Extended Data Fig. 5: In vivo effects of FluPol mutations involved in the interface to the Pol II EC.

a,b, Western blots against PA (a), PB2 (b), and tubulin (a,b) for wild type (WT) and mutant FluPol variants transiently expressed in HEK-293T cells. c, The Steady-state levels of NA mRNA, cRNA and vRNA in a minigenome assay setting are measured by ss-RTqPCR. The WT or indicated mutant WSN vRNPs were reconstituted in HEK-293T cells in the presence of the NA vRNA. The PA-K635A mutant was used as a transcription defective control². Total RNAs were analyzed by strand-specific RTqPCR⁴⁶ c, The data are represented as percentages (100%: WT) and as the mean ± s.d. Each point reflects one biological replicate (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001, 2-way ANOVA on log-transformed data with Tukey’s multiple comparison test (black stars) or Dunnett’s multiple comparison test (colored stars) using the WT as a reference. d, Ratio of mRNA/vRNA levels based on measurements in c shown as the mean ± s.d. (N = 4). *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA on log-transformed data with Dunnett’s multiple comparison test using the WT as a reference. e,f, Mutant viruses rescued by reverse genetics were characterized phenotypically by plaque measurement (e) and titration (f) and genotypically by full genome sequencing using Illumina sequencing (expected and actual genotypes are indicated). Each dot represents one plaque (e) or one titration (f), virus titer is depicted as mean ± s.d. (n = 3 technical replicates). *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA one-way ANOVA on log-transformed data with Dunnett’s multiple comparison test using the WT as a reference. Actual genotypes summarize mutations that occurred in the indicated segment and in more than 50% of the sequencing reads relative to WT. For detailed description see Table 1.