks et al., 2004
) and produced novel cleavage products. Thus, eIF4G appears to be a favoured target for viral proteases.
Here, we have re-examined the cleavage of eIF4GI within wild-type (wt) FMDV-infected BHK cells by Lpro and 3Cpro. We observed that sequential cleavage of eIF4GI was induced by the two proteases, with both processing events occurring before peak viral protein synthesis was achieved. The eIF4GI proteins from a variety of mammalian species all appeared to be sensitive to cleavage by FMDV Lpro, but showed different sensitivities to the FMDV 3Cpro. Following rough mapping of the 3Cpro cleavage site, comparison of the different eIF4GI sequences suggested a single potential cleavage site for 3Cpro. We have shown that modification of a single amino acid at this site within the human eIF4GI sequence can determine the sensitivity of this protein to FMDV 3Cpro.
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METHODS |
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Transient expression assays within cells infected with the recombinant vaccinia virus vTF7-3, which expresses the T7 RNA polymerase (Fuerst et al., 1986), were performed as described previously (Roberts et al., 1998
). Briefly, BHK cells or human A293 cells were infected with vTF7-3 and transfected, by using 8 µg lipofectin (Life Technologies), with plasmid DNA (2·5 µg) that encoded poliovirus (PV) 2Apro (pA
802), FMDV Lbpro (pLb), FMDV 3Cpro (pSKRH3C, with the FMDV IRES or pSK3C) or swine vesicular disease virus (SVDV) 2Apro (pGEM3Z/J1), as used previously (Roberts et al., 1998
; Belsham et al., 2000
; Sakoda et al., 2001
). After 20 h, cell extracts were prepared and analysed by SDS-PAGE and immunoblotting as described above.
Plasmid construction, manipulation and analysis.
All DNA manipulations were performed by using standard methods (Sambrook et al., 1989) or those described by the manufacturers.
Total RNA was extracted by using Trizol (Life Technologies) from the following tissue culture cell lines: BHK (hamster), IBRS-2 (pig), MDBK (bovine), RK13 (rabbit) and LK98 (sheep) or equine embryonic lung cells (kindly supplied by the Animal Health Trust, UK). The RNA was used to produce cDNA by using Moloney murine leukaemia virus reverse transcriptase (Promega) with the primer 4Grt (Table 1). Using this cDNA as a template, PCR was performed with primers 4Gfor and 4Grev (Table 1
) and Pfu DNA polymerase (Promega) to generate a product of approximately 600 bp. The primers corresponded to sequences that are conserved between the murine and human eIF4GI sequences and included sequences to provide flanking EcoRI and NotI restriction sites (Table 1
). PCR products were phosphorylated in vitro and ligated into vector pT7Blue (Novagen). Following transformation of Escherichia coli DH5
, colonies were obtained and plasmid DNA preparations were screened by digestion with EcoRI and NotI. The inserts were sequenced in both directions by using standard primers on a Beckman CEQ8000 machine. The BHK cell eIF4GI cDNA insert was excised by using EcoRI and NotI and ligated into a similarly digested plasmid to generate pGEXBHK, which expressed a GST/eIF4GI/FLAGCAT fusion protein cassette. The vector used had been modified from the GST/eIF4GII/FLAG constructs that were described by Gradi et al. (2003
, 2004)
. A T7 promoter sequence (from complementary oligonucleotides T7for and T7rev; Table 1
) and the FMDV IRES were introduced upstream of the GST-coding sequence. Furthermore, the CAT-coding sequence (prepared by PCR with primers CATfor and CATrev; Table 1
) was introduced downstream of and in frame with the FLAG epitope, to increase the size of the C-terminal cleavage product. Plasmid pGEXBHK was transfected into vTF7-3-infected cells, either alone or with plasmids encoding the viral proteases described above. After 20 h, cell extracts were prepared and incubated with anti-FLAG M2agarose affinity resin (Sigma). Bound proteins were analysed by SDS-PAGE and immunoblotting using rabbit anti-CAT antibodies (Eppendorf5 Prime) and peroxidase-labelled anti-rabbit Ig with chemiluminescence reagents (both from Amersham Biosciences).
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RESULTS |
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eIF4GI proteins from different species show different sensitivities to FMDV 3Cpro
The studies described above and previously (Belsham et al., 2000) show clearly that endogenous eIF4GI expressed within BHK cells is highly susceptible to cleavage by FMDV 3Cpro. To test the generality of this process, FMDV 3Cpro and Lbpro were expressed within other cell types. It was observed that FMDV Lbpro induced cleavage of human eIF4GI within A293 cells, but no cleavage of eIF4GI was observed in these cells when FMDV 3Cpro was expressed (Fig. 3
). Similar results were also observed in COS cells, derived from African green monkey kidney (data not shown). In contrast, both FMDV Lbpro and 3Cpro induced cleavage of murine eIF4GI from 3T3 cells (data not shown), as seen for hamster eIF4GI in BHK cells (Fig. 3
).
