Fig 2: Antiviral roles of RNase L and G3BP1 during SeV infection. (A and B) WT cells were infected with SeV (40 HAU/ml) for 24 h, fixed, and stained with G3BP1 and antibody against SeV (A), and avSGs were purified as described in Materials and Methods (B). The avSG core proteins were immunoprecipitated with G3BP1 antibody, and the immune complex was analyzed for the presence of PKR, Rig-I, OAS, and RNase L by immunoblot analysis. Pellet and SG core fractions were probed for expression of G3BP1 and PKR, Rig-I, OAS, and RNase L. Nonspecific lanes were cropped to generate the images, and the boundaries are indicated. The data are representative of results from two experiments. (C to E) WT, G3BP1 KO, RNase L KO, or U3A (STAT1-defective) cells were infected with SeV (40 HAU/ml) for the indicated times, and viral titers were estimated by determining copy numbers of SeV genomic RNA strands in supernatants by qRT-PCR, (F to J) Expression of SeV antigens was detected using anti-Sendai virus antibody (F and G), and IFN-β mRNA levels were measured by qRT-PCR and normalized to GAPDH mRNA levels (H to J). The data represent means + SE for the results of three independent experiments. *, P < 0.01; **, P < 0.001; ***, P < 0.0001; n.s, not significant.

Fig 3: PKR clusters are autonomous and recruit PB components. (A) Representative immunofluorescence images showing that mRuby-PKR clusters and G3BP1, an SG component, do not colocalize. Right: Plot of signal intensity of mRuby-PKR clusters (red) or G3BP1 immunostaining (green) as a function of distance. The ROI used for metrics is indicated with a white line on the micrograph crops. Scale bars: 10 µm. (B) Time-lapse micrographs showing colocalization and subsequent demixing of mRuby-PKR and GFP-Dcp1a, a PB component. Scale bar: 10 µm. (C) Quantification of the data in B (mean and 95% confidence interval bands; N = 3 experiments, n = 30 cells). (D) Violin plots showing the total number of cells with mRuby-PKR clusters after administration of poly I:C (blue), poly I:C and CHX (red), and poly I:C and 4E-T RNAi (green); N = 3 experiments, n > 2,000; one-way ANOVA. (E) Violin plots showing the number of mRuby-PKR clusters per cell over time in cells treated with poly I:C (blue), poly I:C and CHX (red), and poly I:C and 4E-T RNAi (green); N = 3 experiments, n > 200. (F) Representative micrographs showing that mRuby-PKR clusters recruit the PB component Edc3 after depletion of PBs with CHX and 4E-T RNAi. Scale bar: 10 µm.

Fig 4: Cytosolic L1 RNP foci are poised to be stress granules that co-localize with multiple small DDX6 foci AWT and Dicer_KO mESCs were treated with 0.5 mM sodium arsenite (NaAsO2) for 20 min or left untreated prior to fixation with formaldehyde. Maximum intensity projections across Z stacks of example images from indicated mESCs immunostained for G3BP1 (red) and MOV10 (green) with nuclei stained with DAPI (blue).BRepresentative Western blots showing low AGO2 protein levels in Dicer_KO as compared to WT mESCs (right side). LAMINB1 served as loading control. On the left, maximum intensity projections across Z stacks of example images from indicated mESCs stained for L1 Tf RNA FISH (red) combined with immunostaining for a resident protein of P-bodies, DDX6 (green) and nuclei stained with DAPI (blue). The gray square marks position of the inset. Yellow arrow heads point to cytoplasmic foci where L1 RNA and DDX6 protein co-localize. Data information: (A and B) are representative images of three independent experiments. Bar graphs represent mean ± SD. 94–150 cells per cell line were analyzed for each experiment. Scale bar 5 µm. Source data are available online for this figure.

Fig 5: Activation of RNase L induces antiviral stress granule formation. (A) HT1080 cells were transfected with 2-5A (10 μM) for 8 h, and RNase L-mediated cleavage of rRNA (arrows) was analyzed on RNA chips using the Agilent Bioanalyzer 2100; RIN, RNA integrity number. (B) Cells were fixed and stained with G3BP1 and the indicated antiviral proteins. The magnified images correspond to the boxed regions. (Right) Intensity profiles of G3BP1 and antiviral proteins along the plotted lines, as analyzed by Image J line scan analysis. (C) The percentages of cells forming stress granules were quantitated. (D) RNase L KO cells were either mock transfected or transfected with FLAG-WT-RNase L or FLAG-R667A-RNase L and immunostained for G3BP1 and FLAG. (Right) Intensity profiles of G3BP1 and FLAG along the plotted lines as analyzed by Image J line scan analysis. (E) HT1080 cells were treated with H2O2 (1 mM) for 3 h, and the percentages of cells forming stress granules were quantitated. (F) The cells were immunostained with G3BP1 and the indicated antiviral proteins. (Right) Intensity profiles of G3BP1 and antiviral proteins along the plotted lines as analyzed by Image J line scan analysis. All the experiments included at least 100 cells from three replicates. Scale bars, 10 μm. The data are representative of at least three independent experiments. *, P < 0.01; **, P < 0.001. A.U., arbitrary units.