The assembly of stress granules during foot-and-mouth disease virus infection is uncoupled from activation of cellular intrinsic antiviral signalling

Figures

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  Fig 1. Inhibiting G3BP1 condensation blocks poly(I:C)-induced foci. (A) PK-15 cells transfected with increasing concentrations of poly(I:C) for 6 hours. Sodium arsenite treatment (1 mM for 1 hour) was used as positive control. Cells were analysed by immunofluorescence for the SG markers G3BP1 (gold) and eIF4GI (magenta), and a F-actin marker phalloidin (cyan). Nuclei were stained with DAPI. Scale bars represent 10 μm. (B) The percentage of cells displaying G3BP1-foci was quantified by manual counting of at least 100 cells per replicate. The average size of foci per cell and the number of foci per cell were quantified using the G3BP1 channel and ‘Analyse Particles’ plugin from ImageJ. Results shown as mean ± SEM, n=3, representing the average of at least 30 cells per condition. **p<0.01, *** p<0.001, ****p<0.0001; using ordinary one-way ANOVA with Šidák’s multiple comparison post-test to 50 ng poly(I:C). (C) PK-15 cells transfected with poly(I:C) for 6 hours following treatment with G3BP1 condensation inhibitor (G3Ib) or its enactive enantiomer (G3Ib’). Cells were analysed by immunofluorescence for the SG markers G3BP1 (gold), innate immune response activation markers IRF3 or IRF1…

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  Fig 2. FMDV induces the formation of transient G3BP1-foci during infection. (A-B) PK-15 cells were infected with FMDV for 3.5 hours. Sodium arsenite treatment (1 mM, 1 hour) and poly(I:C) transfection (500 ng) were used as positive controls. Cells were analysed by immunofluorescence for the SG markers G3BP1 (gold) and FMDV 3A (magenta). Nuclei were stained with DAPI. Scale bars represent 10 μm. The percentage of cells displaying G3BP1-foci was quantified by manual counting of cells of at least 100 cells per replicate. The average size of foci per cell and the number of foci per cell were quantified using the G3BP1 channel and ‘Analyse Particles’ plugin from ImageJ. Results shown as mean ± SEM of at least three independent biological replicates, representing the average of at least 30 cells per condition. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; using ordinary one-way ANOVA with Šidák’s multiple comparison post-test. (C) Representative western blot from at least three independent experiments for eIF4GI and G3BP1 cleavage, viral infection (FMDV 3A), and integrated stress response activation (phosphorylation of eIF2α and puromycin). Molecular weights are indicated on the right.…

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  Fig 3. Preventing G3BP1 condensation does not interfere with FMDV replication. (A-C) PK-15 cells were infected with FMDV for 3.5 hours following treatment with G3BP1 condensation inhibitor (G3Ib) or its enactive enantiomer (G3Ib’). (A) Cells were analysed by immunofluorescence for the SG markers G3BP1 (gold) and FMDV 3A (magenta). Nuclei were stained with DAPI. Scale bars represent 10 μm. (B) The percentage of cells displaying G3BP1-foci was quantified by manual counting of at least 100 cells per replicate. The average size of foci per cell and the number of foci per cell were quantified using the G3BP1 channel and ‘Analyse Particles’ plugin from ImageJ. Results shown as mean ± SEM of at least three independent biological replicates, representing the average of at least 30 cells per condition. *p<0.05; **p<0.01; ***p<0.001; using ordinary one-way ANOVA with Šidák’s multiple comparison post-test. (C) Representative western blot from at least three independent experiments for eIF4GI and G3BP1 cleavage, viral infection (FMDV 3A), and integrated stress response activation (phosphorylation of eIF2α and puromycin). Molecular weights are indicated on the right. (D) PK-15 cells were infected…

