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

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.

S evere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), so named by the International Committee on Taxonomy of Viruses, and the consequent severe coronavirus disease 2019 (COVID- 19) have now become the most serious public health problem worldwide 1,2 . The virus mainly infects the respiratory tract of humans, causing fever, dry cough, fatigue, shortness of breath, body aches, diarrhea, and other symptoms 3,4 . A small number of critically ill patients may progress to acute respiratory distress syndrome (ARDS), metabolic acidosis, septic shock, and clotting dysfunction, or even death in severe cases 5,6 . The infection and transmission of SARS-CoV-2 has caused great harm to human health and social development. Therefore, the pathogenesis of SARS-CoV-2 is an urgent scientific problem to be solved.
SARS-CoV-2 infects human respiratory system and causes severe inflammatory responses 7,8 . Evidence shows that a dysregulated innate immune response contributes to the clinical presentation of patients with severe COVID-19 9 . Inflammasomes are an important part of the innate immune system that can recognize cellular stresses and infections 10,11 . Inflammasomes are named according to different sensing proteins and there are four main types as follows: NLRP1, NLRP3, NLRC4, and AIM2 12 . Among them, the NLRP3 inflammasome has important functions in RNA virus infection 13,14 . NLRP3 inflammasome consists of a sensor protein (NLR family PYRIN domain containing-3, NLRP3), an adaptor protein (apoptosis-associated speck-like protein containing a caspase recruitment domain, ASC), and an effector protein (Caspase-1) 15 . NLRP3 protein contains three domains: Pyrin domain (PYD), Nucleotide-binding domain, and Leucine-rich repeat domain 14 . During activation of the NLRP3 inflammasome, NLRP3 PYD interacts with ASC PYD, promotes ASC oligomer formation, and provides a platform for caspase-1 activation. Active caspase-1 is formed by autocatalytic cleavage, which then catalyzes proteolytic processing of pro-interleukin (IL)-1β into mature IL-1β 16 . Excessive IL-1β stimulates systemic inflammation responses by activating various signaling pathways, such as nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase pathways 17,18 . As a result, large amounts of cytokines are released, including IL-6, tumor necrosis factor (TNF), interferon (IFN)-α, IFN-β, and transforming growth factor-β 18 , which can lead to a "cytokine storm" in acute inflammatory diseases. Studies have reported that inflammasomes are induced upon SARS-CoV-2 infection and are associated with COVID-19 severity 19,20 .
SARS-CoV-2 belongs to the β-genus Coronavirus in the Coronaviridae family 21 . The spherical viral particle encapsulates its genome, an unsegmented, positive-sense single-stranded RNA with a size of~30 kb 21 . The viral genome is enclosed by a nucleocapsid (N) protein, which is buried inside phospholipid bilayers and is covered by a spike (S) protein. The membrane (M) protein and the envelope (E) protein are located among the S proteins in the virus envelope. In addition, the virus encodes a series of accessory proteins (ORF3a, ORF6, ORF7a, ORF7b, ORF8, and ORF10) 21,22 . Previous studies have found that SARS-CoV, which also belongs to the coronavirus subfamily, can activate NLRP3 inflammasome 23,24 . Further studies have revealed that E and ORF3a of SARS-CoV activate NLRP3 inflammasome by changing the K + ion permeability of plasma membrane and the production of mitochondrial reactive oxygen species 23,24 . The specific molecular mechanism by which SARS-CoV-2 activates NLRP3 inflammasomes is unclear.
Here we show that SARS-CoV-2 N protein induces proinflammatory cytokines through promoting the assembly and activation of the NLRP3 inflammasome. The N protein aggravates lung injury and accelerates death in acute inflammation mouse models through facilitating the NLRP3 inflammasome activation. We also show that SARS-CoV-2 N protein promotes the assembly of the NLRP3 inflammasome through direct interaction with NLRP3 protein. Moreover, SARS-CoV-2 N-induced lung injury and cytokine production are blocked by MCC950 (a specific inhibitor of the NLRP3) and Ac-YVAD-cmk (an inhibitor of the caspase-1). Compared with NLRP3 +/+ mice, Ninduced lung injury and cytokine production are also suppressed in NLRP3 −/− mice. This work thereby identifies a molecular mechanism by which SARS-CoV-2 infection causes ARDS, provides evidence that links the NLRP3 inflammasome and lung injury, and suggests that MCC950 and Ac-YVAD-cmk might function as potential therapeutic agents for the prevention and treatment of COVID-19.
