Figure 7.
FXRs promote viral replication. (A and B) Signals from the ribosome component RPL7a (A) and translation initiation factor EIF4G3 (B) are detected at FXR1 punctate structures after 24 h SARS-CoV-2 infection with MOI = 1. Images are representative of at least 10 cells. Bars: 5 μm; insets, 2 μm. (C) After 24 h SARS-CoV-2 infection with MOI=1, ACE2-HeLa cells were treated with 10 μg/ml puromycin for 30 min. Puromycin signals colocalize with FXR1 foci. Images are representative of at least 10 cells. Bar: 5 μm; inset, 2 μm. (D and E) Levels of viral subgenomic E RNA are significantly decreased in cells depleted of FXRs compared with control cells after SARS-CoV-2 infection with MOI = 0.1 (D) or MOI = 1 (E). Quantification data are presented as mean ± SD (n = 3). *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. (F) Puromycin incorporation assays with ACE2-HeLa cells show that signals from anti-puromycin antibody are dramatically suppressed upon SARS-CoV-2 infection for 12 h. After 24 h infection, slightly recovered anti-puromycin signals are detected in NC-treated but not siFXRs cells. Immunoblotting shows that viral NP levels are suppressed by siFXRs after 12 and 24 h SARS-CoV-2 infection with MOI = 0.1 or 0.5. (G) TEM images show clustered DMVs in control cells (NC), while DMVs (arrow) are only occasionally observed in siFXRs cells. Bars, 500 nm. (H) Immunoblotting shows that FXR1, FXR2, and FMR1 levels are dramatically increased after 8 h SARS-CoV-2 infection with MOI = 1. (I) The schematic model shows that LLPS of FXRs drives clustering of DMVs via interaction with the UBL1 and HVR domains of Nsp3, which further facilitates recruitment of translation machinery for viral replication. Source data are available for this figure: SourceData F7.

FXRs promote viral replication. (A and B) Signals from the ribosome component RPL7a (A) and translation initiation factor EIF4G3 (B) are detected at FXR1 punctate structures after 24 h SARS-CoV-2 infection with MOI = 1. Images are representative of at least 10 cells. Bars: 5 μm; insets, 2 μm. (C) After 24 h SARS-CoV-2 infection with MOI=1, ACE2-HeLa cells were treated with 10 μg/ml puromycin for 30 min. Puromycin signals colocalize with FXR1 foci. Images are representative of at least 10 cells. Bar: 5 μm; inset, 2 μm. (D and E) Levels of viral subgenomic E RNA are significantly decreased in cells depleted of FXRs compared with control cells after SARS-CoV-2 infection with MOI = 0.1 (D) or MOI = 1 (E). Quantification data are presented as mean ± SD (n = 3). *, P < 0.05; ***, P < 0.001; ****, P < 0.0001. (F) Puromycin incorporation assays with ACE2-HeLa cells show that signals from anti-puromycin antibody are dramatically suppressed upon SARS-CoV-2 infection for 12 h. After 24 h infection, slightly recovered anti-puromycin signals are detected in NC-treated but not siFXRs cells. Immunoblotting shows that viral NP levels are suppressed by siFXRs after 12 and 24 h SARS-CoV-2 infection with MOI = 0.1 or 0.5. (G) TEM images show clustered DMVs in control cells (NC), while DMVs (arrow) are only occasionally observed in siFXRs cells. Bars, 500 nm. (H) Immunoblotting shows that FXR1, FXR2, and FMR1 levels are dramatically increased after 8 h SARS-CoV-2 infection with MOI = 1. (I) The schematic model shows that LLPS of FXRs drives clustering of DMVs via interaction with the UBL1 and HVR domains of Nsp3, which further facilitates recruitment of translation machinery for viral replication. Source data are available for this figure: SourceData F7.

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