Fig. 1 | Nature Communications

Fig. 1

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

Fig. 1

Development of PAR-CLIP for IAV NP. a Schematic for IAV NP PAR-CLIP assay. b Effects of 4-SU on IAV replication. Viral replication (MOI = 0.1) in the presence or absence of 4-SU (100 μM) was assessed by performing a growth curve at the indicated times in 293T cells and titered by TCID50 assay in MDCK cells (bottom). Results are the average + s.e.m. of two experiments. NP localization was assessed following treatment and infection of 293T cells by confocal microscopy (top). Immunofluorescence staining for NP (green) was assessed in the presence or absence of 4-SU and counterstained with DAPI to identify cellular nuclei (blue). Scale bar indicates 25 µm. c PAR-CLIP was conducted on 293T cells infected with WT-PR8 in the absence (lanes 1 and 2) or presence of a monoclonal antibody against to IAV NP (lanes 3–6) or viral hemagglutinin protein (HA) (lanes 7–8). The effect of UV cross-linking on binding of RNA to the viral NP is shown in lanes 3–4 (no UV) and lanes 5–6 (with UV). Radioactivity (32P) is visualized by autoradiograph and the presence or absence of NP and cellular ß-actin was done by western blot. The input sample and eluate are loaded in the uneven and even lanes, respectively. The results are representative of four independent experiments. Original western blots and autoradiograph are shown in Supplementary Fig. 7. d Proportion of PAR-CLIP or RNA-seq derived reads mapping to human or IAV genomes (**P < 0.01 by one-way ANOVA with multiple comparisons correction (Kruskal–Wallis test), n = 4). e Length of negative-sense viral RNA (vRNA) aligning reads was determined using FastX Toolkit and the number of reads of a certain length is plotted as a proportion of total vRNA mapping reads. Mean (black line) ± s.e.m. (blue shading) of 4 experiments. f Nucleotide composition of low-NP-binding regions and IAV genome (displayed as average + s.e.m. base composition of all eight gene-segments). No significant differences were detected between groups (one-way ANOVA with multiple comparisons correction (Kruskal–Wallis test))

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