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A single amino acid substitution renders human eIF4GI sensitive to FMDV 3Cpro
On the basis of these considerations, it seems probable that the E/P bond (aa 712713) in the human eIF4GI sequence may determine the resistance of the human protein to FMDV 3Cpro (Fig. 5). Within the BHK eIF4GI sequence at this position, there is an E/T bond. The E/T amino acid pair in the BHK sequence is similar to many cleavage sites that are used by FMDV 3Cpro (e.g. E/G, E/S, E/V; Table 2
). However, a P residue is not found on either side of the bonds that are cleaved by picornavirus proteases and thus the human protein, which contains a P residue at this position, could be expected to be resistant to cleavage.
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DISCUSSION |
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It could be argued that the P713T amino acid substitution is not necessarily at the cleavage site, as the presence of a P residue could induce a conformational change in the protein. However, the proposed BHK cleavage site sequence (E/T) and its flanking sequences are similar to other FMDV 3Cpro cleavage sites (see Table 2
). Furthermore, the lack of other significant amino acid sequence differences in this region of eIF4GI and the correct location relative to the sites cleaved by the FMDV Lbpro, enterovirus 2Apro and HIV-2 proteases suggest strongly that the cleavage of hamster eIF4GI occurs at the E712/T713 bond. It should be noted that the P713T mutant assembled into eIF4F complexes and was still cleaved correctly in cells expressing FMDV Lbpro and enterovirus 2Apro (Fig. 6b and c
). These results argue strongly that the overall conformation of the mutant eIF4GI was not perturbed significantly by this single amino acid substitution. Clearly, a more detailed analysis is required to establish that all known functions of this protein are unperturbed by modification at this site.
The FMDV 3Cpro cleavage site within eIF4GI is upstream of the region that has been shown to interact with the EMCV IRES and with eIF4A (Imataka & Sonenberg, 1997; Lomakin et al., 2000
). Hence, it would be expected that the 3Cpro-generated C-terminal fragment of eIF4GI would remain functional for translation of the FMDV RNA within FMDV-infected BHK cells. This is therefore consistent with the high level of viral protein synthesis that occurs at approximately 3 h p.i., when both Lpro and 3Cpro have modified eIF4G (Belsham et al., 2000
).
The 3Cpro-generated cleavage site is just 6 and 9 aa upstream of the two sites that are recognized by the HIV-2 protease. In a recent study, it was shown that cleavage of eIF4GI by the HIV-2 protease had no effect on EMCV IRES function, as expected (Prévôt et al., 2003). However, it was suggested that ribosome scanning is greatly impaired in vitro under these conditions. It was shown that the HIV-2 protease enhanced utilization of the first FMDV initiation codon and blocked utilization of the second start site (which is probably recognized following ribosome scanning; Belsham, 1992
) within a cell-free translation system. Thus, it was suggested that removal of a region of about 40 aa from the C-terminal cleavage product of eIF4GI that exhibits general RNA-binding properties blocked ribosome scanning. We therefore investigated the effect of FMDV 3Cpro on recognition of the two FMDV start sites within BHK cells. In these experiments, we were not able to detect any significant differential effect of FMDV 3Cpro or the HIV-2 protease on utilization of the two different start sites (data not shown). This may reflect differences in the assay systems used.
The region of about 40 aa in eIF4GI between the site that is cleaved by the enterovirus 2Apro and sites that are cut by the HIV-2 protease does have unusual features. It includes nine G residues and about eight (seven in mouse/hamster and nine in rabbit) P residues (including four G/P pairs), plus six R residues, so that over half of this region is made up of these three different amino acids. As mentioned above, studies by Prévôt et al. (2003) indicated that this region had general RNA-binding properties and this appears to be consistent with the high proportion (about 20 %) of positively charged residues. However, it is interesting to note that the eIF4GII homologue of eIF4GI has a deletion of 12 aa within this region, compared with eIF4GI [see Gradi et al. (2003)
]. Currently, there is no known functional difference between eIF4GI and eIF4GII, despite their extensive sequence diversity.
