Fig. 3 | Nature Communications

Fig. 3

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

Fig. 3

Attenuating mutations in segment 5 impact coordinated genome packaging. a Confocal microscopy images depicting the localization of nucleoprotein (NP) 8 h post infection (hpi). MDCK cells were infected with the indicated virus (white text), and NP was identified using the MAb HB65 and Alexa-488-conjugated goat-anti-mouse secondary antibody by immunofluorescence. Cell nuclei are stained with DAPI. Panels depict the merged image of DAPI and NP staining. Fields are representative of two independent experiments. Scale bar represents 25 µm. b Dual-luciferase reporter assay to assess viral transcription and genome replication, each combination of plasmids was assessed 3–5 times with corresponding the WT-PR8 combination. (n.s., not significant by one-way ANOVA with multiple comparisons correction (Kruskal–Wallis)). c Mean fluorescence intensity (MFI) of NP in virally infected MDCK cells (MOI = 0.05, experiments were performed twice in duplicate) as revealed by flow cytometry. (n.s., not significant by one-way ANOVA with multiple comparisons correction (Kruskal–Wallis)). d Viral titer (TCID50 ml−1) of 4 HA-units of WT-PR8 and mutant viruses. Results are the average + s.e.m. of three viral titrations and two HA assays per infectious virus titration. (*P < 0.05; n.s., not significant by unpaired t test). e Relative abundance of genome segments in purified WT or mutant viruses. All segments were compared to segment 7 (M) 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. (*P < 0.05; n.s., not significant by one-way ANOVA with multiple comparisons correction (Kruskal–Wallis)) f Proportion of infected cells co-expressing of matrix (M) and NP proteins in singly infected MDCK cells (MOI = 0.05) 16 hpi as revealed by flow cytometry. The average percentage of co-expression was calculated from two experiments performed in duplicate. (n.s., not significant by one-way ANOVA with multiple comparisons correction (Kruskal–Wallis))

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