The role of SARS-CoV-2 proteins in regulating the secretion of mature IL-1β were assessed in a reconstructed NLRP3 inflammasome system as described previously 14 . HEK293T cells were cotransfected with plasmids encoding the three components (NLRP3, ASC, and pro-Caspase-1) of the NLRP3 inflammasome and the substrate pro-IL-1β to generate a NLRP3 inflammasome system. The HEK293T-NLRP3 inflammasome system cells were subsequently transfected with plasmids encoding N, M, E, 3a, 6, 7a, 8, and 10 proteins, respectively. Enzyme-linked immunosorbent assay (ELISA) results showed that IL-1β secretion was significantly induced by N protein in this system (Fig. 1d). In addition, phorbol-12-myristate-13-acetate (PMA)-differentiated THP-1 macrophages were transfected with plasmids encoding N, M, E, 3a, 6, 7a, 8, and 10 proteins, and stimulated with Nigericin (a specific activator for the NLRP3 inflammasome). We noted that the secretion of mature IL-1β was significantly induced by Nigericin and the Nigericin-induced mature IL-1β secretion was further remarkably enhanced by the N protein (Fig. 1e). Collectively, these results demonstrated that SARS-CoV-2 N protein plays an important role in the induction of mature IL-1β secretion.
Next, the effect of N protein on the regulation of proinflammatory cytokines and chemokines was assessed in PMAdifferentiated THP-1 macrophages. THP-1 cells were infected with N-Lentivirus (Lentivirus carrying the N gene) and CT-Lentivirus (as a negative control) to generate two cell lines stably expressing N-Lentivirus and CT-Lentivirus, respectively (Fig. 1f). The stable cells were subsequently differentiated into macrophages. qRT-PCR analyses showed that the expression of IL-1β, IL-6, TNF, CXCL10, IL-11, IL-13, CCL2, and CXCL8 mRNAs was notably elevated by Lentivirus-N, whereas the expression of IL-18, 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 × 10 11 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. IL-10, and VEGFA mRNAs was not affected by Lentivirus-N (Fig. 1g). Interestingly, Lentivirus-N-mediated inductions of inflammatory factors were repressed by MCC950 (a specific inhibitor of the NLRP3) (Fig. 1g), indicating that NLRP3 is involved in the induction of proinflammatory cytokines mediated by the N protein.
The role of N protein in the regulation of NF-κB signaling pathway was then determined. Notably, the levels of NLRP3 mRNA ( Supplementary Fig. 1c, top) as well as NLRP3, pro-IL-1β, and pro-IL-18 proteins ( Supplementary Fig. 1c, bottom) were relatively unaffected by N protein. These results implicated that N protein regulates NLRP3, pro-IL-1β, and pro-IL-18 independent on NF-κB signaling pathway. We noticed that IL-1β protein promoted the production of inflammatory factors and cytokines, including IL-6, TNF, CXCL10, IL-11, IL-13, and CCL2 (Supplementary Fig. 1d). Thus, we speculate that N protein promotes NLRP3 inflammasome activation, thereby inducing mature IL-1β secretion, which subsequently plays a key role in the induction of inflammatory factors and cytokines. Taken together, these results demonstrated that SARS-CoV-2 N protein plays a critical role in the induction of proinflammatory cytokines and suggested that N protein may involve in regulating the activation of the NLRP3 inflammasome.
IL-1β maturation and IL-6 production are induced by SARS-CoV-2 N protein. Given the dominant effect of the activator on the expression of the proinflammatory factors, we further determined the role of SARS-CoV-2 N protein in IL-1β maturation. HEK293T-NLRP3 inflammasome system cells were transfected with plasmid encoding SARS-CoV-2 N protein at different concentrations. The results showed that IL-1β secretion (Fig. 2a, top) and IL-1β (p17) cleavage, as well as Caspase-1 (p20) maturation (Fig. 2a, bottom) were induced by N protein in dose-dependent manners. Interestingly, the level of IL-1β protein was significantly induced by the SARS-CoV-2 N protein and slightly enhanced by the SARS-CoV-2 3a protein ( Supplementary Fig. 2), confirming a specific role of N protein in the activation of IL-1β protein.
