Conservation of L and 3C proteinase activities across distantly related aphthoviruses

Tracey M. Hinton1, Natalie Ross-Smith2, Simone Warner1, Graham J. Belsham2 and Brendan S. Crabb3

Department of Microbiology and Immunology and the Co-operative Research Centre for Vaccine Technology, The University of Melbourne, Victoria 3010, Australia1
BBSRC Institute for Animal Health, Pirbright, Woking, Surrey GU24 0NF, UK2
The Walter and Eliza Hall Institute of Medical Research, PO The Royal Melbourne Hospital, Melbourne, Victoria 3050, Australia3

Author for correspondence: Brendan Crabb. Fax +61 3 9347 0852. e-mail crabb{at}wehi.edu.au


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
The foot-and-mouth disease virus (FMDV) leader (L) proteinase is an important virulence determinant in FMDV infections. It possesses two distinct catalytic activities: (i) C-terminal processing at the L/VP4 junction; and (ii) induction of the cleavage of translation initiation factor eIF4G, an event that inhibits cap-dependent translation in infected cells. The only other member of the Aphthovirus genus, equine rhinitis A virus (ERAV), also encodes an L protein, but this shares only 32% amino acid identity with its FMDV counterpart. Another more distantly related picornavirus, equine rhinitis B virus (ERBV), which is not classified as an aphthovirus, also encodes an L protein. Using in vitro transcription and translation analysis, we have shown that both ERAV and ERBV L proteins have C-terminal processing activity. Furthermore, expression of ERAV L, but not ERBV L, in BHK-21 cells resulted in the efficient inhibition of cap-dependent translation in these cells. We have shown that the ERAV and FMDV L proteinases induce cleavage of eIF4GI at very similar or identical positions. Interestingly, ERAV 3C also induces eIF4GI cleavage and again produces distinct products that co-migrate with those induced by FMDV 3C. The ERBV L proteinase does not induce eIF4GI cleavage, consistent with its inability to shut down cap-dependent translation. We have also shown that another unique feature of FMDV L, the stimulation of enterovirus internal ribosome entry site (IRES) activity, is also shared by the ERAV L proteinase but not by ERBV L. The functional conservation of the divergent ERAV and FMDV proteinases indicates the likelihood of a similar and important role for these enzymes in the pathogenesis of infections caused by these distantly related aphthoviruses.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Foot-and-mouth disease virus (FMDV) is arguably the world’s most important pathogen of domesticated animals, capable of causing devastating economic loss to the cattle, sheep and pig industries in particular. It is a member of the genus Aphthovirus in the family Picornaviridae, a classification shared by only one other virus, equine rhinitis A virus (ERAV) (Li et al., 1996 ; Pringle, 1997 ). ERAV, formerly known as equine rhinovirus 1, is endemic in most if not all countries and is emerging as an important cause of acute febrile respiratory disease in horses (Carman et al., 1997 ; Li et al., 1997 ; Plummer, 1962 ; Studdert, 1996 ). The similarity of ERAV to FMDV extends beyond primary sequence identity (Li et al., 1996 ; Wutz et al., 1996 ) to include pathogenic features, such as the primary site of infection (the nasopharynx) and the establishment of a persistent viraemic infection (Studdert, 1996 ), physico-chemical properties (Newman et al., 1973 , 1977 ; Studdert & Gleeson, 1978 ) and functionally similar genetic elements, such as the internal ribosomal entry site (IRES) (Hinton et al., 2000 ). However, there are some important differences between the two viruses. Most significant amongst these is the different natural history and host range of ERAV (which is primarily an infection of horses) and its inability to cause the vesicular lesions that are so central to the pathology and significance of FMDV infections.

Our laboratory is interested in identifying and characterizing the functions of putative pathogenic determinants of ERAV with the long-term view of understanding the genetic elements that govern the common (e.g. virus persistence) and unique (e.g. tropism) biological features of these distantly related aphthoviruses. For example, we have shown that ERAV receptor binding does not occur via an arginine–glycine–aspartic acid (RGD) motif in VP1 as is the case with FMDV, although ERAV VP1 does appear to bind host receptors (Warner et al., 2001 ). On the other hand, the IRES elements of ERAV and FMDV appear to function similarly (Hinton et al., 2000 ). Here, we describe an investigation into the function of the leader (L) and 3C proteinases of ERAV. We have also characterized the function of the L protein of the more distantly related picornavirus equine rhinitis B virus (ERBV) (Studdert, 1996 ; Wutz et al., 1996 ).

