Figure S5.
FXR1 droplets recruit translation factors and are important for viral proliferation, related toFigs. 6 and 7. (A) Ribosomes (magenta) partition into FXR1 liquid droplets (cyan). Bar: 5 μm. (B) Viral RNA (magenta) partitions into FXR1 liquid droplets (cyan). Bar: 5 μm. (C) Nsp3N (green) and viral RNA (magenta) partition into FXR1 liquid droplets (cyan). Bar: 5 μm. (D) Immunostaining with anti-RPS6 shows that ribosome component RPS6 is detected at FXR1 and dsRNA double-positive punctate structures 24 h after SARS-CoV-2 infection with MOI = 1. Images are representative of at least 10 cells. Bar: 5 μm; inset, 2 μm. (E) Control cells (NC) and siFXRs cells were infected with SARS-CoV-2-Spike–packaged pseudoviruses expressing luciferase. The pseudovirus entry efficiency is presented as luciferase activity normalized relative to control levels. N.S., not significant. (F) Compared with FXRs KD cells, reintroducing RNAi-resistant FXR1 significantly increases NP levels after SARS-CoV-2 infection with MOI = 0.5 for 24 h. Quantifications of levels of NP (normalized by Actin levels) are shown. (G–J) Compared with control cells (NC), viral N (G and I) and Spike (H and J) RNA levels are significantly decreased in FXRs KD cells after SARS-CoV-2 infection with MOI = 0.1 (G and H) or MOI = 1 (I and J). Quantitative data are presented as mean ± SD (n = 3). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (K)FXR1, FXR2, and FMR1 mRNA levels are efficiently depleted by siFXRs. Quantitative data are presented as mean ± SD (n = 3). (L–N) Transcription levels of FXR1 (L), FXR2 (M), and FMR1 (N) are dramatically increased upon SARS-CoV-2 infection with MOI = 1. (O) Amino acid alignment of the UBL1 and HVR domains of Nsp3 from SARS-CoV-2, SARS-CoV, MERS, hCoV-229E, and hCoV-OC43. The alignment was generated using SnapGene. Identical residues are indicated with green boxes. The black lines above the SARS-CoV-2 Nsp3 sequence show the two FXR1 interaction regions. (P) GFP-FXR1 colocalizes well with Nsp3/4+ puncta in cells coexpressing mCherry-U+H(SARS)+3C with Flag-Nsp4 (cyan). Images are representative of at least 10 cells. Bar: 5 μm; inset, 2 μm. (Q) In an mCherry-Trap assay, levels of endogenous FXR1 immunoprecipitated by mCherry-U+H(SARS)+3C are comparable with those precipitated by mCherry-Nsp3. (R) Myc-FXR1 (magenta) puncta colocalize well with GFP-nsP3(VEEV) foci. Images are representative of at least 10 cells. Bar: 5 μm; inset, 2 μm. (S and T) Formation of GFP-nsP3(VEEV) foci is comparable in NC and siFXRs-treated cells (S). Images are representative of at least 10 cells. Quantitative data are presented as mean ± SD (NC, n = 32; siFXRs, n = 43) (T). Bars: 5 μm. Source data are available for this figure: SourceData FS5.

FXR1 droplets recruit translation factors and are important for viral proliferation, related to Figs. 6 and 7. (A) Ribosomes (magenta) partition into FXR1 liquid droplets (cyan). Bar: 5 μm. (B) Viral RNA (magenta) partitions into FXR1 liquid droplets (cyan). Bar: 5 μm. (C) Nsp3N (green) and viral RNA (magenta) partition into FXR1 liquid droplets (cyan). Bar: 5 μm. (D) Immunostaining with anti-RPS6 shows that ribosome component RPS6 is detected at FXR1 and dsRNA double-positive punctate structures 24 h after SARS-CoV-2 infection with MOI = 1. Images are representative of at least 10 cells. Bar: 5 μm; inset, 2 μm. (E) Control cells (NC) and siFXRs cells were infected with SARS-CoV-2-Spike–packaged pseudoviruses expressing luciferase. The pseudovirus entry efficiency is presented as luciferase activity normalized relative to control levels. N.S., not significant. (F) Compared with FXRs KD cells, reintroducing RNAi-resistant FXR1 significantly increases NP levels after SARS-CoV-2 infection with MOI = 0.5 for 24 h. Quantifications of levels of NP (normalized by Actin levels) are shown. (G–J) Compared with control cells (NC), viral N (G and I) and Spike (H and J) RNA levels are significantly decreased in FXRs KD cells after SARS-CoV-2 infection with MOI = 0.1 (G and H) or MOI = 1 (I and J). Quantitative data are presented as mean ± SD (n = 3). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. (K)FXR1, FXR2, and FMR1 mRNA levels are efficiently depleted by siFXRs. Quantitative data are presented as mean ± SD (n = 3). (L–N) Transcription levels of FXR1 (L), FXR2 (M), and FMR1 (N) are dramatically increased upon SARS-CoV-2 infection with MOI = 1. (O) Amino acid alignment of the UBL1 and HVR domains of Nsp3 from SARS-CoV-2, SARS-CoV, MERS, hCoV-229E, and hCoV-OC43. The alignment was generated using SnapGene. Identical residues are indicated with green boxes. The black lines above the SARS-CoV-2 Nsp3 sequence show the two FXR1 interaction regions. (P) GFP-FXR1 colocalizes well with Nsp3/4+ puncta in cells coexpressing mCherry-U+H(SARS)+3C with Flag-Nsp4 (cyan). Images are representative of at least 10 cells. Bar: 5 μm; inset, 2 μm. (Q) In an mCherry-Trap assay, levels of endogenous FXR1 immunoprecipitated by mCherry-U+H(SARS)+3C are comparable with those precipitated by mCherry-Nsp3. (R) Myc-FXR1 (magenta) puncta colocalize well with GFP-nsP3(VEEV) foci. Images are representative of at least 10 cells. Bar: 5 μm; inset, 2 μm. (S and T) Formation of GFP-nsP3(VEEV) foci is comparable in NC and siFXRs-treated cells (S). Images are representative of at least 10 cells. Quantitative data are presented as mean ± SD (NC, n = 32; siFXRs, n = 43) (T). Bars: 5 μm. Source data are available for this figure: SourceData FS5.

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