Foot-and-mouth disease virus (FMDV) is a member of the picornavirus family of non-enveloped, positive-sense, single-stranded RNA viruses. Picornavirus assembly involves the multimerisation of a capsid subunit (P1 or P1-2A) into pentameric structures, which further assemble into intact capsids containing the viral RNA genome.

The capsid subunit is co-translationally myristoylated and proteolytically cleaved by a viral protease (3Cpro) to initiate the assembly cascade. A cell-free assay was developed to analyse the requirement for these processes in pentamer assembly. Pentamer assembly was found to be dependent on myristoylation. In these assays, two 3Cpro recognition sites in the P1 protein could be cleaved independently and were both required for the efficient formation of pentamers. In addition, a system was developed for the production of large quantities of purified recombinant capsid precursor that could be used for future structural studies.

Existing studies have shown that molecular chaperones such as heat-shock protein 90 (Hsp90) are required for capsid assembly of other picornaviruses. Pharmacological inhibition of Hsp90 reduced growth of FMDV in cell culture and prevented pentamer assembly in the cell-free assay. Hsp90 was not required for processing, which contrasted with existing models for this part of the picornavirus life cycle.

Upon RNA encapsidation a maturation cleavage occurs on the inside of the capsid generating the structural protein VP4. Upon virus entry into cells, capsids disassemble into pentamers that no longer contain VP4. Pentamers from the disassembly pathway are therefore thought to have different properties than pentamers found on the assembly pathway. A maturation-like cleavage event was engineered into recombinant pentamers. Cleavage of this site altered pentamer sedimentation from that expected of assembly pentamers (14S) to that expected of disassembly pentamers (12S). This confirmed that loss of VP4 from pentamers controlled the switch in the properties of these pentamers.