An Engineered Maturation Cleavage Provides a Recombinant Mimic of Foot-and-Mouth Disease Virus Capsid Assembly-Disassembly

Abstract

Picornavirus capsids are assembled from 60 copies of a capsid precursor via a pentameric assembly intermediate or 'pentamer'. Upon completion of virion assembly, a maturation event induces a final cleavage of the capsid precursor to create the capsid protein VP4, which is essential for capsid stability and entry into new cells. For the picornavirus foot-and-mouth disease virus (FMDV), intact capsids are temperature and acid-labile and can disassemble into pentamers. During disassembly, capsid protein VP4 is lost, presumably altering the structure and properties of the resulting pentamers. The purpose of this study was to compare the characteristics of recombinant "assembly" and "disassembly" pentamers. We generated recombinant versions of these different pentamers containing an engineered cleavage site to mimic the maturation cleavage. We compared the sedimentation and antigenic characteristics of these pentamers using sucrose density gradients and reactivity with an antibody panel. Pentamers mimicking the assembly pathway sedimented faster than those on the disassembly pathway suggesting that for FMDV, in common with other picornaviruses, assembly pentamers sediment at 14S whereas only pentamers on the disassembly pathway sediment at 12S. The reactivity with anti-VP4 antibodies was reduced for the 12S pentamers, consistent with the predicted loss of VP4. Reactivity with other antibodies was similar for both pentamers suggesting that major antigenic features may be preserved between the VP4 containing assembly pentamers and the disassembly pentamers lacking VP4.

Keywords: antigenicity; capsid assembly; foot-and-mouth disease virus; picornavirus.

Figure 1

Assembly and disassembly of FMDV capsids and control of pentamer switching. Black arrows indicate pathway of normal FMDV capsid assembly and disassembly. The green arrow represents the pathway taken by the ‘maturation engineered’ pentamers generated in this study. Processing of P1-2A facilitates folding of P1-2A into an assembly competent protomer (5S) followed by assembly of 5 protomers into a pentameric structure with 14S sedimentation containing intact VP0 protein. Twelve pentamers assemble into intact capsids in a concentration dependent manner and if viral RNA is encapsidated they form virions (146S). Low pH, heat or cell entry trigger the disassembly of intact capsids into disassembly versions of pentamers (12S) containing VP2 and lacking VP4. An introduced maturation cleavage mutation allows conversion of assembly pentamers directly into disassembly pentamers. Created with Biorender.com.

Figure 2

FMDV capsid precursor mat mutant can be cleaved by Pre^pro independently of 3C^pro processing. Cell free expression reactions of wt (top panel) or mat (bottom panel) were either unprocessed (Lane 1), processed with 3C^pro (Lane 2) and then Pre^pro (Lane 3), or processed with Pre^pro (Lane 4) and then 3C^pro (Lane 5) as indicated above the gel image for one hour at 37 °C. Arrows indicate sequential treatment with both proteases. Samples were resolved by SDS-PAGE and visualised by fluorography. The predicted sizes of capsid proteins and precursors are identified to the right of the gel images and the positions of molecular weight markers are indicated to the left.

Figure 3

FMDV mat mutant pentamers and protomers sediment slower after cleavage. Cell free expression reactions of wt (upper panel) and mat (lower panel), were sedimented through sucrose density gradients and the radioactive counts were analysed in 24-equal fractions. Traces correspond to treatments as follows; mock processing (blue), 3C^pro (red), sequential treatment with 3C^pro then Pre^pro (orange) or Pre^pro then 3C^pro (black). Gradient profiles are single data points, representative of the position of pentamer peaks observed on multiple occasions. Arrows indicate peak position of sedimentation markers as determined in [31].

Figure 4

FMDV mat mutant pentamers lack VP4 but are antigenically indistinguishable from wt pentamers. Antibodies were incubated with either wt (red) and mat (orange) pentamers before the addition of either Protein A (GP sera) or Protein G (all other antibodies) magnetic beads. Radioactivity bound to the beads was read by scintillation counting. Pentamer immunoprecipitation is expressed as a proportion of input counts. The data shown are the mean of two independent biological repeats and error bars represent the range of the data.