The aphthovirus L protein is the most N-terminal coding region of the polyprotein. FMDV produces two forms of the L proteinase, Lb and the less abundant Lab. These species differ only at their N termini, the result of initiation at one of two in-frame AUG codons, which are separated by 84 nucleotides (Clarke et al., 1985 ; Sangar et al., 1987 ). FMDV L proteins are papain-like cysteine proteinases, which fold into two globular domains, placing the active residues in a cleft traversing the centre of the molecule (Guarné et al., 1998 , 2000 ). FMDV L proteinases have been shown to possess two distinct activities: (i) self-cleavage at the L/VP4 junction to liberate L from the growing polyprotein; and (ii) inducing cleavage of eukaryotic initiation factor 4G (eIF4G), an integral member of the translation initiation machinery (Devaney et al., 1988 ; Medina et al., 1993 ; Piccone et al., 1995b ; Strebel & Beck, 1986 ). The latter activity inhibits cellular cap-dependent translation initiation. FMDV L protein processes eIF4GI removing the N-terminal domain responsible for binding eIF4E, the cap-binding protein. Truncated eIF4GI retains the ability to bind to the remainder of the eIF4F–eIF3–40S ribosomal subunit complex, which is sufficient for internal initiation of protein synthesis on a picornavirus IRES (Kirchweger et al., 1994 ; Pestova et al., 1996 ). Shut-off of cap-dependent translation initiation may promote the production of viral proteins and also inhibit interferon (IFN)- {alpha}/{beta} production, halting an antiviral response in the infected cell (Chinsangaram et al., 1999 , 2001 ; Mayr et al., 2001 ). Interestingly, deletion of the L protein produces an attenuated virus that is unable to spread efficiently to secondary infection sites, highlighting the fact that this protein is a major pathogenic determinant (Brown et al., 1996 ; Piccone et al., 1995a ). A further feature of FMDV L is its ability to enhance translation initiation from type I picornavirus IRESs present in enteroviruses (Ziegler et al., 1995 ; Roberts et al., 1998 ).

Like FMDV, ERAV appears to utilize two in-frame initiation codons resulting in two forms of the L protein (Hinton et al., 2000 ). However, in contrast to FMDV, the ERAV Lab AUG codon is utilized far more frequently than the Lb AUG. ERAV L proteins are predicted to be of similar length to their FMDV counterparts and have maintained the key catalytic residues and predicted secondary structure of FMDV L proteins (Skern et al., 1998 ). However, these two proteins share only 32% amino acid identity raising doubts as to the likelihood of extensive functional conservation. ERBV is not an aphthovirus but is a member of the related Erbovirus genus (Pringle, 1999 ; Wutz et al., 1996 ). ERBV also encodes an L protein, which is predicted to be of similar length to FMDV Lab. However, ERBV L shares only 18% amino acid identity with FMDV L and is predicted to fold differently (Wutz et al., 1996 ). ERBV L does appear to possess some key catalytic residues, suggesting that it may function as a proteinase (Skern et al., 1998 ).

In addition to extensively processing the viral polyprotein (reviewed by Ryan & Flint, 1997 ), FMDV 3C proteinase also cleaves two important eukaryotic initiation factors, eIF4G and eIF4A (Belsham et al., 2000 ). FMDV 3C is the only picornavirus 3C protein that has been shown to function in this way. ERAV 3C shares 40% amino acid identity with its FMDV counterpart but its function has not yet been characterized.

In this paper we have shown that the distinctive properties of the FMDV L and 3C proteinases are shared by their ERAV counterparts. In contrast, although we demonstrated that ERBV L is a proteinase that cleaves at the L/VP4 junction, this enzyme does not appear to possess the other specific functions attributed to the aphthovirus L proteinases.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Virus growth and metabolic labelling.
ERAV strain 393/76 (Studdert & Gleeson, 1978 ) and ERBV strain 1436/71 (Steck et al., 1978 ) were used throughout this study. Vero, BHK-21 and RK-13 cell monolayers were grown essentially as described elsewhere (Warner et al., 2001 ). For metabolic labelling, Vero cells were cultured in six-well trays (35 mm2 per well) and infected with ERAV at an m.o.i. of 20 TCID50 per cell. At 0, 3, 6 or 9 h post-infection (p.i.), cells were starved of methionine by the addition of methionine-free medium for 1 h, incubated with 30 µCi/ml [35S]methionine (Amersham) for 30 min and the cells lysed in 500 µl SDS–PAGE reducing sample buffer. Samples were separated using 12% SDS–PAGE gels, which were fixed in a 7% acetic acid, 10% methanol solution and treated with Amplify (Amersham). Dried gels were either exposed to Kodak Hyperfilm MP or underwent phosphoimager analysis.