Next, PMA-differentiated THP-1 macrophages were transfected with plasmids encoding SARS-CoV-2 N at different concentrations and treated with Nigericin. We noted that IL-1β secretion was induced by Nigericin and the Nigericin-induced IL-1β secretion was further promoted by N protein in a dosedependent manner ( Supplementary Fig. 3a). Similarly, IL-1β (p17) cleavage and Caspase-1 (p20) maturation were induced by Nigericin, and Nigericin-induced IL-1β (p17) cleavage and Caspase-1 (p20) maturation were further facilitated by N protein in concentration-dependent manners ( Supplementary Fig. 3b). However, we found that the level of mature IL-18 was relatively unaffected by N protein (Supplementary Fig. 3c), although its maturation and production relied on the same cleavage mechanism as IL-1β.
In addition, THP-1 cells stably infected with CT-Lentivirus and N-Lentivirus were differentiated into macrophages, which were then treated with lipopolysaccharide (LPS) plus ATP or LPS plus Nigericin. The results indicated that IL-1β secretion was induced by LPS plus ATP or LPS plus Nigericin, and such inductions were further enhanced significantly by N protein (Fig. 2b, top). Consistently, IL-1β (p17) cleavage was also induced by LPS plus ATP or LPS plus Nigericin, and such inductions were further enhanced by N protein (Fig. 2b, bottom). We noted that the level of mature IL-18 was induced by LPS plus ATP or LPS plus Nigericin, but such inductions were relatively unaffected by N protein (Fig. 2c). Notably, the production of widely described proinflammatory cytokine IL-6 was induced by LPS plus ATP or LPS plus Nigericin, and such induction was also further enhanced by N protein (Fig. 2d). Moreover, IL-6 production was confirmed to be induced by N protein in a dose-dependent manner ( Supplementary Fig. 3d).
Next, granulocyte macrophage-colony stimulating factor (GM-CSF) differentiated mice bone marrow-derived monocytes (BMDMs) were infected with CT-Lentivirus or N-Lentivirus and stimulated with LPS, LPS plus ATP, or LPS plus Nigericin. In the treated mouse BMDMs, IL-1β secretion was activated by LPS plus ATP or LPS plus Nigericin, and such activations were further facilitated notably by N protein (Fig. 2e). Similarly, IL-6 production was also induced by LPS plus ATP or LPS plus Nigericin, and such inductions were further promoted by N protein in treated mice BMDMs (Fig. 2f). Therefore, our results demonstrated that SARS-CoV-2 N protein induces IL-1β maturation and IL-6 production.
Therefore, we further investigated the role of NLRP3 involved in IL-1β maturation and IL-6 production induced by the N protein. THP-1 cells stably infected with CT-Lentivirus and N-Lentivirus were differentiated into macrophages, which were pretreated with MCC950 (a specific inhibitor of the NLRP3) and then stimulated with LPS plus ATP or LPS plus Nigericin. Notably, in the presence of LPS plus ATP or LPS plus Nigericin, secreted IL-1β protein was significantly induced by N protein, but N-induced secreted IL-1β protein was remarkably suppressed by MCC950 (Fig. 2g). Similarly, IL-6 protein was enhanced by N protein, whereas N-enhanced IL-6 protein was also repressed by MCC950 (Fig. 2h). These results suggest that N protein induces IL-1β secretion and IL-6 production depending on NLRP3. Moreover, GM-CSF differentiated BMDMs of wide-type mice and NLRP3 −/− mice were infected with N-Lentivirus and then stimulated with LPS, LPS plus ATP, and LPS plus Nigericin. We noticed that the levels of secreted IL-1β protein (Fig. 2i) and IL-6 protein ( Fig. 2j) induced by N protein were significantly lower in the BMDMs of NLRP3 −/− mice compared with that in BMDMs of wide-type mice (Fig. 2i, j). These data indicate that N protein fails to induce IL-1β protein and IL-6 protein in NLRP3 −/− mice BMDMs, and thereby suggesting that NLRP3 is required for IL-1β secretion and IL-6 production induced by the N protein.
Taken together, our results demonstrate that N protein induces IL-1β maturation and IL-6 production, and reveal that NLRP3 is required for N-induced IL-1β secretion and IL-6 production protein, thereby suggesting that N protein may play an important role in the activation of the NLRP3 inflammasome.
