To identify the active-site residues of the 3C proteinase of foot-and-mouth disease virus (FMDV), we introduced mutations into the 3C coding region and examined the activity of mutant enzymes on various substrates. Based on alignment of FMDV 3C with other picornavirus 3C proteinases and with the trypsin family of serine proteinases, mutations were introduced at residues presumed to be part of the catalytic triad, involved in substrate binding, or present in nonconserved regions. Wild-type and mutant 3C proteins were expressed in Escherichia coli and tested for their ability to cleave synthetic substrates corresponding to different portions of the viral genome. Substitutions at His-46 (catalytic triad), Asp-84 (catalytic triad), or His-181 (substrate binding) produced enzymes unable to process P1, P2, or P3 substrates in trans, whereas a change in the conserved Asp-98 had no effect on enzyme activity. Substitution of Ser for Cys-163 (catalytic triad) yielded an enzyme that retained activity on some substrates, while a substitution of Gly at this position resulted in a completely inactive enzyme. The kinetics of trans processing of translation products from a transcript encoding the P1 and P2 coding regions and the 2C/3A cleavage site with wild-type 3C or a transcript encoding P1 with 3C mutants revealed that the order of cleavage was VP3-VP1, VP0-VP3, VP1-2A, 2C-3A, and 2B-2C. Mutations in 3C that resulted in a partially active enzyme were individually introduced into full-length FMDV cDNA and RNA transcripts were translated in a cell-free system and used to transfect cells. In all cases the virus that was rescued had reverted to the wild-type 3C codon.

The foot-and-mouth disease virus (FMDV) leader (L) protein is involved in autocatalytic cleavage at the L/P1 junction and in the cleavage of translation initiation factor p220, a subunit of the cap-binding protein complex. It has been suggested that this proteinase has homology to the papain-like family of cysteine proteinases, and from this information, we have investigated the active-site residues by introducing specific mutations into the L gene. Mutations of Cys-23 to Ala or His-120 to Leu resulted in enzymes that lacked cis activity at the L/VP4 cleavage site, trans activity on a truncated L-P1 substrate, and p220 cleavage activity. Mutations of Cys-23 to Ser or His-110 to Leu resulted in enzymes that retained some or all cis activity and had reduced p220 cleavage. These mutations were introduced separately into a full-length FMDV cDNA, and RNA transcripts derived from these cDNAs were translated in a cell-free system and transfected into cells. The C23S mutant inefficiently cleaved at the L/P1 junction and within P1, and virus obtained from transfected cells reverted to wild type. The H110L mutant cleaved the L/P1 junction almost as well as the wild-type enzyme, and virus recovered from transfected cells retained the mutation and displayed wild-type viral protein synthesis and host shut-off kinetics.

The foot-and-mouth disease virus (FMDV) Lb gene was cloned into bacterial expression vectors under the control of a T7 RNA polymerase promoter. The Lb protein was expressed in both an in vitro transcription-translation system and in Escherichia coli. In vitro expression of a construct containing the Lb gene fused to a portion of the VP4 and 3D genes demonstrated cis cleavage activity that could be blocked by the thiol protease inhibitor E-64. Lb expressed in E. coli was purified from the soluble fraction by metal chelation chromatography. Purified Lb had trans cleavage activity at the L/P1 junction and cleaved the p220 component of the cap-binding protein complex.

Candidate foot-and-mouth disease (FMD) DNA vaccines designed to produce viral capsids lacking infectious viral nucleic acid were evaluated. Plasmid DNAs containing a portion of the FMDV genome coding for the capsid precursor protein (P1-2A) and wild-type or mutant viral proteinase 3C (plasmids P12X3C or P12X3Cmut, respectively) were constructed. Cell-free translation reactions programmed with pP12X3C (wild-type 3C) and pP12X3C-mut produced a capsid precursor, but only the reactions programmed with the plasmid encoding the functional proteinase resulted in P1-2A processing and capsid formation. Baby hamster kidney (BHK) cells also produced viral capsid proteins when transfected with these plasmids. Plasmid P12X3C was administered to mice by intramuscular, intradermal, and epithelial (gene gun) inoculations. Anti-FMD virus (FMDV) antibodies were detected by radioimmunoprecipitation (RIP) and plaque reduction neutralization assays only in sera of mice inoculated by using a gene gun. When pP12X3C and pP12X3C-mut were inoculated into mice by using a gene gun, both plasmids elicited an antibody response detectable by RIP but only pP12X3C elicited a neutralizing antibody response. These results suggest that capsid formation in situ is required for effective immunization. Expression and stimulation of an immune response was enhanced by addition of an intron sequence upstream of the coding region, while addition of the FMDV internal ribosome entry site or leader proteinase (L) coding region either had no effect or reduced the immune response.