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  Fig 4. Preventing G3BP1 condensation does not interfere with the activation of interferon response during FMDV infection. (A-B) PK-15 cells were infected with FMDV or LL-FMDV for 3.5 hours and analysed by immunofluorescence for the SG markers G3BP1 (gold) and FMDV 3A (magenta). Nuclei were stained with DAPI. Scale bars 10 μm. (B) The percentage of cells displaying G3BP1-foci in infected (i) or bystander (b) cells was quantified by manual counting of at least 100 cells per replicate. The average foci size and the number of foci per cell were quantified using the G3BP1 channel and ‘Analyse Particles’ plugin from ImageJ. Results shown as mean ± SEM of at least three independent biological replicates, representing the average of at least 30 cells per condition. *p<0.05; **p<0.01; ns: non-significant; using ordinary one-way ANOVA with Šidák’s multiple comparison post-test. (C-D) PK-15 cells were infected with LL-FMDV for 3.5 hours following treatment with G3BP1 condensation inhibitor (G3Ib) or its enactive enantiomer (G3Ib’). The analysis was done as in (B). (E) Representative western blot from at least three independent experiments for innate immune response (STAT1 and IRF3…

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  Fig 5. FMDV-induced biocondensates formation are dependent on translation inhibition. PK-15 cells were treated with 1 mM sodium arsenite for 1 h, transfected with 500 ng poly(I:C), or infected with FMDV or LL-FMDV for 3.5 hours, following by a 30-minute treatment with 25 µg/ml cycloheximide treatment prior to harvesting. (A) Cells were analysed by immunofluorescence for the SG markers G3BP1 (gold) and viral protein 3A (magenta). Nuclei were stained with DAPI. Scale bars represent 10 μm. (B) The percentage of cells displaying G3BP1-foci was quantified by manual counting of at least 100 cells per replicate. The average size of foci per cell and the number of foci per cell were quantified using the G3BP1 channel and ‘Analyse Particles’ plugin from ImageJ. Results shown as mean ± SEM of at least three independent biological replicates, representing the average of at least 30 cells per condition. **p < 0.01; ***p < 0.001; ****p < 0.0001; ns: non-significant; using ordinary one-way ANOVA with Šidák’s multiple comparison post-test.

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  Fig 6. Preventing SGs disassembly impairs FMDV replication. (A-C) PK-15 cells were infected with FMDV for 3.5 hours with or without 6.25 µM raloxifene. (A) Cells were analysed by immunofluorescence for the SG markers G3BP1 (gold) and viral protein 3A (magenta). Nuclei were stained with DAPI. Scale bars represent 10 μm. (B) The percentage of cells displaying SGs was quantified by manual counting of cells with G3BP1-foci of at least 100 cells per replicate. The average size of foci per cell and the number of foci per cell were quantified using the G3BP1 channel and ‘Analyse Particles’ plugin from ImageJ. Results shown as mean ± SEM of at least three independent biological replicates, representing the average of at least 30 cells per condition. *p < 0.05; ***p < 0.001; ****p < 0.0001; using ordinary one-way ANOVA with Šidák’s multiple comparison post-test. (C) PK-15 cells were infected with FMDV at a MOI of 0.01 with increasing doses of raloxifene, alongside a mock-infected control, and cell confluence was monitored every hour for 24 hours using an IncuCyte Zoom. Data represents an average of three replicates, normalized to the first time-point measured (1 hpi). (D) Representative western…

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  Fig 7. G3BP1 resistant to 3Cpro cleavage forms persistent SG during infection. (A-B) PK-15 cells were co-transfected with 500 ng of G3BP1 WT or mutant constructs and 500 ng 3Cpro or Lpro for 24 h or (B) transfected with G3BP1 constructs for 24 h and infected with FMDV for an additional 6 h. (A-B) Representative western blot for G3BP1 cleavage following (A) transfection with FMDV proteases or (B) infection. Molecular weights are indicated on the right. (C) PK-15 cells transfected with 3Cpro or Lpro, or it’s catalytically inactive forms (3Cpro-X or Lpro-X), and treated with sodium arsenite (1 mM for 1 h) were imaged for the SG markers G3BP1 (gold) and Caprin1 (cyan), and HA-tagged viral proteins (magenta). White arrows indicate transfected cells. Nuclei were stained with DAPI. Scale bars represent 10 μm. (D) The percentage of cells displaying G3BP1-foci was quantified by manual counting of at least 30 transfected cells per replicate. ***p < 0.001; ns: non-significant; using ordinary one-way ANOVA with Šidák’s multiple comparison post-test. (E) Immunoblotting showing G3BP1 cleavage the same conditions as in (C). (F) Cells were transfected with 500 nm of G3BP1 WT or E284A following by…