Martin D. Ryan

Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence.

The 2A region of the foot-and-mouth disease virus (FMDV) polyprotein is only 16 amino acids in length. During synthesis of the FMDV polyprotein a primary proteolytic processing event occurs between the 2A and 2B regions of the polyprotein. The activity responsible for this cleavage is not known but it is thought that either an unidentified virus-encoded proteinase may be responsible, or that 2A acts as a substrate for a host cell proteinase. A series of recombinant FMDV polyproteins has been constructed in which sequences to the N- or C-terminal side of the 2A region have been deleted. Analysis of the processing of these polyproteins shows that a 19 amino acid sequence spanning 2A is sufficient to mediate polyprotein cleavage at a site immediately C-terminal to 2A, whereas deletions extending into the 2A region prevent cleavage.

Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein.

We describe the construction of a plasmid (pCAT2AGUS) encoding a polyprotein in which a 19 amino acid sequence spanning the 2A region of the foot‐and‐mouth disease virus (FMDV) polyprotein was inserted between the reporter genes chloramphenicol acetyl transferase (CAT) and beta‐glucuronidase (GUS) maintaining a single, long open reading frame. Analysis of translation reactions programmed by this construct showed that the inserted FMDV sequence functioned in a manner similar to that observed in FMDV polyprotein processing: the CAT2AGUS polyprotein underwent a cotranslational, apparently autoproteolytic, cleavage yielding CAT‐2A and GUS. Analysis of translation products derived from a series of constructs in which sequences were progressively deleted from the N‐terminal region of the FMDV 2A insertion showed that cleavage required a minimum of 13 residues. The FMDV 2A sequence therefore provides the opportunity to engineer either whole proteins or domains such that they are cleaved apart cotranslationally with high efficiency.

Virus-encoded proteinases of the picornavirus super-group

IP: 54.70.40.11 On: Sat, 03 Aug 2019 19:03:23 Journal of General Virology (1997), 78, 699–723. Printed in Great Britain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

The cleavage activities of aphthovirus and cardiovirus 2A proteins.

The primary 2A/2B polyprotein cleavage of aphtho-and cardioviruses is mediated by their 2A proteins cleaving C-terminally. Whilst the aphthovirus 2A region is only 16 aa (possibly 18 aa) long, the cardiovirus 2A protein is some 150 aa. We have previously shown that foot-and-mouth disease virus (FMDV) 2A is able to mediate cleavage in an artificial (chloramphenicol acetyltransferase/FMDV 2A/beta-glucuronidase [CAT-2A-GUS]) polyprotein system devoid of any other FMDV sequences with high (approximately 85%), although not complete, cleavage. In this paper we show that insertion of upstream FMDV capsid protein 1 D sequences increases the activity. In addition, we have demonstrated that the cardiovirus Theilers murine encephalomyelitis virus(TME) 2A protein, when linked to GUS in a single ORF, is able to cleave at its own C terminus with high efficiency--if not completely. The C-terminal 19 aa of TME 2A, together with the N-terminal proline residue of protein 2B, were inserted into the CAT/GUS artificial polyprotein system (in a single ORF). This recombinant [CAT-deltaTME2A-GUS] polyprotein was able to mediate cleavage with high (approximately 85%) efficiency--directly comparable to the activity observed when FMDV 2A was inserted. A similar insertion into [CAT-GUS] of the C-terminal 19 aa of the cardiovirus encephalomyocarditis virus (EMC) 2A, together with the N-terminal proline residue of protein 2B, produced a [CAT-delta EMC2A-GUS] polyprotein which also mediated cleavage at approximately 85%. Analysis of the products of expression of these artificial polyproteins in a prokaryotic translation system did not, apparently, reveal any GUS cleavage product.

Differentiating infection from vaccination in foot-and-mouth disease using a panel of recombinant, non-structural proteins in ELISA

A profiling ELISA was developed to detect antibody to the non-structural (NS) proteins Lb, 2C, 3A, 3D, and the polyprotein 3ABC, of foot-and-mouth disease virus (FMDV). The assay was used to examine panels of sera from naive cattle, and from experimentally infected or vaccinated animals. All sera from cattle experimentally infected with any of the seven serotypes of FMDV were positive for antibody to 2C, 3A, 3D and 3ABC, and the majority were positive for Lb. The three categories of sera could be differentiated on the basis of the presence or absence of antibody to the structural and/or NS proteins of FMDV. The assay is simple, rapid and reproducible and can be used to identify previous infection in animals which are seropositive for antibody to the structural proteins of the virus. Validating the assay with field sera demonstrated that antibody to 3ABC, and usually one or more of the other non-structural proteins, was detected only in animals reported to have shown clinical signs of FMD. Vaccinated cattle which had received less than five vaccinations, were frequently positive for antibody to 3D but were negative for antibody to 3ABC. Occasional animals which had received more than ten vaccinations had NS protein antibody profiles which were similar to those seen following infection.

Co-translational, intraribosomal cleavage of polypeptides by the foot-and-mouth disease virus 2A peptide.

