Figure 2
In situ immunofluorescence analysis of capsid proteins and capsids produced in mammalian cells transfected with MVM pSVtk-VP1/VP2 plasmids. (a) Representative in situ immunofluorescence image pairs are shown for cells transfected with wt or representative mutants of Groups 1 (E146A), 2 (K478A) or 3 (Q137K), and for mock-transfected cells as a negative control. In each image pair, the left image corresponds to capsid protein (red fluorescence) and the right image to assembled capsids (green fluorescence). The amounts and avidity of sera and labelled secondary antibodies used to detect either protein or assembled capsid were different, so comparison between signals obtained with different antibodies is not valid. (b) Assembly efficiency for each mutant capsid relative to the wt capsid, for which a reference value of 1 has been asigned (green bar). These values were obtained as previously described55,81. Mutant plasmids and the wt control plasmid were transfected in parallel using the same batch of cells in a same experiment. Assembly efficiency was determined as described in footnote c of Table 1: A large enough number of cells was visualized; the number of those cells that yielded a positive signal (above a sensitivity threshold) when an anti-capsid antibody (green fluorescence) was used was divided by the number of cells that yielded a positive signal (above a sensitivity threshold) when an anti-capsid protein antibody (red fluorescence) was used; and the values obtained for each mutant were normalized. For example: if for a given mutant capsid 80 cells showed green fluorescence and 200 cells showed red fluorescence above a predefined threshold, the absolute assembly efficiency of that mutant capsid was taken as (80/200) × 100 = 40%; if for the for the wt capsid in the same experiment 160 cells showed green fluorescence and 200 cells showed red fluorescence, the absolute assembly efficiency of the wt capsid was (160/200) × 100 = 80%. In this example, the relative assembly efficiency of the mutant capsid compared to the wt capsid would be (40/80) × 100 = 50% Average values were obtained by counting cells in 15–25 fields in each of two independent experiments. Values for mutants of Groups 1, 2, or 3 are respectively indicated by blue, red or yellow bars. Error bars indicate standard deviations (SD). Differences in average values relative to wt that corresponded to ≥1 standard deviation were taken as statistically significant (with a 66% confidence; Table 1).
