Fig. 4

SARS-CoV-2 nucleocapsid protein suppresses cGAS-G3BP1 interaction. a Calu3 cells were infected with SARS-CoV-2 (MOI = 0.1) for 0 or 24 h. Cell lysates were collected, immunoprecipitated with A + G beads along with cGAS antibody, followed by immunoblotting analysis. b N protein-inducible Calu3 cells transfected with scramble (SCR) siRNA or G3BP1 siRNA #1, were treated with increasing concentration of Dox (200 ng/mL and 400 ng/mL) for 24 h with biotin-ISD treatment for 2 h before harvest. Cell lysates were collected, immunoprecipitated with NeutrAvidin agarose resin, followed by immunoblotting analysis. c N protein-inducible Calu3 cells were treated with Dox (200 ng/mL) or left untreated. Cell lysates were collected, immunoprecipitated with A + G beads together with cGAS antibody, followed by immunoblotting analysis. d–f Representative images of confocal microscopy in HeLa cells expressing GFP-cGAS, mCherry-N and CFP-G3BP1 with 100 bp dsDNA stimulation (2 μg/mL) for 12 h before harvest (d). Scale bars indicated 10 μm. The number of GFP-cGAS condensates (e) were analyzed from n = 20 cells and the size of GFP-cGAS condensates per cell (f) were measured from n = 30 condensates. g–i Representative images of confocal microscopy of recombinant GFP-cGAS (10 μM, green), CFP-G3BP1 (10 μM, cyan), mCherry-N (5 μM, red) and Cy5-ISD (2 μM, magenta) incubated in LLPS buffer at 37 °C (g). Scale bars indicated 10 μm. The size of GFP-cGAS condensates (h) was measured from n = 200 condensates and the proportion of cGAS-G3BP1 co-localized condensates/cGAS condensates (i) were analyzed from n = 5 views. j Schematic figure of domain organization of G3BP1 and its domain deletion mutant. k HEK-293T cells were transfected with Flag-empty vector (EV) or Flag-GFP-cGAS along with HA-EV, HA-G3BP1 full length (FL), △RBD, IDR and △NTF2L. Cell lysates were collected, immunoprecipitated with Flag-beads, followed with immunoblotting analysis. l HEK-293T cells were transfected with Flag- EV or Flag-N along with HA-EV, HA-G3BP1 FL, △RBD, IDR and △NTF2L. Cell lysates were collected, immunoprecipitated with Flag-beads, followed with immunoblotting analysis. Data in e, i were expressed as mean ± SD of indicated samples for each condition. Data in f, h were expressed as median and quartiles of indicated samples for each condition. ****P < 0.0001, ns not significant (unpaired two-tailed student’s t-test). Similar results were obtained for 3 independent biological experiments in a–d, g, k, l