title: Assembly and characterization of foot-and-mouth disease virus empty capsid particles expressed within mammalian cells.
abstract: The foot-and-mouth disease virus (FMDV) structural protein precursor, P1-2A, is cleaved by the virus-encoded 3C protease (3C(pro)) into the capsid proteins VP0, VP1 and VP3 (and 2A). In some systems, it is difficult to produce large amounts of these processed capsid proteins since 3C(pro) can be toxic for cells. The expression level of 3C(pro) activity has now been reduced relative to the P1-2A, and the effect on the yield of processed capsid proteins and their assembly into empty capsid particles within mammalian cells has been determined. Using a vaccinia-virus-based transient expression system, P1-2A (from serotypes O and A) and 3C(pro) were expressed from monocistronic cDNA cassettes as P1-2A-3C, or from dicistronic cassettes with the 3C(pro) expression dependent on a mutant FMDV internal ribosome entry site (IRES) (designated P1-2A-mIRES-3C). The effects of using a mutant 3C(pro) with reduced catalytic activity or using two different mutant IRES elements (the wt GNRA tetraloop sequence GCGA converted, in the cDNA, to GAGA or GTTA) were analysed. For both serotypes, the P1-2A-mIRES-3C construct containing the inefficient GTTA mutant IRES produced the highest amount of processed capsid proteins. These products self-assembled to form FMDV empty capsid particles, which have a related, but distinct, morphology (as determined by electron microscopy and reconstruction) from that determined previously by X-ray crystallography. The assembled empty capsids bind, in a divalent cation-dependent manner, to the RGD-dependent integrin αvβ6, a cellular receptor for FMDV, and are recognized appropriately in serotype-specific antigen ELISAs.
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Lindholm, 4771 Kalvehave, Denmark.
authors.forename: Maria
authors.initials: M
authors.lastname: Gullberg
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Lindholm, 4771 Kalvehave, Denmark.
authors.forename: Bartosz
authors.initials: B
authors.lastname: Muszynski
authors.affiliation: Department of Microbiology and Immunology, the Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA.
authors.forename: Lindsey J
authors.initials: LJ
authors.lastname: Organtini
authors.affiliation: Department of Microbiology and Immunology, the Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA.
authors.forename: Robert E
authors.initials: RE
authors.lastname: Ashley
authors.affiliation: Department of Microbiology and Immunology, the Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA.
authors.forename: Susan L
authors.initials: SL
authors.lastname: Hafenstein
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Lindholm, 4771 Kalvehave, Denmark.
authors.forename: Graham J
authors.initials: GJ
authors.lastname: Belsham
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Lindholm, 4771 Kalvehave, Denmark.
authors.forename: Charlotta
authors.initials: C
authors.lastname: Polacek
mesh_terms: D000086042:3C Viral Proteases; D000818:Animals; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D006224:Cricetinae; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics* / Q000302:isolation & purification*; D015870:Gene Expression; D005822:Genetic Vectors; D021621:Imaging, Three-Dimensional; D046911:Macromolecular Substances / Q000378:metabolism; D008854:Microscopy, Electron; D011485:Protein Binding; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D014616:Vaccinia virus / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D022701:Virosomes / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D036022:Capsid Proteins; D046911:Macromolecular Substances; D014764:Viral Proteins; D022701:Virosomes; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases