SARS-CoV-2 N protein promotes NLRP3 inflammasome activation to induce hyperinflammation

Abstract

Excessive inflammatory responses induced upon SARS-CoV-2 infection are associated with severe symptoms of COVID-19. Inflammasomes activated in response to SARS-CoV-2 infection are also associated with COVID-19 severity. Here, we show a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation to induce hyperinflammation. N protein facilitates maturation of proinflammatory cytokines and induces proinflammatory responses in cultured cells and mice. Mechanistically, N protein interacts directly with NLRP3 protein, promotes the binding of NLRP3 with ASC, and facilitates NLRP3 inflammasome assembly. More importantly, N protein aggravates lung injury, accelerates death in sepsis and acute inflammation mouse models, and promotes IL-1β and IL-6 activation in mice. Notably, N-induced lung injury and cytokine production are blocked by MCC950 (a specific inhibitor of NLRP3) and Ac-YVAD-cmk (an inhibitor of caspase-1). Therefore, this study reveals a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation and induces excessive inflammatory responses.

Fig. 1. SARS-CoV-2 N protein induces inflammatory responses.

Transcriptional level of indicated gene in macrophage (a) and dendritic cells (b) infected with SARS-CoV-2 (GSE155106, RNA-Seq data from GEO database) were analyzed and the values were showed in logarithmic form. c HEK293T cells were transfected with plasmids encoding N, M, E, 3a, 6, 7a, 8, and 10 proteins for 24 h. The indicated proteins in cell extract were analyzed by WB. d HEK293T cells were co-transfected with plasmids encoding NLRP3, ASC, pro-Caspase-1, or pro-IL-1β, and transfected with plasmids encoding N, M, E, 3a, 6, 7a, 8, and 10 for 48 h. Supernatants were analyzed by ELISA for IL-1β. e PMA-differentiated THP-1 macrophages were transfected with plasmids encoding N, M, E, 3a, 6, 7a, 8, and 10 proteins for 48 h, and then stimulated with 2 μM Nigericin or DMSO for 2 h. IL-1β in cell supernatants was measured by ELISA. f, g THP-1 cells were stably infected with Lentivirus-CT or Lentivirus-N, differentiated into macrophages. Lentivirus-N macrophages were then treated with MCC950 (0.01 μM) for 1 h. Cell lysates were analyzed by immunoblotting (f). The indicated gene mRNA was quantified by qRT-PCR (g). h, i C57BL/6 genetic background mice were tail vein-injected with 300 μl containing 5 × 10¹¹ vg of AAV-Lung-EGFP (n = 8) or AAV-Lung-N (n = 16); after 2 weeks, they were treated with MCC950 (50 mg/kg) or Ac-YVAD-cmk (8 mg/kg) by intraperitoneal injection for AAV-Lung-N (n = 8) mice; after 3 weeks, two mice for each group were killed and the lungs were collected. The indicated proteins were analyzed by WB (h). The indicated gene mRNAs were quantified by qRT-PCR (i). Mock means healthy donors (a, b), untreated cells (e), and injection of the same dose of PBS (i). Control means transfected with empty plasmids (c, d). Lipo means transfection with reagents Lipo2000 (e). Data are representative of three independent experiments and one representative is shown. Error bars indicate SD of technical triplicates. Values are mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, two-tailed Student’s t-test. Source data are provided as a Source Data file.

Fig. 2. IL-1β maturation and IL-6 production are induced by N protein.

