RNA-induced liquid phase separation of SARS-CoV-2 nucleocapsid protein facilitates NF-κB hyper-activation and inflammation

Fig. 1. N protein of SARS-CoV-2 enhances SARS-CoV-2-triggered NF-κB signaling. a Volcano plot of DEGs comparing SARS-CoV-2 infection cells versus negative control (NC). Calu3 cells were infected SARS-CoV-2 at MOI = 0.05 for 24, 48, and 72 h. RNA-seq was performed on poly(A)-enriched total RNA. ns not significant. b Dot-plot visualization of enriched GO terms showing the upregulated genes from (a). The color of the dots represents the p-value for each enriched GO term, and the size represents the reads count of genes enriched in the total gene set. c IGV browser tracks showing the RNA-seq signals of inflammatory response genes in Calu3 cells at indicated time points post infection. d Quantitative PCR with reverse transcription analysis of NF-κB relative cytokines genes (TNF, IL6, IL1B) of Calu3 cells with SARS-CoV-2 infection at MOI = 0.05 for indicated time points. e Calu3 cells were infected with SARS-CoV-2 at MOI = 0.05 for indicated time points and the lysates were harvested for immunoblot analysis with indicated antibodies. f HEK293T cells were transfected with NF-κB-luciferase (luc) reporter along with Flag- empty vector (EV) or Flag-N protein, followed by transfecting plasmid…

Fig. 2. SARS-CoV-2 N protein promotes NF-κB signaling at TAK1 and IKK complex level. a Schematic overview of NF-κB signaling pathway. b HEK293T cells were transiently transfected with NF-κB luciferase reporter, Flag-empty vector (EV) and increasing amount of Flag-N protein together with plasmids expressing IRAK1, TRIF, RIPK1, TRAF2, TRAF6, TAK1 and TAB1, IKKβ and p65. Cell lysates were collected at 36 h post-transfection and luciferase activities were tested. c HEK293T cells were transiently transfected with HA-N protein plasmid along with Flag-empty vector (EV), Flag-TAK1, Flag-TAB1, and Flag-TAB2 plasmids, followed with 1 μg/mL poly(I:C) treatment for 6 h before harvested. Whole-cell lysates were subjected to immunoprecipitation with anti-Flag beads and immunoblot analysis was performed with indicated antibodies. d HEK293T cells were transiently transfected with HA-N protein plasmid along with Flag-EV, Flag-IKKα, Flag-IKKβ, and Flag-NEMO plasmids, followed with 1 μg/mL poly(I:C) treatment for 6 h before harvested. Whole-cell lysates were subjected to immunoprecipitation with anti-Flag beads and immunoblot analysis was performed with indicated antibodies. e Extracts of Calu3 cells…

Fig. 3. SARS-CoV-2 N protein undergoes LLPS to promote NF-κB signaling. a HEK293T cells were transfected with Flag-empty vector (EV) and Flag-N plasmid, treated with 1 μg/mL polyU for 6 h. Cell lysates were collected and treated with DSS for immunoblotting with indicated antibodies. b HEK293T cells were transfected with HA-N along with Flag-EV and Flag-N plasmids, treated with 1 μg/mL polyU for 6 h. Whole-cell lysates were subjected to immunoprecipitation with anti-Flag beads and immunoblot analysis was performed with indicated antibodies. The ratio of the gray value between IP-HA and IP-Flag was determined. c Representative live-cell images (up) of HeLa cells stably expressing EGFP-N protein with 1 μg/mL poly(I:C) or 1 μg/mL polyU treatment for 6 h or left non-treated (NT). Scale bars, 5 μm. Quantitative analysis (down) of N protein droplets (20 cells each sample). d Representative images (left) of fluorescence recovery after photobleaching (FRAP) assay of N protein-polyU droplets in HeLa cells. HeLa cells stably expressing EGFP-N protein with 1 μg/mL polyU treatment for 6 h before bleaching. Bleaching was performed at the indicated time points and the recovery occurred at 37 °C. Scale…

Fig. 4. LLPS of SARS-CoV-2 N protein recruits TAK1 and IKK complex. a Representative image of confocal micrographs of Huh7 cells stably expressing EGFP-N protein, treated with 1 μg/mL poly(I:C) for 6 h, followed by labeling IKKβ and DAPI. Scale bars, 5 μm. b–c Representative images showing TAK1 (b) or IKKβ (c) were distributed into N protein-polyU droplets in vitro. 10 μM mCherry-N protein and 5 μM EGFP-TAK1 (b) or EGFP- IKKβ (c) were mixed with 10 ng/mL polyU and incubated at 37 °C for 10 min. Confocal 5× zoom-in of the area highlighted above was re-photographed. Scale bars, 5 μm. d–e Representative images (d) of confocal microscopy of TRE-N-A549 with 200 ng/mL doxy treated for 24 h or left untreated, followed by 1 µg/mL poly(I:C) treatment for 6 h or left non-treated (NT). Cells were fixed and labeled of TAK1 and IKKβ. Scale bars, 5 μm. Quantitative analysis (e) of the co-localization puncta (10 cells per sample). f HEK293T cells were transiently transfected HA-TAK1, Flag-IKKβ and Myc-N, followed by 1 µg/mL poly(I:C) treatment for 6 h. The whole-cell lysates were collected for immunoprecipitation by anti-Flag beads and immunoblotting by indicated antibodies. The ratio of the gray value…

Fig. 5. CTD domain of SARS-CoV-2 N protein CTD domain is critical for its LLPS and NF-κB regulatory capability. a Domain organization of SARS-CoV-2 N protein and its domain deletion mutants. b HEK293T cells were transfected Flag-N full-length (FL) along with HA-empty vector (EV), HA-N FL, △NTD, △SR, and △CTD. Whole-cell lysates were subjected to immunoprecipitation with anti-HA beads and immunoblot analysis was performed with indicated antibodies. The ratio of the gray value between IP-Flag and IP-HA was determined. c Representative images of live-cell confocal microscopy (up) of HeLa cells expressing EGFP-N FL, △NTD, and △CTD. Immunoblot analysis (down) were performed on the cell lysates with indicated antibodies. d Representative images showing TAK1 was distributed into N protein-poly(I:C) droplets in vitro. 10 µM mCherry-N protein or mCherry-N protein-△CTD, 5 µM EGFP-TAK1 were mixed with 10 ng/mL poly(I:C) and incubated at 37 °C for 10 min. Confocal 5× zoom-in of the area highlighted above was re-photographed. Scale bars, 5 μm. e Lysates of HEK293T cells transfected Flag-TAK1 and HA-EV, HA-N FL, △NTD, △SR, and △CTD, together with 1 µg/mL poly(I:C) for 6 h, were performed…