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.

Cell culture propagation of foot-and-mouth disease virus: adaptive amino acid substitutions in structural proteins and their functional implications

“…The most commonly described amino acid exchanges replace a positively charged lysine with a glutamate (the anion of glutamic acid) with a single negative charge, but its replacement with an uncharged asparagine or a positively charged arginine has also been observed [50,52,[60][61][62]. Amino acid exchanges at position 210 inhibit the cleavage of the VP1-2A product [60][61][62]. They have been reported for FMDV serotype O, serotype A, and SAT 1 and are always linked to the E83K substitution, also in VP1 [50, Fig.…”

Emergency Services of Viral RNAs: Repair and Remodeling

“…Gasparyan, E. V. Khitrina, M. S. Kolesnikova, A. P. Gmyl, and V. I. Agol, unpublished data). Even prevention of viral polyprotein cleavage at a canonical site may be relatively well tolerated, as demonstrated with engineered foot-and-mouth disease virus (FMDV) mutants having amino acid substitutions preventing disruption of the bond between the VP1 and 2A moieties normally cleaved by the viral 3C pro protein (239,240). The 2A protein of this virus is composed of merely 18 amino acids, and capsids of the viable progeny of the mutants contained the VP1-2A fusions instead of VP1.…”

Use of recombinant capsid proteins in the development of a vaccine against the foot-and-mouth disease virus

“…37 Thus the assays are suitable for the diagnosis of a primary index case and for use in an ongoing outbreak. However, these assays are not designed to discriminate between serotypes of FMDV and each of the assays can fail to detect particular be thought that this cleavage was dependent on packaging of the genome, however, it has become apparent that it occurs within assembled "empty" capsid particles (lacking the RNA genome) too [22][23][24] but the mechanism is not known. The P2 and P3 regions of the polyprotein include the NSPs.…”

“…23,24,51,57 These systems generate empty capsid particles which can be detected by sucrose gradient analysis and by electron microscopy. The structure of these particles has been determined by 3D-reconstructions from electron micrographs 23 and by X-ray crystallography.…”

“…Sequences for the HA and FLAG epitope tags were inserted into the VP1 within the virus capsid without adverse effect on virus growth. 77 In an alternative strategy, it has been shown that the cleavage of the VP1/2A junction (by 3C pro ) is not required for virus viability 24,78 and the modification of the cleavage site can result in the formation of "self-tagged" virus particles which contain the VP1-2A fusion protein. Thus no "foreign" sequences are added to the genome but the presence of the 2A attached to the VP1 can be readily detected (using anti-2A antibodies).…”