Figure 2: Knockdown of FMRP impairs influenza virus replication in cell culture and in mice.

(a) Protein levels of FMRP in FMRP-stable knockdown 293T cells (shFMRP) or control cells (scrambled). (b) Knockdown of FMRP specifically attenuates influenza virus replication in cell culture. shFMRP cells or control cells were infected with influenza A/WSN/33 (WSN) virus (MOI=0.01); supernatants were then harvested at the indicated time points and subjected to TCID₅₀ assays (mean±s.d. of three independent experiments, **P<0.01, two-tailed Student’s t-test). (c) H&E staining of the lungs of WT and FMRP^−/− mice infected with 10⁴ TCID₅₀ of PR8 virus on day 3 p.i. (magnification × 400). Scale bar, 100 μm. (d) Histological scoring of H&E-stained lung sections as shown in (c) (mean±s.d., *P<0.05, two-tailed Student’s t-test). WT (n=3), FMRP^−/− (n=3) and WT mock-infected (n=3). (e) Left: immunohistochemical detection of NP in the lungs of WT or FMRP^−/− mice infected with PR8 virus on day 3 p.i. (magnification × 400). Scale bar, 100 μm. Right: statistical analysis of positively immunostained cells (mean±s.d., n=3, *P<0.05, two-tailed Student’s t-test). (f) Viral lung titres of WT and FMRP^−/− mice on days 3 and 6 p.i. (mean±s.d., n=3, *P<0.05, two-tailed Student’s t-test, see also Supplementary Fig. 4). (g,h) Infected WT (n=11) and FMRP^−/− mice (n=23) were monitored for 14 days for weight loss (g) and survival (h). Control groups received PBS only (WT, n=4 and FMRP^−/−, n=4). Survival in FMRP^−/− mice was significantly enhanced compared with WT animals (mean±s.d., **P<0.01, two-tailed Student’s t-test). None of the PBS-inoculated control groups lost weight and all survived. Data are representative of two independent experiments.