Comparison of eIF4GI sequences downstream of the eIF4E-binding site and around the viral protease cleavage sites demonstrated that the proteins from different mammalian species are closely related, but there are small variable regions. The cleavage sites in eIF4GI that are used by the different viral proteases appear to cluster around these variable regions of eIF4GI (Fig. 5). On the basis of the derived amino acid sequences, it is possible to predict that each of the eIF4GI proteins examined would be susceptible to cleavage by FMDV Lpro, but 3Cpro cleavage appears to be restricted (among the species examined) to the hamster and mouse sequences. Thus, this cleavage does not appear to be important for FMDV infection within bovine, ovine or porcine cells. It is interesting to note that eIF4GI within BHK cells is also cleaved by the equine rhinitis A virus (ERAV) 3Cpro (Hinton et al., 2002
). ERAV is the only aphthovirus other than FMDV. However, on the basis of the eIF4GI-coding sequence (Fig. 5
), it appears that the equine eIF4GI should also be resistant to the aphthovirus 3Cpro.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Belsham, G. J. (1992). Dual initiation sites of protein synthesis on foot-and-mouth disease virus RNA are selected following internal entry and scanning of ribosomes in vivo. EMBO J 11, 11051110.[Abstract]
Belsham, G. J. & Jackson, R. J. (2000). Translation initiation on picornavirus RNA. In Translational Control of Gene Expression Monograph 39, pp. 869900. Edited by N. Sonenberg, J. W. B. Hershey & M. B. Mathews. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Belsham, G. J., McInerney, G. M. & Ross-Smith, N. (2000). Foot-and-mouth disease virus 3C protease induces cleavage of translation initiation factors eIF4A and eIF4G within infected cells. J Virol 74, 272280.
Bradley, C. A., Padovan, J. C., Thompson, T. L., Benoit, C. A., Chait, B. T. & Rhoads, R. E. (2002). Mass spectrometric analysis of the N terminus of translational initiation factor eIF4G-1 reveals novel isoforms. J Biol Chem 277, 1255912571.
Byrd, M. P., Zamora, M. & Lloyd, R. E. (2002). Generation of multiple isoforms of eukaryotic translation initiation factor 4GI by use of alternate translation initiation codons. Mol Cell Biol 22, 44994511.
Clark, A. T., Robertson, M. E. M., Conn, G. L. & Belsham, G. J. (2003). Conserved nucleotides within the J domain of the encephalomyocarditis virus internal ribosome entry site are required for activity and for interaction with eIF4G. J Virol 77, 1244112449.
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 cap-binding protein complex. J Virol 62, 44074409.[Medline]
Fuerst, T. R., Niles, E. G., Studier, F. W. & Moss, B. (1986). Eukaryotic transient expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proc Natl Acad Sci U S A 83, 81228126.[Abstract]
Gingras, A.-C., Raught, B. & Sonenberg, N. (1999). eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem 68, 913963.[CrossRef][Medline]
Gradi, A., Imataka, H., Svitkin, Y. V., Rom, E., Raught, B., Morino, S. & Sonenberg, N. (1998). A novel functional human eukaryotic translation initiation factor 4G. Mol Cell Biol 18, 334342.
Gradi, A., Svitkin, Y. V., Sommergruber, W., Imataka, H., Morino, S., Skern, T. & Sonenberg, N. (2003). Human rhinovirus 2A proteinase cleavage sites in eukaryotic initiation factors (eIF) 4GI and eIF4GII are different. J Virol 77, 50265029.
Gradi, A., Foeger, N., Strong, R., Svitkin, Y. V., Sonenberg, N., Skern, T. & Belsham, G. J. (2004). Cleavage of eukaryotic translation initiation factor 4GII within foot-and-mouth disease virus-infected cells: identification of the L-protease cleavage site in vitro. J Virol 78, 32713278.
Guarné, A., Tormo, J., Kirchweger, R., Pfistermueller, D., Fita, I. & Skern, T. (1998). Structure of the foot-and-mouth disease virus leader protease: a papain-like fold adapted for self-processing and eIF4G recognition. EMBO J 17, 74697479.
Hinton, T. M., Ross-Smith, N., Warner, S., Belsham, G. J. & Crabb, B. S. (2002). Conservation of L and 3C proteinase activities across distantly related aphthoviruses. J Gen Virol 83, 31113121.
Imataka, H. & Sonenberg, N. (1997). Human eukaryotic translation initiation factor 4G (eIF4G) possesses two separate and independent binding sites for eIF4A. Mol Cell Biol 17, 69406947.[Abstract]
Kirchweger, R., Ziegler, E., Lamphear, B. J. & 8 other authors (1994). Foot-and-mouth disease virus leader proteinase: purification of the Lb form and determination of its cleavage site on eIF-4. J Virol 68, 56775684.[Abstract]
Kolupaeva, V. G., Pestova, T. V., Hellen, C. U. T. & Shatsky, I. N. (1998). Translation eukaryotic initiation factor 4G recognizes a specific structural element within the internal ribosome entry site of encephalomyocarditis virus RNA. J Biol Chem 273, 1859918604.
Kolupaeva, V. G., Lomakin, I. B., Pestova, T. V. & Hellen, C. U. T. (2003). Eukaryotic initiation factors 4G and 4A mediate conformational changes downstream of the initiation codon of the encephalomyocarditis virus internal ribosomal entry site. Mol Cell Biol 23, 687698.
