Characterisation and enrichment of foot-and-mouth disease virus replication complexes

Foot-and-mouth disease virus (FMDV) is an animal pathogen, which continues to pose a global threat regardless of the economic status of a nation. FMDV has a positive-sense RNA genome that is replicated via negative-sense intermediates in virus-produced replication complexes. These are assembled from viral RNA and proteins, in association with host cell factors. However, the precise composition of the FMDV replication complex remains undefined and a better understanding of the organisation and composition of this complex could provide novel therapeutic targets. Understanding the composition of the FMDV replication complex and the host factors has remained challenging due to the technical difficulties in isolating such structures from infected cells. Here, a combination of immunoprecipitation, ultracentrifugation and cellular electron microscopy (EM) is used to investigate the composition and morphology of replication complexes in the context of a sub-genomic replicon model. The 2C protein of FMDV plays many roles within the viral lifecycle, one of these is thought to be in the establishment and maintenance of the replication complex. The interactome of 2C protein was investigated by immunoprecipitation using a novel antibody reagent reactive against the C-terminus of 2C. Co-immunoprecipitation of 3AB, 3ABB and 3CD with 2C was observed to occur in a detergent-sensitive manner. Mass spectrometry comparing mock- to replicon-transfected BHK-21 cells following anti-2C immunoprecipitation identified 190 proteins which were >2-fold enriched in the presence of replication. Among these were >80 proteins normally localised to the endoplasmic reticulum (ER).