title: 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.
abstract: The foot-and-mouth disease virus (FMDV) capsid protein precursor, P1-2A, is cleaved by 3C(pro) to generate VP0, VP3, VP1, and the peptide 2A. The capsid proteins self-assemble into empty capsid particles or viruses which do not contain 2A. In a cell culture-adapted strain of FMDV (O1 Manisa [Lindholm]), three different amino acid substitutions (E83K, S134C, and K210E) were identified within the VP1 region of the P1-2A precursor compared to the field strain (wild type [wt]). Expression of the O1 Manisa P1-2A (wt or with the S134C substitution in VP1) plus 3C(pro), using a transient expression system, resulted in efficient capsid protein production and self-assembly of empty capsid particles. Removal of the 2A peptide from the capsid protein precursor had no effect on capsid protein processing or particle assembly. However, modification of E83K alone abrogated particle assembly with no apparent effect on protein processing. Interestingly, the K210E substitution, close to the VP1/2A junction, completely blocked processing by 3C(pro) at this cleavage site, but efficient assembly of "self-tagged" empty capsid particles, containing the uncleaved VP1-2A, was observed. These self-tagged particles behaved like the unmodified empty capsids in antigen enzyme-linked immunosorbent assays and integrin receptor binding assays. Furthermore, mutant viruses with uncleaved VP1-2A could be rescued in cells from full-length FMDV RNA transcripts encoding the K210E substitution in VP1. Thus, cleavage of the VP1/2A junction is not essential for virus viability. The production of such engineered self-tagged empty capsid particles may facilitate their purification for use as diagnostic reagents and vaccines.
authors: National Veterinary Institute, Technical University of Denmark, Lindholm, Kalvehave, Denmark.
authors: Maria
authors: M
authors: Gullberg
authors: Charlotta
authors: C
authors: Polacek
authors: Anette
authors: A
authors: Bøtner
authors: Graham J
authors: GJ
authors: Belsham
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002213:Capsid / Q000378:metabolism*; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004252:DNA Mutational Analysis; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics / Q000502:physiology*; D050296:Microbial Viability; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D019065:Virus Assembly
chemical_list: D036022:Capsid Proteins; D050505:Mutant Proteins; D011498:Protein Precursors; C442501:VP1 protein, Foot-and-mouth disease virus; D014764:Viral Proteins; C057421:virus protein 2A
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