SARS-CoV-2 N protein interacts with NLRP3 protein. The role of SARS-CoV-2 N protein in the regulation of NLRP3 inflammasome was then investigated. Initially, the interaction of N protein with the NLRP3 inflammasome components, NLRP3, ASC, and Caspase-1, was explored. Co-immunoprecipitation (Co-IP) assays showed that N protein only interacted with NLRP3 protein and failed to interact with ASC protein or Caspase-1 protein in HEK293T and A549 cells (Fig. 3a, b). Reciprocal Co-IP assays further conformed that NLRP3 protein interacted with N protein (Fig. 3c, d), but failed to interact with 3a protein (Supplementary Fig. 4a, b) in HEK293T and A549 cells. Importantly, we demonstrated that purified His-N protein could directly bind to NLRP3 protein (Fig. 3e). There are four types of ASC-dependent inflammasomes (NLRP1, NLRP3, NLRC4, and AIM2) that have been reported 12 . Here we determined the role of N protein in the regulation of NLRP1, NLRP3, NLRC4, and AIM2 inflammasomes. Co-IP assays showed that N protein only interacted with the NLRP3 protein, but failed to interact with the NLRP1, NLRC4, or AIM2 proteins (Supplementary Fig. 5a). Notably, we showed that N protein could induce IL-1β production in the presence of NLRP3, but failed to promote IL-1β production in the presence of NLRP1, NLRC4, or AIM2 ( Supplementary Fig. 5b). These results demonstrate that N protein specifically and directly triggers NLRP3. Moreover, confocal microscope analyses showed that in HEK293T and A549 cells, N protein and NLRP3 protein were co-localized in the cytoplasm (  specifically interacts with NLRP3 protein to regulate the NLRP3 inflammasome. Sequence of N protein involved in NLRP3 inflammasome activation. The sequences of SARS-CoV-2 N protein involved in the interaction with NLRP3 protein were identified by evaluating progressive truncated mutants of N protein, N1-N8 (Fig. 4a). Co-IP results showed that NLRP3 interacted with N1 (1-340), N5 (90-420), N6 (180-420), and N7 (260-420), but failed to interact with N2 (1-260), N3 (1-180), N4 (1-90), and N8 (340-420) in HEK293T cells (Fig. 4b, c) and A549 cells (Fig. 4d), suggesting that the domain containing 260aa-340aa of N protein is involved in the interaction with NLRP3.
Next, the effect of N protein progressive truncations (N1-N8) on the activation of the NLRP3 inflammasome was evaluated. The HEK293T-NLRP3 inflammasome system cells were transfected with plasmids encoding N protein or its truncated mutants N1-N8, respectively. ELISA assays showed that IL-1β secretion, IL-1β p17 cleavage, and Caspase-1 p20 maturation in the cell supernatants were significantly induced by the N1, N5, N6, and N7, but not affected by N2, N3, N4, and N8 (Fig. 4e), indicating that the domain containing 260aa-340aa of N protein is involved in the activation of the NLRP3 inflammasome. Taken together, we demonstrated that the sequence 260aa-340aa of SARS-CoV-2 N interacts with NLRP3 to activate the NLRP3 inflammasome.
Interaction of NLRP3 with ASC is promoted by N protein. As SARS-CoV-2 N interacts with NLRP3, we would like to know the role of N in regulating the components of the NLRP3 inflammasome. Initially, the effect of N protein on the expression of NLRP3, Caspase-1, and ASC was examined. The results showed that the protein levels of NLRP3, Caspase-1, and ASC were not influenced by N protein in HEK293T cells (Fig. 5a). However, N and ASC could immunoprecipitate with each other in the presence of NLRP3 in HEK293T cells (Fig. 5b, c) and A549 cells (Fig. 5d, e), suggesting that N, NLRP3, and ASC three proteins together might form a complex N-NLRP3-ASC. Interestingly, the interaction of NLRP3 and ASC was enhanced by N protein in 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.  dose-dependent manners in HEK293T cells (Fig. 5f) and A549 cells (Fig. 5g), but 3a protein had no influence in the interaction of NLRP3 and ASC compared with N protein ( Supplementary  Fig. 6a, b). Co-IP results also demonstrated that N protein interacted with endogenous ASC in the presence of NLRP3 and the interaction was promoted by stimulating with Nigericin in PMA-differentiated THP-1-N cells (Fig. 5h). Taken together, these results suggested that SARS-CoV-2 N promotes the interaction of NLRP3 with ASC.
