The switch between acute and persistent paramyxovirus infection caused by single amino acid substitutions in the RNA polymerase P subunit
Fig 3
Single amino acid (nucleotide) change determines whether PIV5 has an acute or persistent phenotype.
Panel a) Monolayers of A549 cells were either mock infected or infected with rPIV5-W3:P(S157) or rPIV5-W3:P(F157) at 10 pfu/cell. At the times indicated the cells were metabolically labelled for 1h with [35S]-L-methionine. Polypeptides present in the total cell extracts were separated by electrophoresis through a 4–12% SDS-PAG, and the labelled polypeptides visualized using a phosphorimager. Panel b) At the time of labelling images of the monolayers were taken using a phase contrast microscope. Note the lack of a cpe at 72h p.i. in cells infected the rPIV5-W3:P(S157) and almost complete death in cells infected with rPIV5-W3:P(F157). Panel c) Monolayers of A549 cells were either mock infected or infected with rPIV5-W3:P(S157) or rPIV5-W3:P(F157) at 10 pfu/cell and at the times indicated washed once and the % cell viability monitored using PrestoBlue cell viability reagent. Data shown represents mean values (n = 6 replicates; error bars = SD). Panel d) Plaques of rPIV5-W3:P(S157) and rPIV5-W3:P(F157) formed on monolayers of A549 at 4 days p.i. Plaques were either visualised by immunostaining the monolayers or staining the monolayers with crystal violet. Panel e) One step growth curve of A549 cells infected with either rPIV5-W3:P(S157) or rPIV5-W3:P(F157). Values for all times points were statistically significant (the P values in a T-test for each time point are shown). Data shown represents mean values (n = 6 replicates; error bars = SD) and the figure is representative of 3 independent experiments.