{blacksquare} Plasmid construction.
Plasmids were constructed using standard techniques with only slight modifications (Sambrook et al., 1989 ). The plasmids pA-NTR+5'L and pB-NTR+5'L contained most of the first half of the ERAV and ERBV genomes, respectively (S. Warner & B. S. Crabb, unpublished data). These were subjected to PCR amplification in the presence of the relevant oligonucleotides (Table 1) using 35 cycles of 94 °C for 45 s, 54 °C for 30 s and 68 °C for 45 s, and using Taq HiFi according to the manufacturer’s instructions (Gibco BRL). PCR products were digested with NcoI and XhoI before ligation into similarly digested pET-28a plasmid. These plasmids included pEA/L, pEA/L-VP4, pEA/ {Delta}L-VP4, pEB/L-VP4 and pEA/3C. pB-NTR+5'L was renamed pEB/L-VP1 for use in experiments described in this paper. Construction of pEB/ {Delta}L-VP2 involved digestion of pEB/L-VP1 with XbaI/SacI and ligation into NheI/SacI-digested pET-28a (note that digestion with XbaI and NheI produce compatible ends). The C-terminal extension of pEA/L-VP4 to produce pEA/L-VP1 involved digestion of pA-NTR+5'L with SpeI/XhoI and ligation into similarly digested pEA/L-VP4. Inserts were sequenced by primer extension and BigDye chain termination chemistry (Applied Biosystems). pET.EA(1–965) and pET.EB(1–920) have been described previously (Hinton & Crabb, 2001 ) and in this paper have been renamed pCAT/EA/GFP and pCAT/EB/GFP, respectively. Both plasmids contain the CAT and GFP reporter genes separated by either the full ERAV or ERBV 5' NTR in the pET-28a backbone. Expression of CAT was used as a measure of cap-dependent translation.


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Table 1. Oligonucleotides used to construct L and 3C protein plasmids

 
{blacksquare} In vitro translation assays.
Coupled transcription and rabbit reticulocyte lysate (RRL) translation reactions were performed essentially according to the manufacturer’s instructions (Promega). Briefly, 0·5 µg (except where indicated) of plasmid DNA and [35S]methionine (10 µCi) were added to an RRL reaction mixture and incubated at 30 °C for 90 min. If a single plasmid was to be compared with samples containing two plasmids, 0·25 µg of parental vector (pET-28a) was added to the mix such that the final amount of DNA in all reactions was 0·5 µg. Samples were separated on 15% SDS–PAGE gels, fixed for 30 min in a 7% acetic acid, 10% methanol solution and incubated for 30 min in 1M sodium salicylate, 50% methanol for fluorography. Dried gels were analysed using a phosphoimager (Molecular Dynamics Inc.) and/or using X-ray film.

{blacksquare} Transient expression assays to assess inhibition of cap-dependent translation.
For T7-mediated transcription, 0·5 µg of plasmid DNA was transfected into BHK-21 cells that had been infected with the recombinant vaccinia virus vTF7-3, which expresses T7 RNA polymerase (Fuerst et al., 1986 ). Lipofectamine Plus reagent (Life Technologies)-mediated transfection was performed as described previously (Hinton et al., 2000 ). Inhibition of cap-dependent translation was monitored by co-transfection of pCAT/EA/GFP or pCAT/EB/GFP with the respective L protein expression vector. Cell extracts were prepared 20 h post-transfection. Each experiment was performed at least twice. Transfected cells were harvested by trypsin treatment, resuspended in PBS and separated for relevant assays. CAT activity was determined by thin-layer chromatography as directed by the manufacturer (Promega). GFP expression was monitored by FACS analysis as described previously (Hinton et al., 2000 ). For each sample, the GFP:CAT value represented the ratio of total GFP fluorescence (mean) to CAT activity (% acetylation in a linear range) from an equivalent number of cells.

{blacksquare} Transient expression assays to assess IRES activation.
Transient expression assays were performed as described previously (Roberts et al., 1998 ). Briefly, BHK-21 cells (35 mm dishes) were infected with vTF7-3 (Fuerst et al., 1986 ) and transfected with 2 µg of the dicistronic reporter plasmids of the form pGEM-CAT/IRES/LUC (Roberts et al., 1998 ) alone or together with 0·5 µg of the test plasmids using lipofectin (8 µg; Life Technologies). Luciferase (LUC) activity in the samples was detected using a luciferase assay kit (Promega) and a luminometer. When required, the FMDV Lb proteinase was expressed from plasmid pLb (Medina et al., 1993 ).

