Effect of replacement E8K in VPg on the specific infectivity of V19–4 (R55W).
A, Plaque assay with cells transfected with RNA transcripts of WT or the indicated mutant constructs. At 2 hours post–transfection, the cell culture supernatants were removed and the cells were covered with semisolid agar. Plaques were visualized either at 48 or 72 hours post–transfection. Note that substitutions R55W in 2C and E8K in VPg enhanced the plaque-forming capacity of V19–4. B, Specific infectivity (calculated as the ratio of the number of PFUs obtained to the ng of RNA used in the electroporation) for WT and the indicated V19–4 mutants. C, Total and extracellular FMDV RNAs measured at 6 hours after electroporation of BHK–21 cells with WT and the indicated V19–4 mutant RNAs. D, Amount of viral polymerase (3D) determined by metabolic labelling after electroporation of BHK–21 cells with the indicated RNAs, and densitometry of the relevant ptotein band. The results are expressed as percentage of the amount measured following electroporation with WT RNA, which is taken as 100%. Values in B, C, D are the average of 3 determinations; standard deviations are given. Procedures are described in Materials and Methods.