Abstract
Foot-and-mouth disease virus (FMDV) is one of the most extensively studied animal pathogens because it remains a major threat to livestock economies worldwide. However, the dynamics of FMDV infection are still poorly understood. The application of reverse genetics provides the opportunity to generate molecular tools to further dissect the FMDV life cycle. Here, we have used reverse genetics to determine the capsid packaging limitations for a selected insertion site in the FMDV genome. We show that exogenous RNA up to a defined length can be stably introduced into the FMDV genome, whereas larger insertions are excised by recombination events. This led us to construct a recombinant FMDV expressing the fluorescent marker protein, termed iLOV. Characterization of infectious iLOV-FMDV showed the virus has a plaque morphology and rate of growth similar to the parental virus. In addition, we show that cells infected with iLOV-FMDV are easily differentiated by flow cytometry using the inherent fluorescence of iLOV and that cells infected with iLOV-FMDV can be monitored in real-time with fluorescence microscopy. iLOV-FMDV therefore offers a unique tool to characterize FMDV infection in vitro, and its applications for in vivo studies are discussed.
Highlights
Foot-and-mouth disease virus (FMDV) is the aetiological agent of foot-and-mouth disease (FMD) of cloven-hoofed animals
During translation of the FMDV polyprotein, an intra-ribosomal self-processing event occurs at the C terminus of 2A separating the region containing the capsid proteins (P1) from most of the non-structural proteins. 2A is subsequently cleaved from P1 at its N-terminal by the 3C protease, which is responsible for the majority of the remaining proteolytic cleavages in the FMDV polyprotein
The work investigates the packaging limitations of the FMDV capsid for a specific insertion site and explains why previous attempts to construct FMDV-encoding fluorescent proteins, such as GFP, failed
Summary
Foot-and-mouth disease virus (FMDV) is the aetiological agent of foot-and-mouth disease (FMD) of cloven-hoofed animals. An internal ribosome entry site facilitates translation of the FMDV genome, yielding a polyprotein that is subsequently processed to a number of intermediate products and 12 mature proteins: the nonstructural auto-proteinase (Lpro); the structural proteins VP4 (1A), VP2 (1B), VP3 (1C) and VP1 (1D) and the remaining non-structural proteins (nsp) 2A, 2B, 2C, 3A, 3B, 3Cpro and 3Dpol (Forss et al, 1984; Rueckert, 1996). During translation of the FMDV polyprotein, an intra-ribosomal self-processing event occurs at the C terminus of 2A separating the region containing the capsid proteins (P1) from most of the non-structural proteins. 2A is subsequently cleaved from P1 at its N-terminal by the 3C protease, which is responsible for the majority of the remaining proteolytic cleavages in the FMDV polyprotein. During translation of the FMDV polyprotein, an intra-ribosomal self-processing event occurs at the C terminus of 2A separating the region containing the capsid proteins (P1) from most of the non-structural proteins. 2A is subsequently cleaved from P1 at its N-terminal by the 3C protease, which is responsible for the majority of the remaining proteolytic cleavages in the FMDV polyprotein.
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