Cell extracts were analysed by SDS–PAGE and immunoblotting. Specific proteins were recognized using the following antibodies: sheep anti-eIF4GI (C terminus) (Li et al., 2001 ), rabbit anti-CAT (5prime-3prime Inc.) and goat anti-LUC (Promega). Detection on X-ray film was achieved using the appropriate peroxidase-labelled anti-species antibodies (Amersham Pharmacia Biotech) and chemiluminescence reagents.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Infection with ERAV leads to a reduction in host cell protein synthesis
Infection of cells with FMDV results in the rapid inhibition of host cell protein synthesis (Belsham et al., 2000 ; Chinsangaram et al., 1999 ). To investigate if this is also the case with ERAV, Vero cells were infected at a high m.o.i. (20 TCID50 per cell) and pulse-labelled with [35S]methionine at various times p.i. The percentage reduction in protein synthesis was calculated by quantification of the radioactivity in the 36–200 kDa range for each time-point and compared with that in the 1 h lane. From this analysis, it was evident that by 7 h p.i. with ERAV, host cell protein synthesis had reduced to almost half the level seen at the 1 h time-point (Fig. 1). No further decrease was observed at a later time-point. Hence, ERAV infection resulted in a pronounced inhibition of host protein synthesis in these cells, although this inhibition appeared to be less rapid than that observed in FMDV-infected cells, in which complete shutdown is observed within 4 h (Belsham et al., 2000 ). Complete shutdown of host protein synthesis during ERAV infection was not observed. Viral proteins (indicated by arrows in Fig. 1) were first observed at 7 h with a substantial increase in viral protein synthesis seen by 10 h. Cytopathic effect was also observed for ERAV at 7 h p.i. with complete cell death evident by 24 h. No inhibition of host protein synthesis was seen in RK-13 cells infected with ERBV (data not shown), although an inability to culture this virus to a sufficiently high titre made interpretation of this result difficult.



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Fig. 1. ERAV infection of Vero cells results in a rapid reduction in host protein synthesis. Cells were pulse-labelled with [35S]methionine at 1, 4, 7 and 10 h p.i., lysed and samples were resolved on a 12% SDS–PAGE gel, which was subjected to fluorography. Samples from infected cells labelled overnight (O/N) and mock-infected cells were also analysed. The percentage reduction in host protein synthesis was determined by quantification of the radioactivity in the 36–200 kDa range of each lane (dashed boxes) by phosphoimager analysis. Most strongly labelled viral proteins were below 36 kDa (arrows).

 
The rapid reduction in host protein synthesis seen with ERAV is consistent with eIF4GI processing, as has been reported to occur in ERAV-infected cells (Wutz et al., 1996 ). In FMDV-infected cells, the initial cleavage of eIF4GI is mediated by the L proteinase. As indicated above, the L proteins of ERAV and ERBV show low amino acid identity to the FMDV L proteinase (Fig. 2).



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Fig. 2. Alignment of the L proteinase amino acid sequences from FMDV (O1K), ERAV and ERBV. Identical residues are shaded and key catalytic site residues are indicated (stars). The predicted folding of the FMDV L protein is shown above the sequence (adapted from Skern et al., 1998 ; Guarné et al., 1998 ). The methionine residues found at the N terminus of the various Lab and Lb forms of L proteinase are underlined and italicized.

 
Autocatalytic cleavage by ERAV and ERBV L proteins
To investigate the role of the L proteins from ERAV and ERBV in the life cycle of these viruses, the properties of the distinct L proteins were analysed. One function of the two forms of the FMDV L proteinase (Lab and Lb) is to process the polyprotein at a site between the C terminus of L and the N terminus of VP4 (Medina et al., 1993 ). We aimed to test whether ERAV and ERBV L proteins functioned similarly. To address this, several plasmids were constructed (Fig. 3), which were designed to express the full-length leader protein from these viruses fused to different lengths of polyprotein in the context that occurs naturally in the viral genome. The plasmid pEA/L-VP1 contained the sequence from AUG2, the major initiating codon of ERAV (Hinton et al., 2000 ), to the fifth encoded protein, VP1 (Fig. 3A). A downstream AUG can also be utilized in ERAV such that, as with FMDV, two forms of the L protein can be synthesized. pEA/L-VP4 contained the ERAV sequence from AUG2 into the second protein, VP4. A third plasmid, pEA/L, was constructed, which contained only the predicted full-length ERAV Lab sequence. These ERAV plasmids did not contain the ERAV IRES. ERBV plasmids for this analysis included pEB/L-VP1, which contained the viral sequence from the poly(C) tract to VP1 and hence contained the ERBV IRES (Fig. 3B). pEB/L-VP4 contained the ERBV sequence from the major initiation codon (AUG2) into VP4 (Hinton & Crabb, 2001 ).



