The region between the KH2 and IDR domains in FXR1 is essential for FXR1-Nsp3 interaction. (A) The schematic shows the domain organization of FXR1. The Nsp3-interacting region (NIR) is located between the KH2 and IDR1 domains. (B and C) Alphafold predictions show that Nsp3 (green) interacts with FXR1 (cyan) via an α-helix (aa V68–I77) in the UBL1 domain (B) and a β-strand (aa 142–146) in the HVR domain (C). Key residues are labeled in the images. (D and E) Cells cotransfected with mCherry-Nsp3(E70A/L71A) or mCherry-Nsp3(Δ142–146) and Nsp4 show a huge abundance of small puncta that fail to colocalize with endogenous FXR1, while WT mCherry-Nsp3 and Nsp4 form big dots and recruit FXR1 (D). Numbers of DMVs are shown as mean ± SD [mCherry-Nsp3, n = 33; mCherry-Nsp3(E70A/L71A), n = 37; mCherry-Nsp3(Δ142–146), n = 30] (E). ****, P < 0.0001. Bars: 5 μm; insets, 2 μm. (F) In a GFP-Trap assay, GFP-FXR1 immunoprecipitates dramatically reduced levels of mCherry-Nsp3(E70A/L71A) compared to WT mCherry-Nsp3. (G) In a GFP-Trap assay, GFP-FXR1(I355A/L358A/K359A) immunoprecipitates dramatically reduced levels of mCherry-Nsp3 compared to WT GFP-FXR1. (H) In a GFP-Trap assay, the interaction between Nsp3 and FXR1 is inhibited by expression of mCherry-Nsp3(Δ142–146) or FXR1(R315D/R317D). Source data are available for this figure: SourceData F5.