a HEK293T cells were co-transfected with plasmids encoding NLRP3, ASC, pro-Caspase-1, and pro-IL-1β, and transfected with plasmids encoding different concentrations of SARS-CoV-2 N for 48 h. Supernatants were analyzed (top) by ELISA for IL-1β. Cell lysates were analyzed (bottom) by immunoblotting. b–d THP-1 cells were stably infected with Lentivirus-CT or Lentivirus-N, differentiated into macrophages. Then stimulated with LPS (1 μg/ml) plus ATP (2.5 mM) or LPS (1 μg/ml) plus Nigericin (2 μM). IL-1β (b, top), IL-18 (c), and IL-6 (d) in supernatants was measured by ELISA. Cell lysates were analyzed (b, bottom) by immunoblotting. e, f GM-CSF-differentiated mice BMDMs were infected with Lentivirus-CT or Lentivirus-N and stimulated with LPS (1 μg/ml), LPS (1 μg/ml) plus ATP (2.5 mM), or LPS (1 μg/ml) plus Nigericin (2 μM). IL-1β (e) or IL-6 (f) in supernatants was measured by ELISA. g, h THP-1 cells were stably infected with Lentivirus-CT or Lentivirus-N, differentiated into macrophages. The cells were treated with MCC950 (0.01 μM) for 1 h and then stimulated with LPS (1 μg/ml) plus ATP (2.5 mM) or LPS (1 μg/ml) plus Nigericin (2 μM). IL-1β protein (g) and IL-6 protein (h) in supernatants were measured by ELISA. i, j GM-CSF-differentiated BMDMs isolated from NLRP3^+/+ mice and NLRP3^−/− mice were infected with Lentivirus-N and stimulated with LPS (1 μg/ml), LPS (1 μg/ml) plus ATP (2.5 mM), or LPS (1 μg/ml) plus Nigericin (2 μM). IL-1β protein (i) and IL-6 protein (j) in supernatants were measured by ELISA. Mock means untreated cells (a–d and g, h). Data are representative of three independent experiments and one representative is shown. Error bars indicate SD of technical triplicates. Values are mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, two-tailed Student’s t-test. Source data are provided as a Source Data file.

Fig. 3. SARS-CoV-2 N protein interacts with NLRP3 protein.

HEK293T cells (a) or A549 cells (b) were co-transfected with HA-SARS-CoV-2-N and Flag-ctrl, Flag-NLRP3, Flag-pro-Caspase-1, or Flag-ASC. Cell lysates were immunoprecipitated using anti-Flag antibody and analyzed using anti-Flag and anti-HA antibody. Cell lysates (40 μg) was used as Input. HEK293T cells (c) or A549 cells (d) were co-transfected with Flag-NLRP3 and HA-SARS-CoV-2-N. Cell lysates were immunoprecipitated using anti-Flag antibody or anti-HA antibody, and analyzed using anti-Flag and anti-HA antibody. Cell lysates (40 μg) was used as Input. e Purified His-CT (10 μg) or His-N (10 μg) was incubated with cell lysates of Flag-NLRP3-transfected HEK293T cells. Cell extracts were incubated with Ni-NTA Agarose beads. Mixtures were analyzed by immunoblotting using anti-NLRP3 or anti-His antibody. Untreated protein includes His-SARS-CoV-2-N (1 μg), His-CT (1 μg), or HEK293T cell lysates were analyzed by immunoblotting (as input). HEK293T cells (f, h) or A549 cells (g, i) were transfected with Flag-SARS-CoV-2-N and HA-NLRP3 or HA-ASC for 24 h. Nucleus marker DAPI (blue), Flag-SARS-CoV-2-N (green), and HA-NLRP3 or HA-ASC (red) were then visualized with confocal microscopy. Flag-Ctrl, Flag-NC, or HA-NC means pcDNA3.1(+)–3× flag or pCAGGS-HA empty plasmid. Data were representative of three independent experiments. Source data are provided as a Source Data file.

Fig. 4. Sequence of N protein involved in NLRP3 inflammasome activation.

a Schematic diagram of wild-type SARS-CoV-2-N protein and truncated mutants N protein (N1 to N8). HEK293T cells (b) or A549 cells (d) were co-transfected with HA-NLRP3 and Flag-Ctrl, Flag-N truncated mutants (N1–N8). Cell lysates were immunoprecipitated using anti-Flag antibody and analyzed using anti-Flag and anti-HA antibody. Cell lysates (40 μg) were used as Input. c HEK293T cells were co-transfected with HA-NLRP3 and Flag-N truncated mutants (N1–N8). Cell lysates were immunoprecipitated using anti-HA antibody, IgG antibody was used as negative control, and analyzed using anti-Flag and anti-HA antibody. Cell lysates (40 μg) was used as Input. e HEK293T cells were co-transfected with plasmids encoding NLRP3, ASC, pro-Casp1, and pro-IL-1β, and transfected with plasmids encoding SARS-CoV-2-N protein and truncated mutants N protein (N1–N8) for 48 h. Supernatants were analyzed by ELISA for IL-1β and by WB for p17 and p20. Flag-ctrl means pcDNA3.1(+)–3× flag empty plasmid. Mock means untreated cells (e). Control means transfected empty plasmid (e). Data are representative of three independent experiments and one representative is shown. Error bars indicate SD of technical triplicates. Values are mean ± SEM. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, two-tailed Student’s t-test. Source data are provided as a Source Data file.