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Fig. 3. ERAV and ERBV L proteins have in cis proteinase activity, which processes the L/VP4 junction. The structures of the ERAV (A) and ERBV (B) L protein expression plasmids are shown. All plasmids contained the T7 RNA polymerase promoter (T7) and terminator (T7 term) as indicated. Amino acid numbers indicated above the diagrams begin from the first utilized AUG in the ERAV and ERBV genomes (Hinton & Crabb, 2001 ; Hinton et al., 2000 ). Predicted molecular masses (in kDa) of the various proteins expected to be synthesized from these plasmids are indicated. Bold arrows indicate the predicted L protein cleavage site between L and VP4. Note that pEB/L-VP1 contains the ERBV IRES. ERAV (C) and ERBV (D) L protein plasmids were analysed in coupled in vitro transcription–translation reactions in the presence of [35S]methionine. Samples were analysed by SDS–PAGE and autoradiography. Cleavage products are labelled to the right of each panel.

 
RNA transcribed from these plasmids was used to programme rabbit reticulocyte lysates in the presence of [35S]methionine. Proteins were separated by SDS–PAGE and subjected to fluorography. ERAV L proteins synthesized from pEA/L-VP1 and pEA/L-VP4 demonstrated in cis self-cleavage activity as evidenced by their co-migration with the full-length ERAV Lab synthesized from pEA/L (Fig. 3C). The VP4-VP1 cleavage product of the expected size was also observed in the pEA/L-VP1 lane. No unprocessed products were observed in any ERAV lane, indicating that the ERAV L protein functions efficiently in C-terminal processing. The 20 kDa species observed probably represented a non-specific degradation rather than Lb, since when the Lb initiation codon in pEA/L-VP4 was modified to AUA it was still produced (data not shown). This species may represent a unique cleavage product of the ERAV L protein.

Using the same approach, ERBV L demonstrated partial self-processing ability, as bands visible at the expected molecular mass for active ERBV L were observed from both pEB/L-VP1 and pEB/L-VP4 plasmids (Fig. 3D). A cleavage product corresponding to the VP4-VP1 species was also observed in the pEB/L-VP1 lane. In contrast to ERAV, unprocessed products corresponding to the L-VP1 and L-VP4 species were visible in the respective lanes, indicating that whilst ERBV L functions in an in cis C-terminal cleavage event, it is less efficient than ERAV L in this system.

FMDV L proteins have also been shown to process the viral polyprotein between L and VP4 in trans (Medina et al., 1993 ). To determine whether the ERAV and ERBV L proteins were similarly active, plasmids designed to express inactive L-VP4 substrate polypeptides were constructed. pEA/{Delta}L-VP4 contained the C-terminal half of ERAV L (which does not encode the predicted catalytic residues) fused to VP4 (Fig. 3A). pEB/{Delta}L-VP2 contained the C-terminal end of ERBV L fused to downstream viral sequences up to the third viral protein, VP2 (Fig. 3B). RNA transcripts from these plasmids were used to programme RRLs, either individually or together with transcripts from pEA/L-VP4 or pEB/L-VP4, which encoded active L proteinases. When the inactive L protein transcripts were analysed in the absence of L proteinase expression, bands representing full-length unprocessed substrates were observed at 18 kDa for ERAV or 43 kDa for ERBV (indicated by the asterisks in Fig. 4). ERAV L was able to process the ERAV substrate in trans as the 18 kDa uncleaved product was absent when these proteins were co-expressed (Fig. 4, lane 3). The cleavage products, expected to migrate at 8·1 and 7·8 kDa, were too small to be detected on this gel. ERBV L proteinase synthesized from pEB/L-VP4 also demonstrated an ability to cleave homologous substrate in trans with the 43 kDa ERBV substrate processed to the expected 35 kDa product (Fig. 4, band 4). ERBV substrate was not completely processed by ERBV L, consistent with the partial activity seen in cis. An unexpected band of 13 kDa (Fig. 4, band 3) was also visible and probably represented a non-specific cleavage product.



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Fig. 4. In trans cleavage of L/VP4 substrates by ERAV and ERBV L proteinases. L proteinase expression plasmids (pEA/L-VP4 and pEB/L-VP4) were analysed by in vitro transcription–translation either alone or together with the substrate plasmids pEA/{Delta}L-VP4 and pEB/{Delta}L-VP2 in the presence of [35S]methionine. When a plasmid was to be assayed alone, an equal amount of parental vector pET-28a was added. Samples were analysed on SDS–PAGE and autoradiography. Asterisks indicate the band corresponding to the full-length substrates. Various cleavage products referred to in the text are numbered.

 
To determine whether ERAV and ERBV L proteinases were capable of processing heterologous substrates, plasmids expressing the active L proteinases from pEA/L-VP4 and pEB/L-VP4 were analysed using in vitro transcription–translation reactions in the presence of the vectors pEB/{Delta}L-VP2 and pEA/ {Delta}L-VP4, respectively. ERAV L proteinase was able to process the ERBV substrate; however, this occurred at a different position to that targeted by ERBV L (Fig. 4, bands 1 and 2), indicating different cleavage specificities between the two virus L proteins. The ERBV L proteinase did not appear to process the ERAV substrate efficiently (Fig. 4, lane 8).

