Fig. 4: NS2 expression suppresses replication of PB1 lacking certain genomic regions.

a Reanalysis of data from Fig. 3. Comparing variants of identical length, the frequency at which a region is, or is not, observed under each condition was determined, and the median across all lengths for a given replicate and region presented. The dotted line separates regions whose absence enhances replication (above) from those whose absence reduces replication (below). Asterisks indicate a significant effect that was greater than 2-fold, one-sample two-tailed t-test, Benjamini-Hochberg corrected FDR < 0.1. b How the absence of the indicated region in (a) influences replication upon NS2 expression across a range of different lengths. Points shown if there were at least 100 measurements across all conditions. Statistics performed as in (a), with a FDR cutoff of 0.05. c Each indicated deletion was generated in a PB1_177:385 background, co-transfected with a barcoded PB1 400nt competitor with, or without, NS2, and its replication was compared against the parental background at 24 h post-transfection by qPCR. Asterisks indicate that a given deletion reduces replication, one-tailed one-sample t-test, p < 0.05. Points represent mean and standard deviation, n = 3. d Variants from (c) were co-transfected with a barcoded PB1 400nt competitor and rescued into virions using infectious virus. A549 cells were infected with this population at a genome-calibrated MOI of 25 for 8 hours. The change in competitor and variant frequencies in vRNA during viral replication was determined by qPCR. Asterisks indicate reduced replication of the indicated variant when compared to the parental, two-tailed two-sample t-test, Benjamini-Hochberg corrected FDR < 0.05. e Positions in a full-length PB1 alignment defined as variable or non-variable by calculating the fraction of nucleotides at a position that do not match the major variant. At the chosen cutoff, 2.5%, a position is considered variable if at least 2.5% of sequences do not match the major variant. This cutoff was chosen to match the inflection point on our curve. f Frequency of variable sites within the given regions of PB1 as defined in (e). Significant difference between categories tested using Fisher’s exact test. Source data are provided as a Source Data file.