DMV clustering depends on the UBL1 and HVR domains in Nsp3. (A) The schematic shows the domain organization of Nsp3 deletion mutants. (B and C) Cells with coexpression of mCherry-Nsp3(ΔUBL1), Nsp3(ΔHVR), or Nsp3(ΔUBL1ΔHVR) and Nsp4-GFP contain a large number of small Nsp3/4⁺ puncta that fail to colocalize with endogenous FXR1, while full-length mCherry-Nsp3 forms big puncta with Nsp4, which colocalize well with FXR1 foci (C). Numbers of DMVs are shown as mean ± SD (mCherry-Nsp3, n = 33; mCherry-Nsp3(ΔUBL1), n = 22; mCherry-Nsp3(ΔHVR), n = 30; mCherry-Nsp3(ΔUBL1ΔHVR), n = 41) (B). ****, P < 0.0001. Bars: 5 μm; insets, 2 μm. (D) TEM images show that coexpressing Nsp3(ΔUBL1), Nsp3(ΔHVR), or Nsp3(ΔUBL1ΔHVR) with Nsp4 induces DMV dispersion throughout the cytoplasm, while clustered DMVs are present in cells expressing full-length (FL) Nsp3 and Nsp4. Bars: 500 nm; insets, 200 nm. (E) In a GFP-Trap assay, GFP-FXR1 immunoprecipitates full-length mCherry-Nsp3, but not mCherry-Nsp3(ΔUBL1), mCherry-Nsp3(ΔHVR), or mCherry-Nsp3(ΔUBL1ΔHVR). (F) Deleting either the UBL1 or HVR domain of Nsp3N slightly reduces the entry of Nsp3 into FXR1 condensates, while deleting both domains completely eliminates Nsp3 from FXR1 droplets. Bars: 5 μm. Source data are available for this figure: SourceData F4.