SARS-CoV-2 N protein facilitates NLRP3 inflammasome assembly. The role of SARS-CoV-2 N protein in regulating the assembly of the NLRP3 inflammasome was then explored. Localization of NLRP3 as a speck structure in the cytoplasm is an indicator of inflammasome complex formation 27 . In HEK293T cells (Fig. 6a, top) and A549 cells (Fig. 6a, bottom), NLRP3 alone was diffusely distributed in the cytoplasm, whereas in the presence of SARS-CoV-2 N protein, NLRP3 co-localized with N and formed specks. However, in the presence of SARS-CoV-2 3a protein, NLRP3 was co-localized with 3a, but failed to form specks (Fig. 6a). We noted that N2 (1aa-260aa) (Fig. 6b) and N8 (340aa-420aa) (Fig. 6c) were distributed in both the nucleus and cytoplasm, and failed to co-localize with NLRP3 and NLRP3 failed to form specks in the cytoplasm of HEK293T cells (Fig. 6b, c, top) and A549 cells (Fig. 6b, c, bottom). Confocal microscope showed that in PMA-differentiated THP-1 macrophages, N protein alone was diffusely distributed in the cytoplasm (Fig. 6d); endogenous NLRP3 protein alone also diffusely located in the cytoplasm (Fig. 6e, top). However, in the presence of N protein, endogenous NLRP3 protein aggregated and formed specks in the cytoplasm (Fig. 6e, bottom). Collectively, these data suggested that SARS-CoV-2 N protein may promote the formation of the NLRP3 inflammasome complex.
Moreover, we explored the effect of N protein on the assembly of the NLRP3 inflammasome complex. HEK293T cells (Fig. 7a) and A549 cells (Fig. 7b) were co-transfected with plasmids as indicated. In the presence of GFP and NLRP3, GFP was distributed in both the cytoplasm and nucleus, NLRP3 was diffusely distributed in the cytoplasm (Fig. 7a (a-e) and Fig. 7b  (a-e)). In the presence of N and NLRP3, NLRP3 was co-localized with N and formed speck structures (Fig. 7a (f-j) and Fig. 7b  (f-j)). In the presence of GFP and ASC, GFP was distributed in both the cytoplasm and nucleus, ASC was distributed in the nucleus and cytoplasm, and formed small ring structures (Fig. 7a  (k-o) and Fig. 7b (k-o)). When N and ASC presented together, N and ASC were not co-localized (Fig. 7a (p-t) and Fig. 7b (p-t)). Notably, in the presence of GFP, ASC, and NLRP3, NLRP3 and ASC were co-localized in the cytoplasm to form "ring-like" structures ( Fig. 7a (u-y) and Fig. 7b (u-y)). Importantly, when N, ASC, and NLRP3 expressed together, the three proteins were obviously co-localized to form "sphere-like" structures ( Fig. 7a  (z-ad) and Fig. 7b (z-ad), and Supplementary Movies 4 and 5). However, in the presence of 3a, ASC, and NLRP3 together, the three proteins were co-localized, but failed to form "sphere-like" structures ( Fig. 7a (ae-ai) and Fig. 7b (ae-ai)).
Previous studies demonstrated that the NIMA-related kinase 7 (NEK7) is important to license NLRP3 inflammasome activation through directly binding to NLRP3 29 . Here we investigated that the role of NEK7 in NLRP3 inflammasome activation is mediated by SARS-CoV-2 N protein. Small interfering RNAs specifically targeting NEK7 were generated. The results showed that NEK7 mRNA ( Supplementary Fig. 8a-c, top) and NEK7 protein ( Supplementary Fig. 8a-c, bottom) were significantly attenuated by si-NEK7−3 but relatively unaffected by si-NEK7−1 and si-NEK7−2 in THP-1 cells (Supplementary Fig. 8a-c). THP-1 cells stably infected N-Lentivirus were differentiated into macrophages and then transfected with si-NEK7−3 and treated with Nigericin. Notably, IL-1β protein was induced by N protein and significantly stimulated by Nigericin, whereas N-promoted and Nigericininduced IL-1β proteins were relatively not affected by si-NEK7−3 ( Supplementary Fig. 8d). Similarly, N-promoted oligomerization of ASC in THP-1 cells (Supplementary Fig. 8e) and N-facilitated interaction of NLRP3-ASC were not influenced by si-NEK7−3 in THP-1 cells (Supplementary Fig. 8f), HEK293T cells (Supplementary Fig. 8g), and A549 cells (Supplementary Fig. 8h). These results indicated that NEK7 is not required for N proteinmediated NLRP3 inflammasome activation and NLRP3-ASC interaction. Taken together, these data demonstrated that SARS-CoV-2 N protein, but not SARS-CoV-2 3a protein, promotes the assemble of the NLRP3 inflammasome complex.
