Fig. 6

SARS-CoV-2 nucleocapsid protein LLPS is crucial for inhibition of cGAS-mediated IFN-I signaling. a HEK-293T cells were expressed with Flag-GFP-cGAS, HA-G3BP1, along with Myc-N-FL, △NTD, △SR or △CTD. Cell lysates were collected, immunoprecipitated with Flag-beads, followed by immunoblotting. b Calu3 cells expressed with Flag-N full length (FL), △NTD, △SR or △CTD were stimulated with biotin-ISD (2 μg/mL) for 2 h before harvest. Cell lysates were collected, immunoprecipitated with NeutrAvidin agarose resin, followed by immunoblotting analysis. c Calu3 cells expressed with Flag-N FL, △NTD, △SR or △CTD were transfected with mCherry plasmid (2 μg/mL) as plasmid DNA for 2 h before harvest. Cell lysates were collected, immunoprecipitated with A + G beads together with cGAS antibody, followed by qRT-PCR analysis of extracted DNA to detect cGAS-bound plasmid DNA (mCherry) abundance. d In vitro cGAMP processing ability of recombinant cGAS (10 μM) incubated with recombinant G3BP1 (10 μM), recombinant N protein (5 μM) or recombinant N-△CTD (5 μM) and ISD (2 μM), along with ATP. (e) Immunoblotting analysis of N protein-inducible Calu3 cells or N-△CTD-inducible Calu3 cells treated with Dox (200 ng/mL) along with ISD (2 μg/mL) stimulation for indicated time points. f HEK-293T cells were transfected with IFNβ-luc, TK-luc along with increasing concentration of FL N protein or N-△CTD. Cell lysates were collected and luciferase analysis was performed. Data in c, d and f were expressed as mean ± SEM of 3 independent biological experiments. *P < 0.05, ****P < 0.0001, ns not significant (unpaired two-tailed student’s t-test). Similar results were obtained for 3 independent biological experiments in a, b, e