ERAV L proteinase inhibits cap-dependent translation
FMDV L proteinase activity inhibits host cell protein synthesis by specific shutdown of cap-dependent translation initiation (Devaney et al., 1988 ; Medina et al., 1993 ). To determine whether ERAV and ERBV L proteinases function similarly, these proteinases were expressed in the presence of dicistronic RNAs synthesized from pCAT/EA/GFP and pCAT/EB/GFP, respectively. From these RNAs, translation of the CAT sequence is cap-dependent while translation of the GFP sequence is directed by the IRES elements from ERAV and ERBV, respectively (Hinton & Crabb, 2001 ; Hinton et al., 2000 ). When transfected into vTF7-3-infected BHK-21 cells, both plasmids induced strong CAT and GFP expression (data not shown). Co-transfection of the pCAT/EA/GFP with the ERAV L proteinase plasmid pEA/L-VP4 resulted in a dramatic decrease in CAT activity and hence a 10–40-fold increase in the GFP:CAT ratio in two independent assays (Fig. 5A). A minor increase in GFP expression was observed; however, compared with the decrease observed in CAT expression, this was insignificant to the resultant ratio. This is consistent with the specific inhibition of cap-dependent translation. A similar result was also observed when these experiments were performed in Vero cells, a cell line permissible for ERAV infection (Fig. 5A).



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Fig. 5. Specific inhibition of cap-dependent translation initiation by ERAV and ERBV L proteinases. vTF7-3-infected BHK-21 cells were co-transfected with ERAV L proteinase and ERAV bicistronic plasmids (A) or ERBV L proteinase and ERBV bicistronic plasmids (B). Parental pET-28a plasmid was used as a control. Each experiment was performed in two different cell types as indicated below the panels and on two different days (Assays 1 and 2).

 
In contrast, the ERBV L proteinase expressed from pEB/L-VP4 produced only a small decrease in CAT activity synthesized from pCAT/EB/GFP. This resulted in a 0·5–3-fold increase in the GFP:CAT ratio indicating that ERBV L was unable to substantially inhibit cap-dependent translation initiation (Fig. 5B). Transfection of these ERBV plasmids into an ERBV-permissive cell line, RK-13, produced similar results (Fig. 5B).

ERAV L protein activation of poliovirus and coxsackie B4 virus IRESs
FMDV L and the 2A proteinase of enteroviruses have the ability to stimulate poliovirus and coxsackie virus IRES activity (Roberts et al., 1998 ). To examine if this property is shared by ERAV and ERBV L proteinases, the relevant expression plasmids were co-transfected into BHK-21 cells together with pGEM-CAT/IRES/LUC bicistronic vectors described previously (Roberts et al., 1998 ). These plasmids express mRNAs with CAT requiring cap-dependent translation initiation and luciferase (LUC) driven by either the PV (pGEM-CAT/P25'/LUC), CBV4 (pGEM-CAT/CB4/LUC) or FMDV IRES (pGEM-CAT/FMD/LUC). Co-transfection of plasmids expressing FMDV L (pLb; Medina et al., 1993 ) and PV 2A (pA {Delta}802; (Kaminski et al., 1990 ) were also performed as controls. Cell lysates were assayed for CAT and LUC expression by Western blot (Fig. 6A, B) and for LUC expression activity (Fig. 6C). Expression of the ERAV L protein from both pEA/L-VP4 and pEA/L enhanced PV IRES expression twofold and CBV4 IRES activity fourfold, similar to FMDV L. In contrast, ERBV L was unable to enhance the activity of either the PV or CBV4 IRES. Neither ERAV L nor ERBV L proteinases stimulated FMDV IRES activity in BHK-21 cells, as observed here and previously with the FMDV L proteinase. This approach also provided further evidence for the ability of ERAV but not ERBV L to inhibit cap-dependent translation initiation with a reduction in CAT expression seen in the presence of ERAV L, FMDV L and PV 2A but not ERBV L (Fig. 6A, B).



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Fig. 6. Activation of PV and CBV4 IRESs by ERAV but not ERBV L proteinases. (A) Cells were co-transfected with plasmids expressing either the ERAV L, ERBV L, FMDV L or poliovirus 2A protein together with the bicistronic construct pGEM-CAT/P25'/LUC, which expresses CAT by a cap-dependent mechanism and LUC by PV IRES-directed translation initiation. Cell extracts were separated by SDS–PAGE, transferred to a membrane and probed with anti-CAT or anti-LUC antibodies as indicated to the left. (B) As in (A) except that the bicistronic plasmid used to co-transfect cells (pGEM-CAT/CB4/LUC) possessed the coxackie B4 virus IRES. (C) Quantification of IRES activation by the ERAV L, ERBV L, FMDV L and poliovirus 2A proteinases. Results are the mean (+SD) of at least three separate experiments.