Lamphear, B. J. & Rhoads, R. E. (1996). A single amino acid change in protein synthesis initiation factor 4G renders cap-dependent translation resistant to picornaviral 2A proteases. Biochemistry 35, 1572615733.[CrossRef][Medline]
Lamphear, B. J., Yan, R., Yang, F., Waters, D., Liebig, H.-D., Klump, H., Kuechler, E., Skern, T. & Rhoads, R. E. (1993). Mapping the cleavage site in protein synthesis initiation factor eIF-4 of the 2A proteases from human coxsackievirus and rhinovirus. J Biol Chem 268, 1920019203.
Li, W., Ross-Smith, N., Proud, C. G. & Belsham, G. J. (2001). Cleavage of translation initiation factor 4AI (eIF4AI) but not eIF4AII by foot-and-mouth disease virus 3C protease: identification of the eIF4AI cleavage site. FEBS Lett 507, 15.[CrossRef][Medline]
Lomakin, I. B., Hellen, C. U. T. & Pestova, T. V. (2000). Physical association of eukaryotic initiation factor 4G (eIF4G) with eIF4A strongly enhances binding of eIF4G to the internal ribosomal entry site of encephalomyocarditis virus and is required for internal initiation of translation. Mol Cell Biol 20, 60196029.
López de Quinto, S. & Martínez-Salas, E. (2000). Interaction of the eIF4G initiation factor with the aphthovirus IRES is essential for internal translation initiation in vivo. RNA 6, 13801392.
Mader, S., Lee, H., Pause, A. & Sonenberg, N. (1995). The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4 and the translational repressors 4E-binding proteins. Mol Cell Biol 15, 49904997.[Abstract]
Medina, M., Domingo, E., Brangwyn, J. K. & Belsham, G. J. (1993). The two species of the foot-and-mouth disease virus leader protein, expressed individually, exhibit the same activities. Virology 194, 355359.[CrossRef][Medline]
Ohlmann, T., Prévôt, D., Décimo, D., Roux, F., Garin, J., Morley, S. J. & Darlix, J.-L. (2002). In vitro cleavage of eIF4GI but not eIF4GII by HIV-1 protease and its effects on translation in the rabbit reticulocyte lysate system. J Mol Biol 318, 920.[CrossRef][Medline]
Piccone, M. E., Rieder, E., Mason, P. W. & Grubman, M. J. (1995). The foot-and-mouth disease virus leader proteinase gene is not required for viral replication. J Virol 69, 53765382.[Abstract]
Prévôt, D., Décimo, D., Herbreteau, C. H., Roux, F., Garin, J., Darlix, J.-L. & Ohlmann, T. (2003). Characterization of a novel RNA-binding region of eIF4GI critical for ribosomal scanning. EMBO J 22, 19091921.
Roberts, P. J. & Belsham, G. J. (1995). Identification of critical amino acids within the foot-and-mouth disease virus leader protein, a cysteine protease. Virology 213, 140146.[CrossRef][Medline]
Roberts, L. O., Seamons, R. A. & Belsham, G. J. (1998). Recognition of picornavirus internal ribosome entry sites within cells; influence of cellular and viral proteins. RNA 4, 520529.
Ryan, M. D. & Flint, M. (1997). Virus-encoded proteinases of the picornavirus super-group. J Gen Virol 78, 699723.
Sakoda, Y., Ross-Smith, N., Inoue, T. & Belsham, G. J. (2001). An attenuating mutation in the 2A protease of swine vesicular disease virus, a picornavirus, regulates cap- and internal ribosome entry site-dependent protein synthesis. J Virol 75, 1064310650.
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sangar, D. V., Newton, S. E., Rowlands, D. J. & Clarke, B. E. (1987). All foot and mouth disease virus serotypes initiate protein synthesis at two separate AUGs. Nucleic Acids Res 15, 33053315.[Abstract]
Stassinopoulos, I. A. & Belsham, G. J. (2001). A novel proteinRNA binding assay: functional interactions of the foot-and-mouth disease virus internal ribosome entry site with cellular proteins. RNA 7, 114122.
Willcocks, M. M., Carter, M. J. & Roberts, L. O. (2004). Cleavage of eukaryotic initiation factor eIF4G and inhibition of host-cell protein synthesis during feline calicivirus infection. J Gen Virol 85, 11251130.
Yan, R., Rychlik, W., Etchison, D. & Rhoads, R. E. (1992). Amino acid sequence of the human protein synthesis initiation factor eIF-4. J Biol Chem 267, 2322623231.
Zamora, M., Marissen, W. E. & Lloyd, R. E. (2002). Multiple eIF4GI-specific protease activities present in uninfected and poliovirus-infected cells. J Virol 76, 165177.
Received 5 May 2004;
accepted 10 June 2004.