Fig. 3: nsp6 expression remodels LD distribution, and promotes LD-DMV contact, facilitating lipid flux from LDs to DMV. | Nature Communications

Fig. 3: nsp6 expression remodels LD distribution, and promotes LD-DMV contact, facilitating lipid flux from LDs to DMV.

From: Coronaviral nsp6 hijacks ERAD machinery to facilitate lipolysis and supply membrane components for DMV growth

Fig. 3

a TEM of HeLa-mCEACAM1 cells treated with 200 μM OA (12 h) and infected with MHV-A59 (MOI = 5, 10 h). White arrowheads indicate LD-DMV contacts. Scale bar, 500 nm; * indicates DMV. b Colocalization analysis of LDs, nsp6, and DMVs. HeLa cells were co-transfected with GFP-nsp3-nsp4-HA and Flag-nsp6 and treated with OA for 12 h. Scale bar, 10 μm (n = 16 cells). c HeLa cells were co-transfected with GFP-nsp3 and HA-nsp4, together with or without mCherry-nsp6. The cells then were fixed and analyzed by TEM (n = 62 DMV structures for LD-DMV, n = 53 DMV structures for mito-DMV). Scar bar, 500 nm; * indicates DMV. d 17Cl-1 cells were pre-treated with 50 μM Atglistatin or CAY10499 overnight and then infected with MHV-A59 for 12 h. Scale bar, 20 μm. dsRNA MFI was analyzed using Image J software (n = 8 cells). e 17Cl-1 cells were infected with MHV-A59 at MOI of 0.01 for 2 h and then treated with Atglistatin or CAY10499 at indicated concentrations for 12 h. f Huh-7 cells were infected with HCoV-229E at MOI of 0.01 for 2 h and were treated with CAY10499 at indicated doses. (g) Vero-E6 cells were infected with SARS-CoV-2 at MOI of 0.1 for 2 h and then treated with CAY10499 with indicated doses for 48 h. h HeLa cells co-expressing GFP-nsp3-nsp4 and Flag-nsp6 (6 h) were treated with NG-497 (50 μM, 24 h), followed by a chase in serum-free DMEM (0.5 h) prior to confocal microscopy. Scale bar, 10 μm (n = 14 cells). i 17Cl-1 cells were infected with MHV (MOI = 5, 2 h) and treated with 50 μM CAY10499 (10 h) prior to processing for TEM. Scale bar, 500 nm; * indicates DMV (n = 15 cells). All data are representative of three independent experiments with similar results (a–i). Graphs are presented as mean with SD, N = 3 biological replicates in panel (e, f), 9 replicates (left) and 6 replicates (right) in (g). The unpaired two-tailed Student’s t test was applied. Inter-organelle distance was quantified using Velox software (v3.9.0). See also Supplementary Fig. 3. Source data are provided with this paper.

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