Search

Several reports have previously shown that expression of the foot-and-mouth disease virus (FMDV) capsid precursor protein encoding region P1-2A together with the 3C protease (P1-2A/3C) results in correct processing of the capsid precursor into VP0, VP1 and VP3 and formation of FMDV capsid structures that are able to induce a protective immune response against FMDV challenge after immunization using naked DNA constructs or recombinant viruses. To elucidate whether bovine herpesvirus 1 (BHV-1) might also be suitable as a viral vector for empty capsid generation, we aimed to integrate a P1-2A/3C expression cassette into the BHV-1 genome, which, however, failed repeatedly. In contrast, BHV-1 recombinants that expressed an inactive 3C protease or the P1-2A polyprotein alone could be easily generated, although the recombinant that expressed P1-2A exhibited a defect in direct cell–cell spread and release of infectious particles. These results suggested that expression of the original, active FMDV 3C protease is not compatible with BHV-1 replication. This conclusion is supported by the isolation of recombinant BHV-1/3C*, which contained mutations within the 3C ORF (3C* ORF)—probably introduced spontaneously during generation of BHV-1/3C*—instead of the authentic 3C ORF contained in the transfer plasmids. Within the 3C* ORF, the codons for glycine 38 and phenylalanine 48 were both substituted by codons for serine. The resulting 3C* protease exhibits a highly reduced activity for proteolytic processing of the P1-2A polyprotein and thus might be a good candidate for the generation of live attenuated FMDV variants. [ABSTRACT FROM AUTHOR]

Abstract: The 3C protease from foot-and-mouth disease virus (FMDV 3Cpro) is critical for viral pathogenesis, having vital roles in both the processing of the polyprotein precursor and RNA replication. Although recent structural and functional studies have revealed new insights into the mechanism and function of the enzyme, key questions remain that must be addressed before the potential of FMDV 3Cpro as an antiviral drug target can be realised. [Copyright &y& Elsevier]

The article discusses the interplay between infection and immunity in cattle that have contracted the foot-and-mouth disease virus (FMDV). Topics include a detailed explanation of FMDV and its symptoms such as lameness, low weight, and decreased milk production and an overview of the research by N. Juleff and colleagues and their research methodology, such as the use of molecular techniques to search for viral traces in the mouth and throat tissues of infected cows.

Productive picornavirus infection requires the hijacking of host cell pathways to aid with the different stages of virus entry, synthesis of the viral polyprotein, and viral genome replication. Many picornaviruses, including foot-andmouth disease virus (FMDV), assemble capsids via the multimerization of several copies of a single capsid precursor protein into a pentameric subunit which further encapsidates the RNA. Pentamer formation is preceded by co- and posttranslational modification of the capsid precursor (P1-2A) by viral and cellular enzymes and the subsequent rearrangement of P1-2A into a structure amenable to pentamer formation. We have developed a cell-free system to study FMDV pentamer assembly using recombinantly expressed FMDV capsid precursor and 3C protease. Using this assay, we have shown that two structurally different inhibitors of the cellular chaperone heat shock protein 90 (hsp90) impeded FMDV capsid precursor processing and subsequent pentamer formation. Treatment of FMDV permissive cells with the hsp90 inhibitor prior to infection reduced the endpoint titer by more than 10-fold while not affecting the activity of a subgenomic replicon, indicating that translation and replication of viral RNA were unaffected by the drug. IMPORTANCE FMDV of the Picornaviridae family is a pathogen of huge economic importance to the livestock industry due to its effect on the restriction of livestock movement and necessary control measures required following an outbreak. The study of FMDV capsid assembly, and picornavirus capsid assembly more generally, has tended to be focused upon the formation of capsids from pentameric intermediates or the immediate cotranslational modification of the capsid precursor protein. Here, we describe a system to analyze the early stages of FMDV pentameric capsid intermediate assembly and demonstrate a novel requirement for the cellular chaperone hsp90 in the formation of these pentameric intermediates. We show the added complexity involved for this process to occur, which could be the basis for a novel antiviral control mechanism for FMDV. [ABSTRACT FROM AUTHOR]

