Differential gene expression in porcine SK6 cells infected with wild-type and SAP domain-mutant foot-and-mouth disease virus

ABSTRACT Over the last few years, an essential RNA structure known as the cis-acting replicative element (cre) has been identified within the protein-coding region of several picornaviruses. The cre, a stem-loop structure containing a conserved AAACA motif, functions as a template for addition of U residues to the protein primer 3B. By surveying the genomes of representatives of several serotypes of foot-and-mouth disease virus (FMDV), we discovered a putative cre in the 5′ untranslated region of the genome (contiguous with the internal ribosome entry site [IRES]). To confirm the role of this putative cre in replication, we tested the importance of the AAACA motif and base pairing in the stem in FMDV genome replication. To this end, cre mutations were cloned into an FMDV replicon and into synthetic viral genomes. Analyses of the properties of these replicons and genomes revealed the following. (i) Mutations in the AAACA motif severely reduced replication, and all viruses recovered from genomes containing mutated AAACA sequences had reverted to the wild-type sequence. (ii) Mutations in the stem region showed that the ability to form this base-paired structure was important for replication. Although the cre was contiguous with the IRES, the mutations we created did not significantly reduce IRES-mediated translation in vivo. Finally, the position of the cre at the 5′ end of the genome was shown not to be critical for replication, since functional replicons and viruses lacking the 5′ cre could be obtained if a wild-type cre was added to the genome following the 3Dpol coding region. Taken together, these results support the importance of the cre in replication and demonstrate that the activity of this essential element does not require localization within the polyprotein-encoding region of the genome.

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Pathogenesis of wild-type and leaderless foot-and-mouth disease virus in cattle.

Journal of Virology ◽

10.1128/jvi.70.8.5638-5641.1996 ◽

1996 ◽

Vol 70 (8) ◽

pp. 5638-5641 ◽

Cited By ~ 85

Author(s):

C C Brown ◽

M E Piccone ◽

P W Mason ◽

T S McKenna ◽

M J Grubman

Keyword(s):

Disease Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Wild Type ◽

Mouth Disease Virus ◽

Foot And Mouth

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Recombinant AAV vectors containing the foot and mouth disease virus 2A sequence confer efficient bicistronic gene expression in cultured cells and rat substantia nigra neurons

Gene Therapy ◽

10.1038/sj.gt.3301469 ◽

2001 ◽

Vol 8 (11) ◽

pp. 864-873 ◽

Cited By ~ 66

Author(s):

S Furler ◽

J-C Paterna ◽

M Weibel ◽

H Büeler

Keyword(s):

Gene Expression ◽

Substantia Nigra ◽

Disease Virus ◽

Cultured Cells ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Rat Substantia Nigra

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Integrin αvβ8 Functions as a Receptor for Foot-and-Mouth Disease Virus: Role of the β-Chain Cytodomain in Integrin-Mediated Infection

Journal of Virology ◽

10.1128/jvi.78.9.4533-4540.2004 ◽

2004 ◽

Vol 78 (9) ◽

pp. 4533-4540 ◽

Cited By ~ 102

Author(s):

Terry Jackson ◽

Stuart Clark ◽

Stephen Berryman ◽

Alison Burman ◽

Stephanie Cambier ◽

...

Keyword(s):

Disease Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Productive Infection ◽

Wild Type ◽

Virus Binding ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Β Chain

ABSTRACT Field isolates of foot-and-mouth disease virus (FMDV) have been shown to use three αv integrins, αvβ1, αvβ3, and αvβ6, as cellular receptors. Binding to the integrin is mediated by a highly conserved RGD motif located on a surface-exposed loop of VP1. The RGD tripeptide is recognized by several other members of the integrin family, which therefore have the potential to act as receptors for FMDV. Here we show that SW480 cells are made susceptible to FMDV following transfection with human β8 cDNA and expression of αvβ8 at the cell surface. The involvement of αvβ8 in infection was confirmed by showing that virus binding and infection of the transfected cells are inhibited by RGD-containing peptides and by function-blocking monoclonal antibodies specific for either the αvβ8 heterodimer or the αv chain. Similar results were obtained with a chimeric αvβ8 including the β6 cytodomain (αvβ8/6), showing that the β6 cytodomain can substitute efficiently for the corresponding region of β8. In contrast, virus binding to αvβ6 including the β8 cytodomain (αvβ6/8) was lower than that of the wild-type integrin, and this binding did not lead to infection. Further, the αvβ6 chimera was recognized poorly by antibodies specific for the ectodomain of αvβ6 and displayed a relaxed sequence-binding specificity relative to that of wild-type integrin. These data suggest that the β6 cytodomain is important for maintaining αvβ6 in a conformation required for productive infection by FMDV.