N protein induces lung injury in mice by activating NLRP3 inflammasome. The biological role of SARS-CoV-2 N protein in activating the NLRP3 inflammasome was then assessed. As the main clinical symptom of COVID-19 is acute lung injury 5 , the "cytokine storm" is considered to be an important cause of acute lung injury 26 . Therefore, we explored the effect of N protein on lung injury using an AAV-lung-N C57BL/6 mouse model. C57BL/6 mice were subjected to tail vein injection with AAV-Lung-EGFP or AAV-Lung-N. The results indicated that the protein levels of IL-1β and IL-6 in the sera were significantly higher in AAV-N-infected mice than that in AAV-CT-infected mice ( Supplementary Fig. 9a, b). In addition, C57BL/6 mice injected with AAV-Lung-EGFP or AAV-Lung-N were subjected to intraperitoneal injection with or without Ac-YVAD-cmk and MCC950. We noted that the protein levels of IL-1β and IL-6 were much higher in the sera of AAV-N-infected mice as compared to AAV-CT-infected mice. However, AAV-N-mediated inductions of IL-1β and IL-6 proteins were repressed by Ac-YVAD-cmk ( Supplementary Fig. 9c, d) and MCC950 (Fig. 8a, b). We noticed that unlike IL-1β and IL-6, the IL-18 protein was not affected by AAV-N in the sera pretreated with MCC950 (Fig. 8c). Notably, immunohistochemical fluorescence analyses showed that IL-1β protein (Fig. 8d) and IL-6 protein (Fig. 8e) were highly expressed in the lungs of mice carrying AAV-Lung-N, whereas such activations were attenuated by Ac-YVAD-cmk and MCC950 (Fig. 8d, e). Interestingly, hematoxylin and eosin staining analyses indicated that inflammatory lesions and tissue injuries were obvious in the lungs of mice carrying AAV-Lung-N, but such pathological changes were repressed by the administration of Ac-YVAD-cmk and MCC950 (Fig. 8f). These results suggest that the NLRP3 inflammasome is required for the development of mice lung injury induced by the N protein.
The role of NLRP3 in N-mediated mice lung injury was further determined by in C57BL/6 NLRP3 −/− mice. Knockout of NLRP3 gene in C57BL/6 NLRP3 −/− mice was confirmed by genotyping using mouse tail DNA samples ( Supplementary Fig. 9e). C57BL/6 NLRP3 +/+ and C57BL/6 NLRP3 −/− mice were tail vein-injected with AAV-Lung-EGFP (control) and AAV-Lung-N, respectively. The results confirmed that N protein was specifically expressed in the lung of mice injected with AAV-Lung-N, but not detected in the lung of mice injected with AAV-Lung-EGFP ( Supplementary  Fig. 9f). ELISA results showed that IL-1β protein and IL-6 protein were significantly induced by AAV-N in the sera of NLRP3 +/+ mice, but not affected by AAV-N in the sera of NLRP3 −/− mice (Fig. 9a, b), indicating that NLRP3 is essential for N-mediated activation of IL-1β and IL-6 proteins. We noticed that unlike IL-1β and IL-6, IL-18 protein was not affected by AAV-N in both NLRP3 +/+ and NLRP3 −/− mice (Fig. 9c). In addition, immunohistochemical fluorescence analyses showed that IL-1β and IL-6 proteins were highly expressed in the lungs of NLRP3 +/+ mice carrying AAV-Lung-N, whereas such activations were decreased in the lungs of NLRP3 −/− mice carrying AAV-Lung-N (Fig. 9d,  e). Hematoxylin and eosin staining analyses indicated that inflammatory lesions and tissue injuries were obvious in the lungs of NLRP3 +/+ mice carrying AAV-Lung-N, but such pathological changes were repressed in the lungs of NLRP3 −/− mice carrying AAV-Lung-N (Fig. 9f). Collectively, these results suggested that N protein induces a systemic inflammation and lung injury in mice via activating the NLRP3 inflammasome.
Moreover, C57BL/6 mice were tail vein-injected with AAV-Lung-EGFP or AAV-Lung-N, treated with phosphate-buffered saline (PBS) or LPS, and intraperitoneally injected with or without MCC950 or Ac-YVAD-cmk, as indicated. Interestingly, the survival rates were reduced in mice treated with LPS; relatively unchanged between mice treated with LPS plus injected with AAV-Lung-EGFP and mice treated with LPS; further downregulated significantly in mice treated with LPS plus injected with AAV-Lung-N; and recovered in mice treated with LPS plus injected with AAV-Lung-N and treated with MCC950 (Fig. 8g) or Ac-YVAD-cmk ( Supplementary Fig. 9g). Taken together, we reveal a pathological mechanism of COVID-19, in which SARS-CoV-2 N protein promotes inflammation and lung injury via promoting the activation of the NLRP3 inflammasome (Fig. 10).

