Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease

Abstract

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 virally encoded proteases. 10 of the 13 cleavages are performed by the highly conserved 3C protease (3C pro ), making the enzyme an attractive target for antiviral drugs. We have developed a soluble, recombinant form of FMDV 3C pro , determined the crystal structure to 1.9-Å resolution, and analyzed the cleavage specificity of the enzyme. The structure indicates that FMDV 3C pro adopts a chymotrypsin-like fold and possesses a Cys-His-Asp catalytic triad in a similar conformation to the Ser-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 3C pro discriminates only weakly in favor of P1-Gln over P1-Glu, in contrast to other 3C pro enzymes that strongly favor P1-Gln. The relaxed specificity may be due to the unexpected absence in FMDV 3C pro of an extended ␤-ribbon that folds over the substrate binding cleft in other picornavirus 3C pro structures. Collectively, these results establish a valuable framework for the development of FMDV 3C pro inhibitors.

Processing of the VP1/2A Junction Is Not Necessary for Production of Foot-and-Mouth Disease Virus Empty Capsids and Infectious Viruses: Characterization of “Self-Tagged” Particles

“…It is interesting that in earlier studies (21), it was shown that truncation of P1-2A (removing part of the VP1 sequence) blocked all processing by 3C pro in vitro. The results obtained with the K210E mutant confirm that P2-Lys in the VP1/2A junction is an important determinant of 3C pro specificity (27,28). Previously, replacement of P2-Lys by P2-Ala had been shown to abrogate VP1/2A cleavage using a peptide cleavage assay (27).…”

“…Thus, the processing between VP1 and 2A appeared to be the slowest of the 3C pro -mediated cleavages within the P1-2A precursor. In contrast, the VP1/2A cleavage site was found to be the most rapidly processed protein junction in peptide cleavage assays using short synthetic substrates spanning eight residues on either side of all 10 3C pro cleavage sites (27). However, peptides corresponding to the VP2/VP3, 2C/3A, 3A/3B1, 3B1/3B2, and 3B2/3B3 junctions were uncleaved under these experimental conditions.…”

“…The VP1/2A junction for serotype O and A FMDVs is generally PxKQ/xLNF; thus, it has a Gln residue at the P1 position, which, together with the P4-Pro (P), P2-Lys (K), and P4=-Phe (F) residues, represent the most important determinants of 3C pro specificity at this site (26)(27)(28). Studies of aligned FMDV 3C pro cleavage sequences for over 100 strains of the virus (including representatives of all seven serotypes) reveal that sites with P1-Gln have a strong selectivity for P2-Lys, which suggests that recognition of the P1 residue by 3C pro is influenced by the residue in position P2 (26).…”

Foot-and-Mouth Disease Virus Assembly: Processing of Recombinant Capsid Precursor by Exogenous Protease Induces Self-Assembly of Pentamers In Vitro in a Myristoylation-Dependent Manner

“…2) which contained two substitutions (C95K and C142L) which greatly improved solubility while retaining activity of the enzyme (8,9).…”

“…This problem had hindered attempts to solve the crystal structure of the enzyme but was overcome by the introduction of mutations at the surface of the protein which allowed it to maintain solubility and proteolytic activity (8,9). This modified protease was able to process capsid precursor proteins to near completion, albeit at a low rate, presumably due to reduced activity compared to the wild-type enzyme (capsid precursor polyprotein would normally be processed to completion by native protease in infected cells).…”