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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 arginineglycineaspartic 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 eIF4FeIF340S 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)-
/
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 manufacturers 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/
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/
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(1965) and pET.EB(1920) 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.
|
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.
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 SDSPAGE 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
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/
L-VP4 contained the C-terminal half of ERAV L (which does not encode the predicted catalytic residues) fused to VP4 (Fig. 3A
). pEB/
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.
|
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 1040-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
).
|
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
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
).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 Theilers murine encephalomyelitis virus is involved in the inhibition of IFN- /
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-
/
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 |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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. Journal of Virology 74, 272-280.
Brown, C. C., Piccone, M. E., Mason, P. W. & McKenna, T. S.-C. (1996). Pathogenesis of wild-type and leaderless foot-and-mouth disease virus in cattle. Journal of Virology 70, 5638-5641.[Abstract]
Carman, S., Rosendal, S., Huber, L., Gyles, C., McKee, S., Willoughby, R. A., Dubovi, E., Thorsen, J. & Lein, D. (1997). Infectious agents in acute respiratory disease in horses in Ontario. Journal of Veterinary Diagnostic Investigation 9, 17-23.[Medline]
Chinsangaram, J., Mason, P. W. & Grubman, M. J. (1998). Protection of swine by live and inactivated vaccines prepared from a leader proteinase-deficient serotype A12 foot-and-mouth disease virus. Vaccine 16, 1516-1522.[Medline]
Chinsangaram, J., Piccone, M. E. & Grubman, M. J. (1999). Ability of foot-and-mouth disease virus to form plaques in cell culture is associated with suppression of alpha/beta interferon. Journal of Virology 73, 9891-9898.
Chinsangaram, J., Koster, M. & Grubman, M. J. (2001). Inhibition of L-deleted foot-and-mouth disease virus replication by alpha/beta interferon involves double-stranded RNA-dependent protein kinase. Journal of Virology 75, 5498-5503.
Clark, M. E., Hammerle, T., Wimmer, E. & Dasgupta, A. (1991). Poliovirus proteinase 3C converts an active form of transcription factor IIIC to an inactive form: a mechanism for inhibition of host cell polymerase III transcription by poliovirus. EMBO Journal 10, 2941-2947.[Abstract]
Clarke, B. E., Sangar, D. V., Burroughs, J. N., Newton, S. E., Carroll, A. R. & Rowlands, D. J. (1985). Two initiation sites for foot-and-mouth disease virus polyprotein in vivo. Journal of General Virology 66, 2615-2626.[Abstract]
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 complex. Journal of Virology 62, 4407-4409.[Medline]
Falk, M. M., Grigera, P. R., Bergmann, I. E., Zibert, A., Multhaup, G. & Beck, E. (1990). Foot-and-mouth disease virus protease 3C induces specific proteolytic cleavage of host cell histone H3. Journal of Virology 64, 748-756.[Medline]
Fuerst, T. R., Niles, E. G., Studier, W. & Moss, B. (1986). Eukaryotic transient-expression system based on recombinant vaccinia virus that synthesizes bacteriophage T7 RNA polymerase. Proceedings of the National Academy of Sciences, USA 83, 8122-8126.[Abstract]
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 Journal 17, 7469-7479.
Guarné, A., Hampoelz, B., Glaser, W., Carpena, X., Tormo, J., Fita, I. & Skern, T. (2000). Structural and biochemical features distinguish the foot-and-mouth disease virus leader proteinase from other papain-like enzymes. Journal of Molecular Biology 302, 1227-1240.[Medline]
Hinton, T. M. & Crabb, B. S. (2001). The novel picornavirus equine rhinitis B virus contains a strong type II internal ribosomal entry site which functions similarly to that of encephalomyocarditis virus. Journal of General Virology 82, 2257-2269.
Hinton, T. M., Li, F. & Crabb, B. S. (2000). Internal ribosomal entry site-mediated translation initiation in equine rhinitis A virus: similarities to and differences from that of foot-and-mouth disease virus. Journal of Virology 74, 11708-11716.
Kaminski, A., Howell, M. T. & Jackson, R. J. (1990). Initiation of encephalomyocarditis virus RNA translation: the authentic initiation site is not selected by a scanning mechanism. EMBO Journal 9, 3753-3759.[Abstract]
Kirchweger, R., Ziegler, E., Lamphear, B. J., Waters, D., Liebig, H. D., Sommergruber, W., Sobrino, F., Hohenadl, C., Blaas, D., Rhoads, R. E. & Skern, T. (1994). Foot-and-mouth disease virus leader proteinase: purification of the Lb form and determination of its cleavage site on eIF-4 gamma. Journal of Virology 68, 5677-5684.[Abstract]
Krausslich, H. G., Nicklin, M. J., Toyoda, H., Etchison, D. & Wimmer, E. (1987). Poliovirus proteinase 2A induces cleavage of eucaryotic initiation factor 4F polypeptide p220. Journal of Virology 61, 2711-2718.[Medline]
Li, F., Browning, G. F., Studdert, M. J. & Crabb, B. S. (1996). Equine rhinovirus 1 is more closely related to foot-and-mouth disease virus than to other picornaviruses. Proceedings of the National Academy of Sciences, USA 93, 990-995.
