Fig 2: Infection with RSV Induces Cytoplasmic FUS Assemblies and Is Toxic in Mutant FUS-Expressing Cells(A) RSV infection of SH-SY5Y cells leads to the appearance of clusters of cells with swollen nuclei and cytoplasmic SGs. Representative images of WT cells 24 h post-infection are shown.(B) Upregulation of antiviral markers in RSV-infected WT cells as analyzed by qRT-PCR. n = 4, ****p < 0.0001 (Student’s t test).(C) RSV-inoculated cells develop FUS-positive SGs (?NLS4_ho and ?NLS10_het lines) and FAs (?NLS11_het line). Cells were analyzed 24 h post-infection.(D) More cells develop SGs in FUS?NLS cultures compared with WT cells upon RSV infection. The proportion of SG-containing cells was quantified 24 h after infection. *p < 0.05, ****p < 0.0001 (one-way ANOVA with Dunnett’s test).(E) RSV infection is more toxic for FUS?NLS lines compared with WT cells. Equal numbers of cells were seeded on coverslips. Cells were fixed for analysis at the indicated time points. FUS-positive SGs are still detectable in RSV-infected FUS?NLS lines 48 h post-infection (insets).(F) RSV-infected human patient fibroblasts bearing P525L mutation develop FUS-positive SGs 24 h post-inoculation.(G) FUS mRNA is upregulated in WT and FUS?NLS cells in response to RSV infection. FUS expression was analyzed by qRT-PCR 24 h post-inoculation. n = 4, *p < 0.05 (Student’s t test).(H) Mutant FUS assemblies contain optineurin and nucleocytoplasmic transport factors TNPO1 and Nup107. Cells were fixed and stained 24 h post-inoculation.In all panels, cells were infected with RSV strain A2 at a multiplicity of infection (MOI) of 10 and analyzed at the indicated time points. In (B), (D), and (G), data are represented as mean ± SEM. In (A), (C), (E), (F), and (H), representative images are shown. Scale bars, (A) 50 µm; (C), (F), and (H) 10 µm; and (E) 100 µm.