Fig. 5. Interaction of NLRP3 with ASC is promoted by N protein.

a HEK293T cells were co-transfected with Flag-Ctrl or Flag-SARS-CoV-2-N plus HA-NLRP3, HA-pro-Casp1, or HA-ASC for 36 h, the indicated proteins in cell extract were analyzed by WB. HEK293T cells (b) or A549 cells (d) were co-transfected with HA-SARS-CoV-2-N, HA-NLRP3, and Flag-ASC for 24 h. Cell lysates were immunoprecipitated using anti-Flag antibody and analyzed using anti-NLRP3, anti-ASC, and anti-N antibody. HEK293T cells (c) or A549 cells (e) were co-transfected with Flag-SARS-CoV-2-N, HA-NLRP3, and HA-ASC for 24 h. Cell lysates were immunoprecipitated using anti-Flag antibody and analyzed using anti-NLRP3, anti-ASC, and anti-N antibody. Cell lysates (40 μg) was used as Inputs. HEK293T cells (f) or A549 cells (g) were co-transfected with different concentration of Flag-SARS-CoV-2-N plus HA-NLRP3 and Flag-ASC for 24 h. Cell lysates were immunoprecipitated using anti-NLRP3 antibody and analyzed using anti-NLRP3, anti-ASC, anti-GAPDH, and anti-Flag antibody. h THP-1 macrophages were stably infected with Lentivirus-CT or Lentivirus-N, and stimulated with 2 μM Nigericin or DMSO for 2 h. Cell lysates were immunoprecipitated using anti-NLRP3 antibody and analyzed using anti-NLRP3, anti-ASC, anti-GAPDH, and anti-N antibody. Flag-ctrl means pcDNA3.1(+)–3× flag empty plasmid. Data are representative of three independent experiments. Source data are provided as a Source Data file.

Fig. 6. N protein facilitates NLRP3 aggregation and ASC oligomerization.

a HEK293T cells (top) or A549 cells (bottom) were transfected with Flag-NC/HA-NLRP3, Flag-N/HA-NLRP3, or Flag-3a/HA-NLRP3 for 24 h. The sub-cellular locations of NLRP3 (red), Nucleus marker DAPI (blue), and SARS-CoV-2 N or SARS-CoV-2 3a (green) were visualized with confocal microscopy. b HEK293T cells (top) or A549 cells (bottom) were transfected with Flag-N2+HA-NC or Flag-N2+HA-NLRP3 for 24 h. The sub-cellular locations of NLRP3 (red), Nucleus marker DAPI (blue), and SARS-CoV-2-N8 (green) were visualized with confocal microscopy. c HEK293T cells (top) or A549 cells (bottom) were transfected with Flag-N8+HA-NC or Flag-N8+HA-NLRP3 for 24 h. The sub-cellular locations of NLRP3 (red), Nucleus marker DAPI (blue), and SARS-CoV-2-N8 (green) were visualized with confocal microscopy. d, e PMA-differentiated THP-1 macrophages were stably infected with Lentivirus-CT or Lentivirus-N. The sub-cellular locations of SARS-CoV-2-N (green, d) Nucleus marker DAPI (blue), and NLRP3 (red, e) were visualized with confocal microscopy. f THP-1 macrophages were stably infected with Lentivirus-CT or Lentivirus-N, and stimulated by LPS (1 μg/ml) plus ATP (2.5 mM) or LPS (1 μg/ml) plus Nigericin (2 μM). ASC oligomerization was analyzed by immunoblotting. g GM-CSF differentiated mice BMDMs were infected with Lentivirus-CT or Lentivirus-N and stimulated with LPS (1 μg/ml), LPS (1 μg/ml) plus ATP (2.5 mM), or LPS (1 μg/ml) plus Nigericin (2 μM). ASC oligomerization was analyzed by immunoblotting. h HEK293T cells were co-transfected with plasmids encoding NLRP3, ASC, pro-Caspase-1, or pro-IL-1β, and transfected with plasmids encoding SARS-CoV-2-N protein and truncated mutants N protein (N1 to N8) for 48 h, ASC oligomerization was analyzed by immunoblotting. Flag-NC or HA-NC means pcDNA3.1(+)–3× flag or pCAGGS-HA empty plasmid (a–c). Mock means untreated cells (f, h). Control means transfected empty plasmid (h). Data are representative of three independent experiments. Source Data are provided as a Source Data file.

