Fig. 3

Importance of individual aa substitutions for the replication and cytotoxicity PD-SK. (A) Schematic representation of generated PD-H mutant variants. The following mutants were generated. PD-K4 (A3T), PD-K1 (E596V, E768D), PD-K2A (Y940C), PD-K2B (N1027D), PD-K2AB (Y940C, N1027D), PD-K1-2AB (E596V, E768D, Y940C, N1027D) and PD-SK (A3T, E596V, E768D, Y940C, N1027D), the parenthesis shows the respective aa exchanges compared to PD-H. Colored columns in the scheme represent the location of nucleotide substitutions compare to PD-H. (B) Virus growth kinetics. Upper panel, Colo320 cells were infected with PD-H, PD-K4, PD-K1, PD-K2A, PD-K2B, PD-K2AB, PD-K1-2AB and PD-SK at MOI 0.1. Virus titers were determined by plaque assay at indicated time points. Shown are mean values ± SEM. Significance compared to PD-H, * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. Lower table, replication of the virus variants after 24 h and 48 h relative to PD-H. (C) Plaque sizes. Plaque sizes of indicated viruses were determined by plaque assays on HeLa cell monolayers. Upper panel – shows representative images of viral plaques (white dots) Lower panel - Graphical representation of plaque diameter. Shown are mean values ± SEM. Significance compared to PD-H, ** p < 0.01; **** p < 0.0001, for n = 50 plaques. (D) Cell viability. Colo320 cells were infected with indicated viruses at MOI 0.1 and 1; cell viability was measured 24 h (upper diagram) and 48 h (lower diagram) after infection. Shown are mean values ± SEM. Significance compared to PD-H, ** p < 0.01; *** p < 0.001; **** p < 0.0001