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Contribution of a Multifunctional Polymerase Region of Foot-and-Mouth Disease Virus to Lethal Mutagenesis

Journal of Virology ◽

10.1128/jvi.01119-18 ◽

2018 ◽

Vol 92 (20) ◽

Cited By ~ 4

Author(s):

Ignacio de la Higuera ◽

Cristina Ferrer-Orta ◽

Elena Moreno ◽

Ana Isabel de Ávila ◽

María Eugenia Soria ◽

...

Keyword(s):

Amino Acid ◽

Disease Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Wild Type ◽

Wild Type Enzyme ◽

Lethal Mutagenesis ◽

Mouth Disease Virus ◽

Foot And Mouth ◽

Localization Signal

ABSTRACTViral RNA-dependent RNA polymerases (RdRps) are major determinants of high mutation rates and generation of mutant spectra that mediate RNA virus adaptability. The RdRp of the picornavirus foot-and-mouth disease virus (FMDV), termed 3D, is a multifunctional protein that includes a nuclear localization signal (NLS) in its N-terminal region. Previous studies documented that some amino acid substitutions within the NLS altered nucleotide recognition and enhanced the incorporation of the mutagenic purine analogue ribavirin in viral RNA, but the mutants tested were not viable and their response to lethal mutagenesis could not be studied. Here we demonstrate that NLS amino acid substitution M16A of FMDV serotype C does not affect infectious virus production but accelerates ribavirin-mediated virus extinction. The mutant 3D displays polymerase activity, RNA binding, and copying processivity that are similar to those of the wild-type enzyme but shows increased ribavirin-triphosphate incorporation. Crystal structures of the mutant 3D in the apo and RNA-bound forms reveal an expansion of the template entry channel due to the replacement of the bulky Met by Ala. This is a major difference with other 3D mutants with altered nucleotide analogue recognition. Remarkably, two distinct loop β9-α11 conformations distinguish 3Ds that exhibit higher or lower ribavirin incorporation than the wild-type enzyme. This difference identifies a specific molecular determinant of ribavirin sensitivity of FMDV. Comparison of several polymerase mutants indicates that different domains of the molecule can modify nucleotide recognition and response to lethal mutagenesis. The connection of this observation with current views on quasispecies adaptability is discussed.IMPORTANCEThe nuclear localization signal (NLS) of the foot-and-mouth disease virus (FMDV) polymerase includes residues that modulate the sensitivity to mutagenic agents. Here we have described a viable NLS mutant with an amino acid replacement that facilitates virus extinction by ribavirin. The corresponding polymerase shows increased incorporation of ribavirin triphosphate and local structural modifications that implicate the template entry channel. Specifically, comparison of the structures of ribavirin-sensitive and ribavirin-resistant FMDV polymerases has identified loop β9-α11 conformation as a determinant of sensitivity to ribavirin mutagenesis.

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Modulation of the electrostatic charge at the active site of foot-and-mouth-disease-virus leader proteinase, an unusual papain-like enzyme

Biochemical Journal ◽

10.1042/bj3630493 ◽

2002 ◽

Vol 363 (3) ◽

pp. 493-501 ◽

Cited By ~ 7

Author(s):

Petra SCHLICK ◽

Jakub KRONOVETR ◽

Bernhard HAMPOELZ ◽

Tim SKERN

Keyword(s):

Disease Virus ◽

Active Site ◽

Foot And Mouth Disease ◽

Electrostatic Charge ◽

Mouth Disease ◽

Initiation Factor ◽

Wild Type ◽

Wild Type Enzyme ◽

Mouth Disease Virus ◽

Foot And Mouth

The leader proteinase (Lpro) of foot-and-mouth-disease virus is an unusual papain-like cysteine proteinase. Synthesized without an N-terminal pro precursor region, it frees itself from the growing polypeptide chain by cleavage at its own C-terminus. It also possesses a unique electrostatic environment around the active site, essentially due to Asp163, which orients the catalytic histidine residue, and Asp164; the equivalent residues in papain are Asn175 and Ser176. The importance of these residues for Lpro activity was examined by site-directed mutagenesis. Replacement of Asp163 with asparagine reduced activity by five-fold towards a hexapeptide substrate and slightly delayed self-processing when expressed in rabbit reticulocyte lysates. However, no effect on the cleavage of the only known cellular substrate of Lpro, eukaryotic initiation factor 4GI (eIF4GI), was observed. In contrast, replacement of Asp164 by either alanine, asparagine or lysine abrogated activity towards the hexapeptide. Furthermore, in all cases, the onset of both self-processing and eIF4GI cleavage were significantly delayed; the reaction rates were also diminished compared with those of the wild-type enzyme. The alanine-substituted enzyme was least affected, followed by those substituted with asparagine and lysine. The double mutant protein in which both aspartate residues were replaced by asparagine was most severely affected; it failed to complete either self-processing or eIF4GI cleavage within 3 h, compared with the 8min required by the wild-type enzyme. Hence, we propose that the electrostatic charge of Asp164, and to a lesser extent that of Asp163, is extremely important for Lpro to attain full activity upon synthesis.

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