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).