Assembly and characterization of foot-and-mouth disease virus empty capsid particles expressed within mammalian cells
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Assembly and characterization of foot-and-mouth disease virus empty capsid particles expressed within mammalian cells. / Gullberg, Maria; Muszynski, Bartosz; Organtini, Lindsey J; Ashley, Robert E; Hafenstein, Susan L; Belsham, Graham J; Polacek, Charlotta.
In: The Journal of general virology, Vol. 94, No. Pt 8, 08.2013, p. 1769-1779.Research output: Contribution to journal › Journal article › Research › peer-review
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T1 - Assembly and characterization of foot-and-mouth disease virus empty capsid particles expressed within mammalian cells
AU - Gullberg, Maria
AU - Muszynski, Bartosz
AU - Organtini, Lindsey J
AU - Ashley, Robert E
AU - Hafenstein, Susan L
AU - Belsham, Graham J
AU - Polacek, Charlotta
PY - 2013/8
Y1 - 2013/8
N2 - 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.
AB - 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.
KW - Animals
KW - Capsid Proteins/genetics
KW - Cell Line
KW - Cricetinae
KW - Cysteine Endopeptidases/genetics
KW - Foot-and-Mouth Disease Virus/genetics
KW - Gene Expression
KW - Genetic Vectors
KW - Imaging, Three-Dimensional
KW - Macromolecular Substances/metabolism
KW - Microscopy, Electron
KW - Protein Binding
KW - Protein Multimerization
KW - Protein Processing, Post-Translational
KW - Vaccinia virus/genetics
KW - Viral Proteins/genetics
KW - Virosomes/genetics
U2 - 10.1099/vir.0.054122-0
DO - 10.1099/vir.0.054122-0
M3 - Journal article
C2 - 23740480
VL - 94
SP - 1769
EP - 1779
JO - Journal of General Virology
JF - Journal of General Virology
SN - 0022-1317
IS - Pt 8
ER -
ID: 257916438