Figure 5. Effect of replacement R55W in 2C in the release of FMDV RNA from cells.

10⁶ BHK–21 cells were electroporated with 8 µg of either V19–4, V3, V15–9 or WT RNAs. Then the cells were transferred to M24–wells. A, Total viral RNA (intracellular and extracellular) and extracellular RNA were quantified by real–time RT–PCR, at the indicated hours post-electroporation (hpe). The amount of FMDV RNA was normalised to the number of cells seeded in the corresponding wells. The viral RNA (total and extracellular) measured just after electroporation was subtracted from each corresponding value. Note that RNAs encoding R55W in 2C are significantly more efficient in promoting release of viral RNA from cells at 6 hours post–transfection than those expressing wild type 2C (p<0.05; paired t–Test). B, Ratio of FMDV RNA released from the cells (ratio of extracellular viral RNA to total RNA) at 6 hours post–electroporation, determined from the data shown in A. Assays in A and B were carried out in triplicate and standard deviations are given. C, Detection of capsid proteins VP3 and VP1 in mutant FMDVs expressing 2C with or without replacement R55W. 10⁶ BHK–21 cells were electroporated with 8 µg of viral RNA. At 3 hours post–electroporation, supernatants were removed and DMEM without Met and Cys, but supplemented with [³⁵S] Met–Cys, was added. At 4 hours post–electroporation, supernatants were removed and cells were collected to analyse the synthesis of the viral capsid proteins VP3 and VP1. D, Extracellular samples of cell cultures electroporated with the indicated mutant FMDV RNAs, labelled as described in C, were collected at 2 (0-2 hours) and 20 (2-24 hours) hours post–labelling (6 and 24 hours post–electroporation). Samples were analysed by SDS–PAGE (15% acrylamide). VP3 and VP1 were detected at early time (4 to 6 hours post–transfection) in the supernatant of cells transfected with 8 µg of V15–9 or V3 encoding 2C with R55W, but not with the same amount of RNA from constructs encoding wild type 2C. At later times (6 to 24 hours post–transfection), VP3 and VP1 were detected in the supernatant of cells transfected with any of the constructs tested. Procedures are further detailed in Materials and Methods.