Deep mutational scanning reveals positively selected sites upon chaperone depletion at a biophysically restrictive temperature.

(A) Deep mutational scanning selection scheme: each mutant NP influenza library was subjected to selection at 37°C and 39°C in both basal and chaperone-depleted (HSF1i) cells at an MOI of 0.1 virions/cell. Libraries were deep sequenced following replication to quantify the selection on each mutant in the library. Mutation differential selection corresponds to the differential selection of a specific NP mutant, relative to wt NP. Site differential selection corresponds to the sum of the mutation differential selection values for all variants at a given NP site. (B) Box plots showing the distributions of net site differential selection in both basal and chaperone-depleted (HSF1i) cells at 37°C and 39°C. Each selection was performed in biological triplicate; box plots are shown for each replicate. The whiskers show 5th and 95th percentiles, boxes show 25th and 75th percentiles, line shows median value, and points show outliers. The significance of deviation of the mean from zero (no selection) was evaluated by a one-sample t test; * and *** indicate FDRs p = 0.0031). For pairwise statistical comparisons, p-values are provided in the statistics section of the Methods. Site differential selection values are provided in S3 Data. (C) Representative sequence logo plots showing amino acid variants at select sites differentially selected upon relative to wt. The size of the amino acid letters corresponds to the magnitude of the mutational differential selection, which is on the same scale for each selection. Amino acids above the black line are more fit in the selection condition compared to the mock-selection condition, amino acids below are less fit, and the black line represents behavior of the wt amino acid in the selection condition. ANOVA, analysis of variance; FDR, false discovery rate; HSF1i, condition in which heat shock factor 1 was inhibited; MOI, multiplicity of infection; NP, nucleoprotein; ns, not significant; wt, wild type.