Fig. 2: Two-dimensional M² analysis and empirical validation of the PSL2 motif.

a, Systematic single-nucleotide mutation and mapping of resulting chemical accessibility reveals interactions in the three-dimensional structure of the RNA. Chemical accessibilities are plotted in gray scale (black, highest SHAPE reactivity), across 59 single mutations at single-nucleotide resolutions of PSL2 element from PR8 strain segment PB2. Reactivity peaks (left to right) correspond to nucleotides from the 5′- to the 3′-end of the PB2 RNA. Nucleotides corresponding to known packaging mutation sites¹¹ are indicated on the left in blue. Red emboldened mutations denote packaging-defective mutant sites predicted by M² analysis. Green emboldened mutations indicate synonymous mutant sites analyzed in b. b, Packaging efficiencies of M²-identified synonymous mutants readout by RT–qPCR. Packaging efficiency represents the percentage of mutant PB2 vRNA packaging relative to parental WT PB2. The results are from two independent experiments in biological duplicate and technical triplicate (n = 4). ^****P < 0.0001, ^*P = 0.0321. c, Previously described synonymous (Syn.) mutants (m757, m745, m55c) are mapped on to the PSL2 structure. Compensatory, nonsynonymous mutations m55c-comp, m745-comp and m757-comp were designed at sites predicted to restore WT PSL2 structure based on SHAPE and M² chemical analyses. Black boxed nucleotides denote compensatory mutation sites. The (−)-sense vRNA orientation is shown. d, Packaging efficiencies of packaging-defective and compensatory mutant viruses. For compensatory mutations where a nonsynonymous change was required, a WT PB2 protein expression plasmid was co-transfected during virus rescue. Values are given as percentage of PB2 vRNA packaging relative to WT PR8 virus. The results are from three independent experiments (n = 3), with assays performed in triplicate. ^****P < 0.0001, ^***P < 0.0005; NS, not significant. e, Virus titer determined by plaque assay. Results are expressed in p.f.u. ml⁻¹, with plaque assays in triplicate (n = 3). ^****P < 0.0001, ^***P = 0.0009, ^**P = 0.0149. All error bars represent mean ± s.d. All statistical analyses were performed by ordinary one-way ANOVA using Dunnett’s multiple comparison test against WT computed in GraphPad Prism 9 software.