 
ERAV L proteinase processing of eIF4GI
The inhibition of host cell protein synthesis observed in FMDV-infected cells is linked to the processing of eIF4G. As ERAV inhibits cap-dependent translation initiation similarly to FMDV, we aimed to determine whether ERAV L was able to process eIF4G in a similar way. pEA/L-VP4 and pEA/L, which express the active ERAV L proteinase, were transfected into vTF7-3-infected BHK-21 cells. This was also performed for the ERBV L plasmid, pEB/L-VP4. FMDV L protein expressed from pLb as described previously (Medina et al., 1993 ) was also transfected as a control. Proteins were extracted at 20 h post-transfection, separated by SDS–PAGE and subjected to Western blotting with anti-eIF4GI antibody. From this analysis, it was apparent that expression of ERAV L produced a C-terminal cleavage product of eIF4GI very similar to that induced by FMDV Lb, indicating that these two proteins induced cleavage at an adjacent or identical place (Fig. 7A). As indicated above, this is consistent with reported cleavage of eIF4GI in ERAV-infected cells. In contrast, ERBV L was unable to induce cleavage of eIF4GI consistent with its inability to efficiently inhibit cap-dependent translation.



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Fig. 7. Cleavage of eIF4GI by ERAV L and ERAV 3C proteinases. (A) Leader protein plasmids for ERAV and ERBV were transfected into vTF7-3-infected BHK-21 cells. Cell extracts were separated by SDS–PAGE, transferred to a PVDF membrane and probed with anti-eIF4GI antibody. FMDV L protein synthesized from the plasmid pLb was used as a positive control. The positions of the unprocessed (eIF4GI) and processed (CPC) eIF4GI fragments are indicated to the right. (B, C) The ERAV 3C-expressing plasmid pEA/3C was transfected into vTF7-3-infected BHK-21 cells. Cell extracts were separated by SDS–PAGE and proteins were transferred to a PVDF membrane and probed with either anti-eIF4GI antibody(B) or anti-c-myc antibody (C). FMDV 3C and FMDV L proteinases expressed from pSKRH3C and pLb, respectively, were used as positive controls. SVDV1D{Delta}–2A{Delta} synthesized from pGEM3Z/JI{Delta} was used as a negative control for proteinase activity and as a positive control for c-myc expression.

 
ERAV 3C processing of eIF4GI
A unique feature of the FMDV 3C proteinase is its ability to process both eIF4G and eIF4A (Belsham et al., 2000 ). To determine whether ERAV 3C shares this function, a plasmid expressing ERAV 3C fused to a C-terminal c-myc tag was constructed (pEA/3C) and transfected. Previously described plasmids pGEM3Z/JI{Delta} expressing an unrelated, inactive c-myc-tagged protein (Sakoda et al., 2001 ), pSKRH3C expressing FMDV 3C (Belsham et al., 2000 ) and pLb were transfected as controls. Cell lysates were prepared at 20 h and proteins were separated on SDS–PAGE, transferred to PVDF membrane and probed with either anti-eIF4GI C terminus antibody or anti c-myc tag antibody. ERAV 3C expression was shown to result in eIF4GI cleavage and to yield a product that co-migrated with that generated by the expression of FMDV 3C. The C-terminal cleavage product of eIF4GI generated by the 3C proteinases migrated slightly faster than that induced by the L proteinases (Fig. 7B). However, ERAV 3C, at least in this myc-tagged form, appeared significantly less active than FMDV 3C. ERAV 3C was shown to be expressed from pEA/3C via detection of the c-myc epitope (Fig. 7C). No eIF4A processing by ERAV 3C synthesized from this plasmid was observed (data not shown).


   Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
In this study we have demonstrated that ERAV L is a proteinase that shares two distinct activities with FMDV L: C-terminal processing, which liberates L from the rest of the polyprotein, and the induction of site-specific cleavage of eIF4GI. Expression of ERAV L effectively inhibits cap-dependent translation initiation (presumably as a result of eIF4GI cleavage) and also stimulates the activity of enterovirus (type I) but not aphthovirus (type II) IRES elements. These two properties are also shared with the unrelated enterovirus 2A proteinases (Roberts et al., 1998 ). It has been argued that these functions reflect two distinct proteolytic activities (Roberts et al., 1998 ; see Belsham & Jackson, 2000 , for full discussion). The conservation of L proteinase function across these distantly related aphthoviruses is consistent with their predicted structural similarity (Guarné et al., 1998 , 2000 ) and indicates the likelihood of a similar role for these enzymes in the pathogenesis of ERAV and FMDV infections.