Discussion
SARS-CoV-2 infection could cause acute lung injury and one of the important reasons is dysregulation of the immune system in the lung 7,8 that results in the release of a large number of inflammatory factors and forms "cytokine storm" 9 . In this study, we found that SARS-CoV-2 infection of macrophages and DCs would promote the expression of abundant cytokines and chemokines by analyzing the GEO database (GSE155106). Next, through screening, we found that SARS-CoV-2 N protein promoted the expression of inflammatory factors by activating NLRP3 inflammasome, resulting in mouse lung injury and exacerbating the death of mice in the sepsis model. Further studies indicated that N protein directly interacted with NLRP3 to promote the assembly of NLRP3 inflammasome and thereby activated NLRP3 inflammasome. Above all, our results demonstrated a novel mechanism by which SARS-CoV-2 N protein induced lung injury through NLRP3 inflammasome.
As an important part of the innate immune system, NLRP3 inflammasome plays a crucial role in the recognition of cell damage and pathogenic microbial infection [10][11][12][13] . Our previous studies found that M protein of Dengue virus caused tissue damage and vascular leakage in mice by activating NLRP3 inflammasome 14 , whereas NS5 protein of Zika virus promoted virus entry into the brain, which aggravated the death of mice by activating NLRP3 inflammasome 30 . Whether NLRP3 inflammasome plays a vital role in COVID-19? In this work, we found that SARS-CoV-2 N protein could promote the expression of inflammatory factors. However, after adding MCC950, a specific inhibitor of the NLRP3 or Ac-YVAD-cmk, an inhibitor of the Caspase-1 31 , the levels of N protein-induced inflammatory factors were restored. N protein, as the main structural protein of SARS- CoV-2, is involved in virus replication, assembly, and immune regulation 32,33 . As the most stably expressed structural protein, it is the gold standard in nucleic acid test of SARS-CoV-2. Recent research has also found that N protein combats host RNA interference-mediated antiviral response through its doublestranded RNA-binding activity 34 and N protein can induce both humoral and cellular immune response after infection 35 . Our results demonstrate that SARS-CoV-2 N protein, as a part of the viral envelop protein, is exited in the cytoplasm to trigger NLRP3 activation prior to viral assembly and increases the risk of activating the acute inflammatory response.
As important inflammatory factors in the innate immune system, IL-1β and IL-6 play important roles in the host defense against the virus 36 . Our previous studies have shown that Dengue virus promotes vascular leakage in mice by inducing IL-1β production 37 . Moreover, IL-1β is a highly potent proinflammatory mediator that induces vasodilation and attracts granulocytes to the inflamed tissues 38 . Similar to the function of IL-1β, IL-6 also plays a role by amplifying the innate immune response by recruiting additional immune mediators 39 . Clinical research found that the expression of IL-1β and IL-6 in serum and alveolar lavage fluid are significantly increased in patients infected with SARS-CoV-2 5 . Our results also demonstrate that the levels of IL-1β and IL-6 in the sera and lung are increased by the lung-specific expression of N protein, but the expressions of IL-1β and IL-6 are significantly decreased by the administration of MCC950, Ac-YVAD-cmk, or in AAV-N-infected NLRP3 −/− mice. More interestingly, in the sepsis mouse model 40 , N protein significantly increases the time of mouse death, whereas the addition of MCC950 or Ac-YVAD-cmk not only delays the time of death but also improves the survival rate of the mice. This suggests that SARS-CoV-2 N protein may cause lung injury through inducing the production of proinflammatory factors such as IL-1β and IL-6.
