title: Determinants of the VP1/2A junction cleavage by the 3C protease in foot-and-mouth disease virus-infected cells.
abstract: The foot-and-mouth disease virus (FMDV) capsid precursor, P1-2A, is cleaved by FMDV 3C protease to yield VP0, VP3, VP1 and 2A. Cleavage of the VP1/2A junction is the slowest. Serotype O FMDVs with uncleaved VP1-2A (having a K210E substitution in VP1; at position P2 in cleavage site) have been described previously and acquired a second site substitution (VP1 E83K) during virus rescue. Furthermore, introduction of the VP1 E83K substitution alone generated a second site change at the VP1/2A junction (2A L2P, position P2' in cleavage site). These virus adaptations have now been analysed using next-generation sequencing to determine sub-consensus level changes in the virus; this revealed other variants within the E83K mutant virus population that changed residue VP1 K210. The construction of serotype A viruses with a blocked VP1/2A cleavage site (containing K210E) has now been achieved. A collection of alternative amino acid substitutions was made at this site, and the properties of the mutant viruses were determined. Only the presence of a positively charged residue at position P2 in the cleavage site permitted efficient cleavage of the VP1/2A junction, consistent with analyses of diverse FMDV genome sequences. Interestingly, in contrast to the serotype O virus results, no second site mutations occurred within the VP1 coding region of serotype A viruses with the blocked VP1/2A cleavage site. However, some of these viruses acquired changes in the 2C protein that is involved in enterovirus morphogenesis. These results have implications for the testing of potential antiviral agents targeting the FMDV 3C protease.
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark.
authors.forename: Thea
authors.initials: T
authors.lastname: Kristensen
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark.
authors.forename: Preben
authors.initials: P
authors.lastname: Normann
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark.
authors.forename: Maria
authors.initials: M
authors.lastname: Gullberg
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark.|Present address: Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases and Clinical Research Centre, Hvidovre Hospital and Department of International Health, Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark.
authors.forename: Ulrik
authors.initials: U
authors.lastname: Fahnøe
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark.
authors.forename: Charlotta
authors.initials: C
authors.lastname: Polacek
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark.
authors.forename: Thomas Bruun
authors.initials: TB
authors.lastname: Rasmussen
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Lindholm, DK-4771 Kalvehave, Denmark.
authors.forename: Graham J
authors.initials: GJ
authors.lastname: Belsham
mesh_terms: D000086042:3C Viral Proteases; D019943:Amino Acid Substitution; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D002213:Capsid / Q000187:drug effects / Q000378:metabolism*; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical; D005536:Foot-and-Mouth Disease / Q000821:virology*; D029544:Foot-and-Mouth Disease Virus / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000235:genetics; D008239:Lysine / Q000235:genetics; D009154:Mutation; D014764:Viral Proteins / Q000378:metabolism*; D019065:Virus Assembly / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D036022:Capsid Proteins; C442501:VP1 protein, Foot-and-mouth disease virus; D014764:Viral Proteins; D018698:Glutamic Acid; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases; D008239:Lysine