Li, F., Drummer, H. E., Ficorilli, N., Studdert, M. J. & Crabb, B. S. (1997). Identification of noncytopathic equine rhinovirus 1 as a cause of acute febrile respiratory disease in horses. Journal of Clinical Microbiology 35, 937-943.[Abstract]
Li, W., Belsham, G. J. & Proud, C. G. (2001). Eukaryotic initiation factors 4A (eIF4A) and 4G (eIF4G) mutually interact in a 1:1 ratio in vivo. Journal of Biological Chemistry 276, 29111-29115.
Mason, P. W., Piccone, M. E., McKenna, T. S., Chinsangaram, J. & Grubman, M. J. (1997). Evaluation of a live-attenuated foot-and-mouth disease virus as a vaccine candidate. Virology 227, 96-102.[Medline]
Mayr, G. A., ODonnell, V., Chinsangaram, J., Mason, P. W. & Grubman, M. J. (2001). Immune responses and protection against foot-and-mouth disease virus (FMDV) challenge in swine vaccinated with adenovirusFMDV constructs. Vaccine 19, 2152-2162.[Medline]
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, 355-359.[Medline]
Newman, J., Rowlands, D. J. & Brown, F. (1973). A physico-chemical sub-grouping of the mammalian picornaviruses. Journal of General Virology 18, 171-180.[Medline]
Newman, J. F. E., Rowland, D. J., Brown, F., Goodridge, D., Burrows, R. & Steck, F. (1977). Physicochemical characterization of two serologically unrelated equine rhinovirus. Intervirology 8, 145-154.[Medline]
Pestova, T. V., Shatsky, I. N. & Hellen, C. U. (1996). Functional dissection of eukaryotic initiation factor 4F: the 4A subunit and the central domain of the 4G subunit are sufficient to mediate internal entry of 43S preinitiation complexes. Molecular and Cellular Biology 16, 6870-6878.[Abstract]
Piccone, M. E., Rieder, E., Mason, P. W. & Grubman, M. J. (1995a). The foot-and-mouth disease virus leader proteinase gene is not required for viral replication. Journal of Virology 69, 5376-5382.[Abstract]
Piccone, M. E., Sira, S., Zellner, M. & Grubman, M. J. (1995b). Expression in Escherichia coli and purification of biologically active L proteinase of foot-and-mouth disease virus. Virus Research 35, 263-275.[Medline]
Plummer, G. (1962). An equine respiratory virus with enterovirus properties. Nature 195, 519-520.[Medline]
Pringle, C. R. (1997). Virus taxonomy 1997. Archives of Virology 142, 1727-1733.
Pringle, C. R. (1999). Virus taxonomy at the XIth International Congress of Virology, Sydney, Australia, 1999. Archives of Virology 144, 2065-2070.[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, 520-529.
Ryan, M. D. & Flint, M. (1997). Virus-encoded proteinases of the picornavirus super-group. Journal of General Virology 78, 699-723.
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. Journal of Virology 75, 10643-10650.
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 Research 15, 3305-3315.[Abstract]
Skern, T., Fita, I. & Guarné, A. (1998). A structural model of picornavirus leader proteinases based on papain and bleomycin hydrolase. Journal of General Virology 79, 301-307.[Abstract]
Steck, F., Hofer, B., Schaeren, B., Nicolet, J. & Gerber, H. (1978). Equine rhinoviruses: new serotypes. In Proceedings of the 4th International Conference on Equine Infectious Diseases , pp. 321-328. Edited by J. T. Bryans & H. Gerber. Basel:Karger Veterinary Publications.
Strebel, K. & Beck, E. (1986). A second protease of foot-and-mouth disease virus. Journal of Virology 58, 893-899.[Medline]
Studdert, M. J. (1996). Equine rhinovirus infections. In Virus Infections of Equines , pp. 213-217. Edited by M. J. Studdert. Amsterdam:Elsevier.
Studdert, M. J. & Gleeson, L. J. (1978). Isolation and characterisation of an equine rhinovirus. Zentralblatt fuer Veterinaermedizin Reihe B 25, 225-237.
van Pesch, V., van Eyll, O. & Michiels, T. (2001). The leader protein of Theilers virus inhibits immediate-early alpha/beta interferon production. Journal of Virology 75, 7811-7817.
Warner, S., Hartley, C. A., Stevenson, R. A., Ficorilli, N., Varrasso, A., Studdert, M. J. & Crabb, B. S. (2001). Evidence that equine rhinitis A virus VP1 is a target of neutralizing antibodies and participates directly in receptor binding. Journal of Virology 75, 9274-9281.
Wutz, G., Auer, H., Nowotny, N., Grosse, B., Skern, T. & Kuechler, E. (1996). Equine rhinovirus serotypes 1 and 2: relationship to each other and to aphthoviruses and cardioviruses. Journal of General Virology 77, 1719-1730.[Abstract]
Yalamanchili, P., Datta, U. & Dasgupta, A. (1997a). Inhibition of host cell transcription by poliovirus: cleavage of transcription factor CREB by poliovirus-encoded protease 3Cpro. Journal of Virology 71, 1220-1226.[Abstract]
Yalamanchili, P., Weidman, K. & Dasgupta, A. (1997b). Cleavage of transcriptional activator Oct-1 by poliovirus encoded protease 3Cpro. Virology 239, 176-185.[Medline]
Ziegler, E., Borman, A. M., Kirchweger, R., Skern, T. & Kean, K. M. (1995). Foot-and-mouth disease virus Lb proteinase can stimulate rhinovirus and enterovirus IRES-driven translation and cleave several proteins of cellular and viral origin. Journal of Virology 69, 3465-3474.[Abstract]
Received 21 March 2002;
accepted 15 July 2002.