Fig. 4 | Nature Communications

Fig. 4

From: Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication

Fig. 4

Synonymous structural mutations in low-NP-binding regions in Segments 1, 2, and 8 attenuated virus replication and genome packaging. ac Segment 1 (a, blue line), 2 (b, red line), and 8 (c, green line) low-NP-binding regions defined by PAR-CLIP analysis. Normalized coverage ± s.e.m. was determined for each nucleotide in the PAR-CLIP (blue, red or green symbols) and RNA-seq (black symbols) libraries (n = 4 each). df Relative focus area of WT-PR8 and segment 1, 2, and 8 mutant viruses. Results are the average focus area + s.e.m. of 3 experiments per virus (>60 foci each). Statistical significance was determined by one-way ANOVA with a multiple comparisons correction (Kruskal–Wallis test). **P < 0.01; *P < 0.05. gi Viral titer (TCID50 ml−1) of 4 HA-units of WT-PR8 and mutant virus. Results are the average of three viral titrations and two HA assays per infectious virus titration (TCID50 assay and HA titration experiments, mean + s.e.m). Statistical significance was determined by unpaired t test. **P < 0.01; *P < 0.05. j Relative abundance of genome segments in purified WT or mutant viruses. All segments were compared to segment 1 (PB2) vRNA and normalized to the average of WT-PR8 values using the 2-ddCt method22. Bars represent the mean of 3–6 independent virus preparations + s.e.m. and statistical significance was determined by one-way ANOVA with a multiple comparisons correction (Kruskal–Wallis test). *P < 0.05. k C57BL/6J mice were inoculated with 103 TCID50 in 30 µL. Lungs were collected 48 hpi, homogenized, and titered. Each dot is a single mouse and the line is the median. Dotted line in k represents the limit of detection and statistical significance was determined by one-way ANOVA with a multiple comparisons correction (Kruskal–Wallis test). (***P < 0.005, *P < 0.05)

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