Fig. 6: Mutations to NS2-dependent sites can influence mRNA/cRNA/vRNA ratios and impact replication during infection.

a Each individual mutant in PB1177:385 was transfected alongside a 400nt internal control and minimal replication machinery with, and without, NS2. The relative frequency of each variant relative to the 400nt control was compared against wild-type under each condition at 24 hours. Asterisks indicate a ratio significantly less than 1, one-tailed one-sample t-test, Benjamini-Hochberg corrected FDR < 0.05, indicating that the parental exhibits an NS2-dependent advantage over the variant. Points represent mean and standard deviation, n = 3. b Quantitative analysis of primer extension in PB1177:385. All values in left two columns corrected against a parental template in the absence of NS2. Dotted line represents that value, points above indicate an increase in that molecular species, below, decrease. Values in the right column represent the ratio of points between the left two columns. Asterisks indicate values that are significantly decreased relative to the parental template, one-tailed t-test with Benjamini-Hochberg corrected FDR < 0.1. Individual replicates and mean presented, n = 3. Similar analyzes for full-length PB1 template presented in Supplementary Fig.14. c Indicated variants were introduced into full-length PB1 bearing inactivating mutations to its start codon and rescued via reverse genetics alongside a 1600nt internal competitor. MDCK-SIAT1 cells expressing PB1 were infected with the resulting mixture at a genome-calibrated MOI of 25 for 14 hours. The frequency of each variant in the mixed population was measured before, and after, viral replication. Values plotted represent the change in frequency during replication only. Asterisks indicate values significantly less than the parental, one-tailed two-sample t-test with Benjamini-Hochberg error correction FDR < 0.05. Source data are provided as a Source Data file.