So what is the biological role of the aphthovirus L proteinases? A mutant FMDV lacking the L proteinase is still viable, so FMDV L activity is not essential for virus replication (Piccone et al., 1995a ). This implies that L proteinase-mediated shutdown of cap-dependent translation is not necessary for translation of the viral polyprotein, although it probably promotes it. However, the ‘leaderless’ FMDV mutant does exhibit a reduced plaque size phenotype in vitro and displays reduced pathogenicity when introduced into cattle and swine (Brown et al., 1996 ; Chinsangaram et al., 1998 ; Mason et al., 1997 ). The proposition that the FMDV L proteinase is an important virulence determinant is strengthened by evidence that L proteinase-mediated eIF4G processing effectively down-regulates the induction of type I interferon synthesis in infected cells and by so doing promotes virus spread (Chinsangaram et al., 1999 , 2001 ). In the wake of these findings, leaderless viruses have been proposed as live-attenuated vaccine candidates for the control of FMD (Chinsangaram et al., 1998 ; Mason et al., 1997 ). Given that ERAV L appears to be functionally analogous to FMDV L, it is likely that an ERAV L proteinase deletion mutant would be both viable and less pathogenic that the wild-type virus. Such a mutant has clear potential as a vaccine to control ERAV infections.

Interestingly, the L protein of ERBV, although a proteinase capable of C-terminal processing (in contrast to the cardiovirus L proteins), was unable to induce eIF4GI cleavage or to efficiently inhibit cap-dependent translation initiation. Currently the role of the ERBV L protein is still unknown. Recent work has shown that the L protein zinc-finger motif of the cardiovirus Theiler’s murine encephalomyelitis virus is involved in the inhibition of IFN- {alpha}/{beta} expression through transcriptional regulation (van Pesch et al., 2001 ). As the two unrelated L proteins from aphthoviruses and cardioviruses result, by separate mechanisms, in the inhibition of IFN- {alpha}/{beta} production, this appears to be very important to the pathogenic process of this broad group of picornaviruses. The enterovirus 2A proteinases also induce cleavage of eIF4GI (Krausslich et al., 1987 ) and hence probably inhibit IFN production. Although a zinc-finger motif similar to the cardioviruses has not been identified in ERBV L, this protein may use another unidentified mechanism to inhibit type I interferon production, possibly via a distinct proteinase activity. Clearly, further investigation into the role of the ERBV L proteinase is required to address this.

Recent studies have shown that a unique feature of the FMDV 3C proteinase is the ability to cleave eIF4G and eIF4A (Belsham et al., 2000 ). This ability has not been observed in any other picornavirus 3C proteins, although processing of other cellular proteins including various transcription factors has been attributed to poliovirus 3C (Belsham et al., 2000 ; Clark et al., 1991 ; Yalamanchili et al., 1997a , b ). FMDV 3C has also been shown to process histone H3 (Falk et al., 1990 ). This study shows that the ERAV 3C protein is also able to cleave eIF4GI, albeit inefficiently, at the same (or a very similar) site targeted by FMDV 3C. Although this cleavage event has not been investigated during ERAV infection, the fact that this event is conserved across the distantly related aphthovirus 3C proteinases and is not observed in any other virus genus is consistent with a biologically relevant role for this processing event.

Although ERAV has been classified as the only non-FMDV member of the aphthovirus genus, it remains quite distantly related. The results in this study show that although there is a relatively low sequence identity between the virus species, there are conserved functions such as the L and 3C proteinase activities that are important to viral pathogenesis. This is consistent with the shared pathogenic properties of the viruses, including primary infection of respiratory epithelial cells and establishment of a persistent viraemic infection. However, there are important differences between the replication strategies adopted by ERAV and FMDV, such as initiation codon usage and receptor binding. Features such as these are presumably responsible for the host range and pathological differences observed between ERAV and FMDV, including, most particularly, the absence of vesicles in ERAV infection. It remains a mystery as to whether the formation of vesicles is a virus function or a host response due to the particular tissues affected.


   Acknowledgments
 
We thank Bernard Moss for the provision of the recombinant vaccinia virus vTF7-3. We thank Carol Hartley, Nino Ficorilli and Michael Studdert for the provision of viruses and for helpful advice throughout the course of this study. T.M.H. and S.W. are the recipients of an Australian Postgraduate Research Award and a Melbourne University Postgraduate Scholarship, respectively, and both receive scholarship support from the CRC for Vaccine Technology. B.S.C. is an International Research Scholar of the Howard Hughes Medical Institute.


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Received 21 March 2002; accepted 15 July 2002.