During co-translational protein import into the endoplasmic reticulum ribosomes are docked onto the translocon. This prevents inappropriate exposure of nascent chains to the cytosol and, conversely, cytosolic factors from gaining access to the nascent chain. We exploited this property of co-translational translocation to examine the mechanism of polypeptide cleavage by the 2A peptide of the foot-and-mouth disease virus. We find that the scission reaction is unaffected by placing 2A into a co-translationally targeted protein. Moreover, the portion of the polypeptide C-terminal to the cleavage site remains in the cytosol unless it contains its own signal sequence. The pattern of cleavage is consistent with the proposal that the 2A-mediated cleavage reaction occurs within the ribosome itself. In addition, our data indicate that the ribosome-translocon complex detects the break in the nascent chain and prevents any downstream protein lacking a signal sequence from gaining access to the endoplasmic reticulum.

Effects of Foot-and-Mouth Disease Virus Nonstructural Proteins on the Structure and Function of the Early Secretory Pathway: 2BC but Not 3A Blocks Endoplasmic Reticulum-to-Golgi Transport

ABSTRACT Infection of cells by picornaviruses leads to the generation of intracellular membrane vesicles. The expression of poliovirus (PV) 3A protein causes swelling of the endoplasmic reticulum (ER) and inhibition of protein trafficking between the ER and the Golgi apparatus. Here, we report that the nonstructural proteins of a second picornavirus, foot-and-mouth disease virus (FMDV), also perturb the secretory pathway. FMDV proteins 3A, 2B, 2C, and 2BC expressed alone in cells were recovered from crude membrane fractions, indicating membrane association. Immunofluorescence microscopy showed that 3A was located in a reticular structure and 2B was located in the ER, while 2C was located in both the ER and the bright punctate structures within the Golgi apparatus. 2BC gave punctate cytoplasmic staining and also caused accumulation of ER proteins in large vesicular structures located around the nuclei. The effect of the FMDV proteins on the trafficking of the vesicular stomatitis virus glycoprotein (G protein) from the ER to the cell surface was determined. Unlike its PV counterpart, the 3A protein of FMDV did not prevent trafficking of the G protein to the cell surface. Instead, surface expression of the G protein was blocked by 2BC, with retention of the G protein in a modified ER compartment staining for 2BC. The results suggest that the nonstructural proteins of different picornaviruses may vary in their ability to perturb the secretory pathway. Since FMDV 2BC can block the delivery of proteins to the cell surface, it may, as shown for PV 3A, play a role in immune evasion and contribute to the persistent infections observed in ruminants.

Use of the 2A sequence from foot-and-mouth disease virus in the generation of retroviral vectors for gene therapy.

We describe the construction of retroviral plasmid vectors in which two genes are linked by a minimum of 96 nucleotides encoding the 2A sequence from the picornavirus foot-and-mouth disease virus (FMDV). Transcription and trans- lation gives rise to a bicistronic mRNA and two independent protein products. The system offers advantages to other alternative ways to create polycistronic mRNAs and can be used in gene therapy delivery vectors.

Site-specific release of nascent chains from ribosomes at a sense codon

ABSTRACT “2A” oligopeptides are autonomous elements containing a D(V/I)EXNPGP motif at the C terminus. Protein synthesis from an open reading frame containing an internal 2A coding sequence yields two separate polypeptides, corresponding to sequences up to and including 2A and those downstream. We show that the 2A reaction occurs in the ribosomal peptidyltransferase center. Ribosomes pause at the end of the 2A coding sequence, over the glycine and proline codons, and the nascent chain up to and including this glycine is released. Translation-terminating release factors eRF1 and eRF3 play key roles in the reaction. On the depletion of eRF1, a greater proportion of ribosomes extend through the 2A coding sequence, yielding the full-length protein. In contrast, impaired eRF3 GTPase activity leads to many ribosomes failing to translate beyond 2A. Further, high-level expression of a 2A peptide-containing protein inhibits the growth of cells compromised for release factor activity and leads to errors in stop codon recognition. We propose that the nascent 2A peptide interacts with ribosomes to drive a highly unusual and specific “termination” reaction, despite the presence of a proline codon in the A site. After this, the majority of ribosomes continue translation, generating the separate downstream product.

Specificity of enzyme−substrate interactions in foot-and-mouth disease virus polyprotein processing

A series of transcripts derived from FMDV cDNA plasmids containing defined regions of the genome were translated in a rabbit reticulocyte lysate system. The products were analysed directly or following incubation with an FMDV-infected cell processing extract. Processing by the L proteinase at the L/1A cleavage site occurred when most of the P1-2A protein was absent. Substitution of sequences upstream of the 2C/3A cleavage site showed that the 3C proteinase was also able to cleave at an entirely novel cleavage site, apparently at K-I amino acid pairs. Cleavage at the 2A/2B site was not only independent of L and 3C proteinases, but was shown to occur when 2A and as few as four 2B N-terminal amino acids were present. Thus, the disparate proteolytic activities responsible for all three primary processing events that give rise to the products L, P1-2A, 2BC, and P3 were highly resistant either to major deletion or substitution of protein sequences adjacent to, or at, the site of cleavage. By contrast, secondary processing in trans was sensitive to changes at remote sites. For example, removal of the C-terminal regions of P1-2A and 2BC precursors impaired their ability to act as substrates for 3C proteinase activity. Processing of P1-2A, particularly of the 1D/2A cleavage site, was enhanced by inclusion of sequences from the 3D region of the genome.