FXRs are recruited to DMV sites through binding to Nsp3. (A) The schematic diagram shows the experimental design for identifying DMV-related factors. Lysates from cells expressing Nsp4-GFP and mCherry-Nsp3 or Nsp4-GFP alone (serving as control) are subjected to GFP-Trap, followed by MS analysis. N, nucleus. (B) The volcano plot from the MS data demonstrates quantitative changes in proteins pulled down by Nsp4-GFP in Nsp3/4-expressing cells compared with cells expressing Nsp4-GFP alone. The horizontal dashed line shows where P value is 0.05 (−log 10 [0.05] = 1.3), and the vertical dashed lines show where the fold change is 2 (log 2 [2] = 1) or 0.5 (log 2 [0.5] = −1). These parameters were used as the threshold cutoff. Upregulated proteins are shown in red and downregulated proteins are shown in blue. FXR1 and FMR1 are labeled. (C) In an mCherry-Trap assay, FXR1, FXR2, and FMR1 are immunoprecipitated by mCherry-Nsp3. (D) In an mCherry-Trap assay, FXR1, FXR2, and FMR1 are not immunoprecipitated by Nsp4-mCherry. (E) The schematic shows the specific localization of Nsp3 and Nsp4 on DMVs and the Nsp3-mediated recruitment of FXRs to DMVs. (F) Endogenous FXR1, FXR2, and FMR1 colocalize well with Nsp3/4⁺ foci in mCherry-Nsp3/Flag-Nsp4-coexpressing HeLa cells. FXR1, FXR2, and FMR1 show diffuse cytoplasmic localization under control conditions. Images are representative of at least 10 cells. Bars: 5 μm; insets, 2 μm. (G) Endogenous FXR1 forms puncta that colocalize well with Nsp3 and dsRNA foci after 24 h SARS-CoV-2 infection with MOI = 1. Images are representative of at least 10 cells. Bar: 5 μm; inset, 2 μm. (H) Endogenous FXR2 and FMR1 form puncta that colocalize well with FXR1 and dsRNA foci after 24 h SARS-CoV-2 infection with MOI = 1. Images are representative of at least 10 cells. Bars: 5 μm; insets, 2 μm. Source data are available for this figure: SourceData F1.