Fig. 3: Mutate–map–rescue analysis reveals new PB2 packaging-defective and compensatory mutant partners.

a, Electropherograms from systematic single-nucleotide mutation SHAPE chemical mapping with rescue (mutate–map–rescue)²¹ analysis of individual and compensatory double mutations to test basepairings from 1D data-guided models and to identify predicted successful synonymous PSL2-defective and compensatory mutant pairs. Chemical accessibilities are plotted in gray scale (black = highest SHAPE reactivity), across 59 single mutations at single-nucleotide resolutions of the PSL2 element from the PR8 strain segment PB2. Reactivity peaks (left to right) correspond to nucleotides from the 5′- to the 3′-end of the PB2 RNA (see Supplementary Fig. 7 for a complete set of M²R pairs). b, Electropherogram of successful double synonymous mutant pair determined by the mutate–map–rescue analysis. c, Mutational design of single mutants m52 (G52U) and m65 (C65A), and the double m52/65-comp rescue pair on the PSL2 structure. d, Packaging efficiency of the synonymous single and double-mutant M²R pair. Values are given as a percentage of PB2 vRNA packaging relative to WT PR8 virus. Results represent two independent experiments with biological duplicates performed in technical triplicate (n = 4), except for m65 performed in biological triplicate (n = 6). Statistical analysis is by ordinary one-way ANOVA using Dunnett’s multiple comparison test against WT. ^*P < 0.0001; NS, not significant. e, Viral titer of the supernatants collected in d in p.f.u. ml⁻¹, plaque assays in biological duplicate (n = 2), except for m65 (n = 3), and performed in technical triplicate. f,g, Percentage of day 0 weight and survival of mice infected with single PSL2-disrupting and compensatory PSL2-restoring double-mutant viruses; 6- to 8-week-old female BALB/c mice (n = 6) were intranasally infected with PR8 WT virus, packaging-defective single mutant viruses, m52 and m745, compensatory double-mutant viruses, m52/65-comp and m745-comp, or PBS control. f, Percentage of day 0 weight. g, Kaplan–Meier survival plot of the individual cohorts depicted in f. All error bars represent mean ± s.d. Statistics and graphs for all figures were generated in GraphPad Prism 9 software.