Abstract: Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release or incomplete inactivation. Non-infectious empty capsids are structural mimics of authentic particles with no associated risk and constitute an alternate vaccine candidate. Capsids self-assemble from the processed virus structural proteins, VP0, VP3 and VP1, which are released from the structural protein precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown to be efficient but linkage of the cognate 3C protease to the C-terminus reduces expression significantly. Inactivation of the 3C enzyme in a P1-2A-3C cassette allows expression and intermediate levels of 3C activity resulted in efficient processing of the P1-2A precursor into the structural proteins which assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine. [Copyright &y& Elsevier]

Foot-and-mouth disease virus (FMDV), a positive sense, single- stranded RNA virus, causes a highly contagious disease in cloven-hoofed livestock. Like other picornaviruses, FMDV has a conserved 2C protein assigned to the superfamily 3 helicases, a group of AAA+ ATPases that has a predicted N-terminal membrane- binding amphipathic helix attached to the main ATPase domain. In infected cells, 2C is involved in the formation of membrane vesicles, where it co-localizes with viral RNA replication complexes, but its precise role in virus replication has not been elucidated. We show here that deletion of the predicted N-terminal amphipathic helix enables overexpression in Escherichia coli of a highly soluble truncated protein, 2C(34-318), that has ATPase and RNA binding activity. ATPase activity was abrogated by point mutations in the Walker A (K116A) and B (D 160A) motifs and Motif C (N207A) in the active site. Unliganded 2C(34-318) exhibits concentration-dependent self-association to yield oligomeric forms, the largest of which is tetrameric. Strikingly, in the presence of ATP and RNA, FMDV 2C(34-318) containing the N207A mutation, which binds but does not hydrolyze ATP, was found to oligomerize specifically into hexamers. Visualization of FMDV 2C-ATP-RNA complexes by negative stain electron microscopy revealed hexameric ring structures with 6-fold symmetry that are characteristic of AAA+ ATPases. ATPase assays performed by mixing purified active and inactive 2C(34-318) subunits revealed a coordinated mechanism of ATP hydrolysis. Our results provide new insights into the structure and mechanism of picornavirus 2C proteins that will facilitate new investigations of their roles in infection. [ABSTRACT FROM AUTHOR]

Abstract: Picornavirus replication is critically dependent on the correct processing of a polyprotein precursor by 3C protease(s) (3Cpro) at multiple specific sites with related but non-identical sequences. To investigate the structural basis of its cleavage specificity, we performed the first crystallographic structural analysis of non-covalent complexes of a picornavirus 3Cpro with peptide substrates. The X-ray crystal structure of the foot-and-mouth disease virus 3Cpro, mutated to replace the catalytic Cys by Ala and bound to a peptide (APAKQ|LLNFD) corresponding to the P5–P5′ region of the VP1-2A cleavage junction in the viral polyprotein, was determined up to 2.5 Å resolution. Comparison with free enzyme reveals significant conformational changes in 3Cpro on substrate binding that lead to the formation of an extended interface of contact primarily involving the P4–P2′ positions of the peptide. Strikingly, the deep S1′ specificity pocket needed to accommodate P1′-Leu only forms when the peptide binds. Substrate specificity was investigated using peptide cleavage assays to show the impact of amino acid substitutions within the P5–P4′ region of synthetic substrates. The structure of the enzyme–peptide complex explains the marked substrate preferences for particular P4, P2 and P1 residue types, as well as the relative promiscuity at P3 and on the P′ side of the scissile bond. Furthermore, crystallographic analysis of the complex with a modified VP1-2A peptide (APAKE|LLNFD) containing a Gln-to-Glu substitution reveals an identical mode of peptide binding and explains the ability of foot-and-mouth disease virus 3Cpro to cleave sequences containing either P1-Gln or P1-Glu. Structure-based mutagenesis was used to probe interactions within the S1′ specificity pocket and to provide direct evidence of the important contribution made by Asp84 of the Cys-His-Asp catalytic triad to proteolytic activity. Our results provide a new level of detail in our understanding of the structural basis of polyprotein cleavage by 3Cpro. [Copyright &y& Elsevier]

