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
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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. / Gullberg, Maria; Polacek, Charlotta; Bøtner, Anette; Belsham, Graham J.
In: Journal of Virology, Vol. 87, No. 21, 11.2013, p. 11591-603.Research output: Contribution to journal › Journal article › peer-review
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TY - JOUR
T1 - Processing of the VP1/2A junction is not necessary for production of foot-and-mouth disease virus empty capsids and infectious viruses
T2 - characterization of "self-tagged" particles
AU - Gullberg, Maria
AU - Polacek, Charlotta
AU - Bøtner, Anette
AU - Belsham, Graham J
PY - 2013/11
Y1 - 2013/11
N2 - 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.
AB - 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.
KW - Amino Acid Substitution
KW - Animals
KW - Capsid/metabolism
KW - Capsid Proteins/genetics
KW - Cell Line
KW - DNA Mutational Analysis
KW - Foot-and-Mouth Disease Virus/genetics
KW - Microbial Viability
KW - Mutant Proteins/genetics
KW - Protein Precursors/genetics
KW - Protein Processing, Post-Translational
KW - Viral Proteins/genetics
KW - Virus Assembly
U2 - 10.1128/JVI.01863-13
DO - 10.1128/JVI.01863-13
M3 - Journal article
C2 - 23966400
VL - 87
SP - 11591
EP - 11603
JO - Journal of Virology
JF - Journal of Virology
SN - 0022-538X
IS - 21
ER -
ID: 257916359