Fig. 2: Serial passaging of mutant WSN viruses with mutations at the FluPol-CTD interface selects for adaptive mutations which restore FluPol binding to the CTD and huANP32A.
A Schematic representation of the passaging experiment. B, C Passaged PA K289A viruses. B Plaque phenotype. Representative images of crystal violet stained plaque assays on reverse genetics (RG), p1, p4 and p8 supernatants are shown. C Short-read next generation sequencing of the viral genome. Second site mutations found in ≥10% of reads in at least one passage are shown (green circles). The fraction of reads showing a given mutation is indicated by the circle area and shades of green (schematic on the left: <25, 25–50, 50–75 and >75 % of reads). D Ribbon diagram representation of FluPol(T) conformation (A/NT/60/1968, PDB: 6RR7)41. Second-site mutations observed during passaging of the PA K289A, R454A, K635A and R638A viruses are indicated in grey, green, blue and pink fonts respectively. E, F FluPol second-site mutations observed during passaging of the PA K289A virus. E Recombinant viruses with PA K289A and the indicated second-site mutations were produced (n = 2). Two independent RG supernatants were titrated, plaque diameters (mm) were measured, each dot represents one plaque (see Fig. S3C). (#) pinhead-sized plaques. F Cell-based FluPol binding and activity assays as in Fig. 1B. Left Y-axis (linear scale): FluPol binding to the CTD (grey bars) and huANP32A (blue bars). Right Y-axis (logarithmic scale): FluPol activity (hatched bars). Luminescence signals are represented as a percentage of WT FluPol. Stars indicate statistical significance when passage N is compared to passage N-1, or RG is compared to WT FluPol (mean ± SD, n = 5, 4, 4, *p < 0.033, **p < 0.002, ***p < 0.001, one-way ANOVA; Tukey’s multiple comparisons test). G For each primary mutation and FluPol genotype observed upon serial passaging, FluPol binding to huANP32A, CTD and DDX5 (x-axis in the left, middle and right panel, respectively) were plotted against the FluPol activity (y-axis). Binding and activity data are represented separately in (F) and Fig. S3E. Combinations of mutations which appeared during passaging of the WSN PA K289A, R454A, K635A and R638A mutant viruses are highlighted in grey, green, blue and pink respectively. (mean ± SD, n = 4, r: Pearson correlation coefficient, *p < 0.033, ***p < 0.001,two-tailed 95% confidence interval). Source data are provided as a Source data file.