Fig. 7. N protein promotes NLRP3 inflammasome assembly.

HEK293T cells (a) and A549 cells (b) were co-transfected with GFP/HA-NLRP3, GFP-N/HA-NLRP3, GFP/Flag-ASC, GFP-N/Flag-ASC, GFP/HA-NLRP3/Flag-ASC, GFP-N/HA-NLRP3/Flag-ASC, and GFP-3a/HA-NLRP3/Flag-ASC for 24 h. The sub-cellular locations of HA-tagged NLRP3 (red), Flag-tagged ASC (yellow), GFP-tagged N or GFP-tagged 3a (green), and nucleus marker DAPI (blue) were visualized with confocal microscopy. GFP-NC means pEGFP-C1 empty plasmid (a, b). Data are representative of three independent experiments. Source data are provided as a Source Data file.

Fig. 8. N protein induces lung injury in mice by activating NLRP3 inflammasome.

a–c C57BL/6 genetic background mice were tail vein-injected with 300 μl containing 5 × 10¹¹ vg of AAV-Lung-EGFP (n = 10) or AAV-Lung-N (n = 16) for 2 weeks and then treated with MCC950 (50 mg/kg) by intraperitoneal injection for AAV-Lung-EGFP (n = 5) or AAV-Lung-N (n = 8) mice. Serum was collected at 3 weeks for each group from the orbit. IL-1β (a), IL-6 (b), or IL-18 (c) in the sera was measured by ELISA. Points represent the value of each serum samples. d–f At 3 weeks, mice were killed and the lungs were collected. Histoimmunofluorescence analysis of IL-1β (d, red) or IL-6 (e, red) in the lung after AAV-CT or AAV-N infection. Scale bar is 200 μm (10×) or 50 μm (40×). Histopathology analysis of lung after AAV-CT or AAV-N infection (f). Red arrows indicated the infiltrated inflammatory cells. Scale bar is 200 μm (10×) or 50 μm (40×). g After 4 weeks, pretreated AAV-Lung-N mice (n = 7) with DMSO, pretreated AAV-Lung-N (n = 7) mice with MCC950, or pretreated AAV-Lung-EGFP (n = 5) with MCC950. After 30 min, mock group (n = 7), AAV-Lung-EGFP group (n = 5), AAV-Lung-N (n = 7) group, and another AAV-Lung-N (n = 7) group were treated with LPS (30 mg/kg) by intraperitoneal injection. Another mock group (n = 3) was intraperitoneally injected with PBS. The mice survival rates were evaluated every 2 h post treatment. Mock means inject the same dose of PBS as other groups (a–h). Data are representative of two independent experiments and one representative is shown. Error bars indicate SD of each serum samples, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, two-tailed Student’s t-test (a–c). One-way ANOVA analysis (g). Source data are provided as a Source Data file.

Fig. 9. NLRP3 has an important function in N protein-induced lung injury.

a–c NLRP3^+/+ C57BL/6 mice or NLRP3^−/− C57BL/6 mice were tail vein-injected with 300 μl containing 5 × 10¹¹ vg of AAV-Lung-EGFP (n = 4) or AAV-Lung-N (n = 7). Serum was collected at 3 weeks for each group from the orbit. IL-1β (a), IL-6 (b), or IL-18 (c) in the sera were measured by ELISA assay. Points represent the value of each serum samples. d, e Histoimmunofluorescence analysis of IL-1β (d, red) or IL-6 (e, red) in the lung after AAV-N infected NLRP3^+/+ mice or NLRP3^−/− mice. Scale bar is 200 μm (10×) or 50 μm (40×). f Histopathology analysis of lung after AAV-N infected NLRP3^+/+ mice or NLRP3^−/− mice. Red arrows indicated the infiltrated inflammatory cells. Scale bar is 200 μm (10×) or 50 μm (40×). Data are representative of two independent experiments and one representative is shown. Error bars indicate SD of each serum samples, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, two-tailed Student’s t-test (a–c). Source data are provided as a Source Data file.

Fig. 10. Model by which N protein induces lung injury by activating NLRP3 inflammasome.

During SARS-CoV-2 infection, the nucleocapsid protein (N) could directly interact with NLRP3 to promote the assembly and activation of NLRP3 inflammasome, thus leading to the production of a large number of inflammatory factors (such as IL-1β, IL-6, TNF, CCL2, and CCL8) and the occurrence of lung injury in mice.