NLRP3 inflammasome is composed of a sensor protein (NLRP3), an adaptor protein (ASC), and an effector protein (Caspase-1) 15 . During the activation of NLRP3 inflammasome, NLRP3 deubiquitination 41 and self-aggregation 27 first occurs, followed by ASC recruitment and oligomerization 28 , and then pro-caspase-1 is cut into active caspase-1 (p20), which can cleave pro-IL-1β and pro-IL-18 to produce mature IL-1β (p17) and IL-18, respectively 16 . Our results prove that SARS-CoV-2 N protein can directly interact with NLRP3 to promote the aggregation of NLRP3 protein and further studies indicate that N protein can accelerate the interaction between NLRP3 and ASC, thus facilitating the oligomerization of ASC. Immunofluorescence experiments prove that the N-NLRP3-ASC can form a complex, leading to the activation of NLRP3 inflammasome. Even more interestingly, a Co-IP experiment indicates that the CTD domain of N protein (260aa-340aa) 33 mediates the interaction with NLRP3. Moreover, missing this domain, the levels of p17, p20, and ASC oligomerization is significantly reduced. This further indicates that the interaction between N and NLRP3 protein is essential for activating the NLRP3 inflammasome. It is worth noting that our results show that N protein has no effect on the expression of IL-18, which also as an effector protein downstream of the NLRP3 inflammasome 15 , seeming a little contradiction. We speculate that maybe N protein had no effect on the mRNA level of IL-18, leading to an extremely low level of protein expression, so the effect of N protein on IL-18 is not detected in our experiment.
In conclusion, our study demonstrate that SARS-CoV-2 nucleocapsid protein (N) can 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 and the occurrence of lung injury in mice. Our data indicate that inhibition of NLRP3 inflammasome can reduce "cytokine storm" and lung injury caused by SARS-CoV-2 infection, suggesting that NLRP3 inflammasome is a potential target for COVID-19 therapy.

Methods
Mice. Wild-type C57BL/6 mice were purchased from Guangdong Medical Laboratory Animal Center, Guangzhou, China. NLRP3 −/− C57BL/6 mice have been bred in our laboratory 14 . Mice were bred and maintained under specific pathogen-free conditions. Six-week-old mice were tail vein-injected with 300 μl of AAV-Lung-EGFP-N (5 × 10 11 vg) or AAV-Lung-EGFP (purchased from OBiO 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.
Technology, Shanghai, China). Sera were collected at 1 week and 3 weeks for all groups from retro-orbital. After 4 weeks, mice were treated with LPS (30 mg/kg) by intraperitoneal injection 40 . Mice were killed and tissues were collected for histoimmunofluorescence or histopathology analysis.
Mouse BMDMs were isolated from 6-week-old male C57BL/6 mice. Cells were cultured in RPMI 1640 medium with 10% fetal bovine serum and 10% GM-CSF conditioned medium from L929 cells for 6 days.
Ethics statement. All animal studies were performed in accordance with the principles described by the Animal Welfare Act and the National Institutes of Health Guidelines for the care and use of laboratory animals in biomedical research. All procedures involving mice and experimental protocols were approved by Institute of Laboratory Animal Science, Jinan University. The animal ethic committee number is 20200828-09.
Cell lines and cultures. Embryonic kidney cell lines (HEK293T), human pulmonary epithelial cell (A549), and human monocytic cell lines (THP-1) were purchased from American Type Culture Collection. HEK293T and A549 cells were grown in Dulbecco's modified Eagle medium and THP-1 cells were grown in RPMI 1640 medium. All the medium was supplemented with 10% fetal calf serum, 100 U/ ml penicillin, and 100 μg/ml streptomycin, and maintained at 37°C in a 5% CO 2 incubator. THP-1 cells were differentiated into macrophages by stimulating with PMA for 12 h. The cells were then cultured in fresh medium for 24 h and stimulated with Nigericin, LPS, or ATP. Supernatants were collected for measurement of IL-6, IL-18, and IL-1β (p17) proteins. Cells were collected for qRT-PCR and immunoblot analysis.
ASC oligomerization analysis. HEK293T cells, PMA-differentiated THP-1 cells, and GM-CSF-differentiated BMDMs were lysed in lysis buffer, gently shaken at 4°C for 30 min, and centrifuged at 3380 × g at 4°C for 10 min. Pellets were washed three times with PBS and re-suspended in 200 μl PBS. DSS (2 mM) was added to re-suspended pellets, which were cross-linked at 37°C for 30 min. The samples were centrifuged at 3380 × g for 10 min, cross-linked pellets were re-suspended in 50 μl 2× SDS loading buffer, boiled for 10 min, and analyzed by western blotting.
Statistical analyses. All experiments were repeated at least three times with similar results. Statistical analysis was carried out using the t-test for two groups and one-way analysis of variance for multiple groups (GraphPad Prism7). The date was considered statistically significant when *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; ns stands for not significant.
Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability
RNA-Seq data accessed are deposited in the GEO database under the accession code GSE155106. All other data are included in the article and Supplemental Information, or available from the authors upon reasonable requests. Source data are provided with this paper.