Fig 2: Domains involved in the G3BPs-NPSARS-CoV-2 association. (A) Schematic diagram of the domains of G3BP1 and SARS-CoV-2 NP. IDR, intrinsically disordered region; RBD, RNA-binding domain. (B) 293T cells were cotransfected with NP (SARS-CoV-2) and G3BP1 or truncated mutants, and immunoprecipitation and immunoblotting were then performed with the indicated antibodies. (C and D) 293T cells coexpressing G3BP1 and NPSARS-CoV-2 or truncated mutants were subjected to immunoprecipitation and immunoblotting with the indicated antibodies.

Fig 3: Differential association between SARS-CoV-2 N protein and G3BP1/2 in Vero and Calu-3 cells (A–D) Vero and Calu-3 cells were mock-infected or infected with SARS-CoV-2 S clade at 0.1 MOI; cell lysates were immunoprecipitated with anti-SARS-CoV-2 N mAb and analyzed by western blot with anti-SARS-CoV-2 N Ab (A), anti-G3BP1 antibody (B), anti-G3BP2 antibody (C), or anti-β-actin antibody, as the input control (D). (E) The band intensities of G3BP1 and G3BP2 were normalized with those of β-actin and the relative band intensities of Co-IPed proteins are shown on the graph. Anti-N mAb, anti-SARS-CoV-2 N mAb (clone 1G10C4 mAb); Anti-N Ab, anti-SARS-CoV-2 N polyclonal Ab.

Fig 4: SARS-CoV-2 NP suppresses G3BP-mediated SG formation. (A) A549 cells were treated with 500 μM sodium arsenite (SA) for 1 h to induce SG assembly, and saline was used as a control. G3BP1 (green) and G3BP2 (red) were labeled to visualize SGs by immunofluorescence staining. The nuclei were stained with DAPI (blue). (B) A549 cells expressing GFP-tagged NPSARS-CoV-2 or GFP-vector were treated with 500 μM SA for 1 h to induce SG assembly. TIA1 (red) and G3BP1 (violet) were labeled to visualize SGs by immunofluorescence staining. The nuclei were stained with DAPI (blue). (C) The average SG number per cell (≥10) in panel B was calculated using ImageJ software. All quantitative data are shown as the means ± SDs. **, P < 0.05. (D) A549 cells transfected with or without a G3BP1/G3BP2 plasmid were infected with SARS-CoV-2 for 24 h and treated with or without 500 μM SA during the last hour. NPSARS-CoV-2 (green) and the SG marker G3BP1 (red) were detected via immunofluorescence staining. The nuclei were stained with DAPI (blue). (E) The average SG number per cell (≥10) in panel D was calculated using ImageJ software. All quantitative data are shown as the means ± SDs. **, P < 0.05.

Fig 5: Interaction between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid (N) protein and Ras-GTPase-activating SH3-domain-binding protein 1 (G3BP1) (A) Co-immunoprecipitation (Co-IP) experiments to identify N protein-binding partners. Calu-3 cells were mock-infected or infected with SARS-CoV-2 S clade at 0.1 multiplicity of infection (MOI); lysates prepared at 72 h post-infection were immunoprecipitated with control antibody (normal IgG) or anti-SARS-CoV-2 N monoclonal antibody (mAb). HC, heavy chain. LC, light chain. (B) Identification of G3BP1. The ∼52.1 kDa protein band interacting with SARS-CoV-2 N protein in Co-IP experiments (A) was in-gel digested with trypsin, and the 18 peptides were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). MS/MS analyses of mass peaks (arrows) reveal the peptide spectra of G3BP1. (C) Co-IP experiments to confirm interaction between SARS-CoV-2 N protein and G3BP1. Co-IPed immunocomplexes from panel (A) were subjected to western blot analysis with the indicated antibodies; anti-β-actin antibody was used as the input control. IP, immunoprecipitation; WB, western blot; Anti-N mAb, anti-SARS-CoV-2 N mAb (clone 1G10C4 mAb); Anti-N Ab, anti-SARS-CoV-2 N polyclonal Ab.