Abstract: Foot-and-mouth disease virus is a highly contagious pathogen that spreads rapidly among livestock and is capable of causing widespread agricultural and economic devastation. The virus genome is translated to produce a single polypeptide chain that subsequently is cleaved by viral proteases into mature protein products, with one protease, 3Cpro, carrying out the majority of the cleavages. The highly conserved nature of this protease across different viral strains and its crucial role in viral maturation and replication make it a very desirable target for inhibitor design. However, the lack of a convenient and high-throughput assay has been a hindrance in the characterization of potential inhibitors. In this article, we report the development of a continuous assay with potential for high throughput using fluorescence resonance energy transfer-based peptide substrates. Several peptide substrates containing the 3C-specific cleavage site were synthesized, varying both the positions and separation of the fluorescent donor and quencher groups. The best substrate, with a specificity constant k cat/K M of 57.6±2.0M−1 s−1, was used in inhibition assays to further characterize the protease’s activity against a range of commercially available inhibitors. The inhibition profile of the enzyme showed characteristics of both cysteine and serine proteases, with the chymotrypsin inhibitor TPCK giving stoichiometric inhibition of the enzyme and allowing active site titration of the 3Cpro. [Copyright &y& Elsevier]

Foot-and-mouth disease virus (FMDV) causes a widespread and economically devastating disease of domestic livestock. Although FMDV vaccines are available, political and technical problems associated with their use are driving a renewed search for alternative methods of disease control. The viral RNA genome is translated as a single polypeptide precursor that must be cleaved into functional proteins by vitally encoded proteases. 10 of the 13 cleavages are performed by the highly conserved 3C protease (3Cpro), making the enzyme an attractive target for antiviral drugs. We have developed a soluble, recombinant form of FMDV 3Cpro, determined the crystal structure to 1.9-Å resolution, and analyzed the cleavage specificity of the enzyme. The structure indicates that FMDV 3Cpro adopts a chymotrypsin-like fold and possesses a Cys-His-Asp catalytic triad in a similar conformation to the Set-His-Asp triad conserved in almost all serine proteases. This observation suggests that the dyad-based mechanisms proposed for this class of cysteine proteases need to be reassessed. Peptide cleavage assays revealed that the recognition sequence spans at least four residues either side of the scissile bond (P4-P4') and that FMDV 3Cpro discriminates only weakly in favor of P1-Gln over P1-Glu, in contrast to other 3Cpro enzymes that strongly favor P1-Gln. The relaxed specificity may be due to the unexpected absence in FMDV 3Cpro of an extended β-ribbon that folds over the substrate binding cleft in other picornavirus 3Cpro structures. Collectively, these results establish a valuable framework for the development of FMDV 3Cpro inhibitors. [ABSTRACT FROM AUTHOR]

The 3C protease (3Cpro) from foot-and-mouth disease virus (FMDV), the causative agent of a widespread and economically devastating disease of domestic livestock, is a potential target for antiviral drug design. We have determined the structure of a new crystal form of FMDV 3Cpro, a chymotrypsin-like cysteine protease, which reveals features that are important for catalytic activity. In particular, we show that a surface loop which was disordered in previous structures adopts a β-ribbon structure that is conformationally similar to equivalent regions on other picornaviral 3C proteases and some serine proteases. This β-ribbon folds over the peptide binding cleft and clearly contributes to substrate recognition. Replacement of Cys142 at the tip of the β-ribbon with different amino acids has a significant impact on enzyme activity and shows that higher activity is obtained with more hydrophobic side chains. Comparison of the structure of FMDV 3Cpro with homologous enzyme-peptide complexes suggests that this correlation arises because the side chain of Cys142 contacts the hydrophobic portions of the P2 and P4 residues in the peptide substrate. Collectively, these findings provide compelling evidence for the role of the β-ribbon in catalytic activity and provide valuable insights for the design of FMDV 3Cpro inhibitors. [ABSTRACT FROM AUTHOR]