Fig. 2

Manipulation of vRNA in low-NP-binding regions that disrupts predicted RNA structure attenuates virus replication. a Normalized coverage ± s.e.m. was determined for each nucleotide in the PAR-CLIP (red symbols) and RNA-seq (black symbols) libraries (n = 4 each). Six regions of interest (ROI) in segment 5 (NP) are highlighted, including two low-NP-binding regions (NP_1410–1495 and NP_22–68) and four intermediate NP-binding regions (NP_145–175, NP_456–490, NP_584–608, and NP_1058–1081). Each ROI is indicated with a colored bar. b Focus area of WT-PR8 and NP mutant IAV. MDCK cells were infected with serial dilutions of the indicated viruses and overlayed with infection media (M0.1B) containing 1% agarose and TPCK-trypsin. Seventy-two hours later cells were fixed, permeabilized, and stained for viral antigen (NP). Focus diameter was determined and normalized to WT-PR8 per experiment. Results are the average + s.e.m. of 3–5 experiments per virus (>60 foci each). c MDCK cells were infected with MOI = 0.001 of the indicated viruses and culture supernatant was collected at indicated time points then titered on MDCK cells. Results are the average ± s.e.m. TCID₅₀ ml⁻¹ of two experiments performed in duplicate. d C57BL/6J mice were inoculated with 10³ TCID₅₀ of the indicated viruses in 30 µL. Lungs were collected, homogenized, and titered on MDCK cells. Each dot is a single mouse and the line is the median. Dotted line in c, d represents the limit of detection. (***P < 0.005; **P < 0.01; n.s. not significant by one-way ANOVA with multiple comparisons correction (Kruskal–Wallis test))