SARS-COV-2 protein NSP9 promotes cytokine production by targeting TBK1

Abstract

SARS-COV-2 infection-induced excessive or uncontrolled cytokine storm may cause injury of host tissue or even death. However, the mechanism by which SARS-COV-2 causes the cytokine storm is unknown. Here, we demonstrated that SARS-COV-2 protein NSP9 promoted cytokine production by interacting with and activating TANK-binding kinase-1 (TBK1). With an rVSV-NSP9 virus infection model, we discovered that an NSP9-induced cytokine storm exacerbated tissue damage and death in mice. Mechanistically, NSP9 promoted the K63-linked ubiquitination and phosphorylation of TBK1, which induced the activation and translocation of IRF3, thereby increasing downstream cytokine production. Moreover, the E3 ubiquitin ligase Midline 1 (MID1) facilitated the K48-linked ubiquitination and degradation of NSP9, whereas virus infection inhibited the interaction between MID1 and NSP9, thereby inhibiting NSP9 degradation. Additionally, we identified Lys59 of NSP9 as a critical ubiquitin site involved in the degradation. These findings elucidate a previously unknown mechanism by which a SARS-COV-2 protein promotes cytokine storm and identifies a novel target for COVID-19 treatment.

Keywords: SARS-CoV-2; TBK1; antiviral immunity; cytokine storm; type I interferon.

Figure 1

NSP9 promotes cellular antiviral innate immune response. (A) Luciferase assay of IFN-β activation in HEK293T cells expressing various vectors and stimulated with VSV for 12 hours. (B) ELISA assay of IFN-β expression in L929 cells expressing various vectors and stimulated with VSV for 12 hours. (C, D) Ifnb, Isg15, Ccl5, Tnf and Il6 mRNA levels in L929 cells transfected with control or NSP9 vectors and stimulated with VSV (C) or SEV (D) for indicated times. (E, F) Immunoblot of lysates of L929 cells transfected with control or NSP9 vectors and infected with VSV (E) or SEV (F) for indicated times. (G) Immunoblot analysis of nuclear and cytoplasmic fractions in L929 cells transfected with control or NSP9 vectors and stimulated with VSV for indicated times. Data are representative of at least three independent experiments (mean ± SEM in C, D). ns > 0.05, **P < 0.01, and ***P < 0.001, two-tailed unpaired Student’s t-test.

Figure 2

NSP9 aggravated the tissue damage and death in vivo. (A) Survival of WT (n = 6 per group) infected intraperitoneally with rVSV or rVSV-NSP9 (5 × 10⁸ PFU/mouse) and monitored for 15 days. (B) ELISA assay of IFN-β in blood of WT mice (n = 6) intraperitoneally injected with rVSV or rVSV-NSP9 (5 × 10⁸ PFU/mouse) for 4 days. (C) Microscopy of hematoxylin-and-eosin-stained lung and spleen sections as in (A). (D) Ifnb, Ccl5, Isg15, Tnf and Il6 mRNA levels in the spleens, livers, and lungs of WT mice (n = 6) intraperitoneally injected with rVSV or rVSV-NSP9 (5 × 10⁸ PFU/mouse) for 4 days. Data are representative of at least three independent experiments (Kaplan–Meier analysis in A and mean ± SEM in D). ***P < 0.001, two-tailed unpaired Student’s t-test.

Figure 3

NSP9 interacts with TBK1. (A) Luciferase assay of IFN-β activation in HEK293T cells expressing various vectors and stimulated with VSV (left) or SEV (right) for 12 hours. (B, C) Luciferase assay of IFN-β (B) and ISRE (C) activation in HEK293T cells expressing various vectors. (D–F) Immunoassay of lysates of HEK293T cells expressing various vectors. (G, H) Direct binding of His–NSP9 with GST-TBK1 (G) or GST-TRAF3 (H). Data are representative of at least three independent experiments (mean ± SEM in A–C). ns > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001, two-tailed unpaired Student’s t-test.

Figure 4

NSP9 promotes the activation of TBK1. (A–C) Cell lysates from HEK293T cells transfected with various vectors for 48h and stimulated with VSV for 12 hours, were treated with Flag-beads to IP Flag-TBK1, then immunoassay of IP solution and input as we described in “MATERIALS AND METHODS” section, to detect whether TBK1 dimerization (A), TBK1-TRAF3 interaction (B), or TBK1-IRF3 interaction (C) were affected by NSP9 and virus infection. (D) Cell lysates from HEK293T cells transfected with various vectors were divided into IP and input part to detect which polyubiquitin chain-linked ubiquitination of TBK1 was influenced by NSP9. (E) Immunoassay of cell lysates from HEK293T cells transfected with various vectors and treated as in A to detect whether TBK1-RNF128 interaction was affected by NSP9. (F, G) Immunoassay of cell lysates from HEK293T cells transfected with various vectors and treated as in (D) to detect whether total ubiquitination (F) or K63-linked (G) of TBK1 was influenced by NSP9. (H) Luciferase assay of IFN-β activation in HEK293T cells expressing various vectors. Data are representative of at least three independent experiments (mean ± SEM in H). **P < 0.01, and ***P < 0.001, two-tailed unpaired Student’s t-test.

Figure 5

MID1 contributed to the degradation of NSP9. (A) Immunoassay of cell lysates from HEK293T cells transfected with NSP9 vector and stimulated with VSV for indicated times. (B) Nsp9 mRNA levels in L929 cells infected with VSV or SEV for indicated times. (C) Immunoassay of cell lysates from HEK293T cells transfected with various vectors and infected with VSV for indicated times, then stimulated with DMSO, MG132 or Chloroquine. (D–I) Immunoassay of cell lysates from HEK293T cells (D, F-H) or L929 cells (E, I) transfected with various vectors and infected with VSV for 12 hours, then stimulated with DMSO or MG132. (J) Immunoblot of lysates of L929 cells transfected with control or NSP9 vectors and infected with VSV for indicated times. (K, L) Ifnb mRNA levels in L929 cells transfected with control or NSP9 vectors and stimulated with VSV (K) or SEV (L) for 36 hours. (M) Immunoassay of lysates of HEK293T cells expressing various vectors and stimulated with DMSO or MG132. Data are representative of at least three independent experiments (mean ± SEM in K, L). ns > 0.05, ***P < 0.001, two-tailed unpaired Student’s t-test.

Figure 6

Lys59 is responsible for NSP9 degradation. (A) Luciferase assay of IFN-β (left) and ISRE (right) activation in HEK293T cells transfected with various vectors. (B) ELISA assay of IFN-β expression in L929 cells transfected with various vectors and stimulated with VSV or SEV for 12 hours. (C, D) Immunoassay of cell lysates from L929 cells transfected with various vectors and infected with VSV (C) or SEV (D) for 12 hours. (E) Immunoassay of cell lysates from HEK293T cells transfected with various vectors and infected with VSV for 12 hours, then stimulated with MG132. (F) Ifnb mRNA levels in L929 cells transfected with control or NSP9 vectors and stimulated with VSV (left) or SEV (right) for 36 hours. (G) Immunoblot of lysates of L929 cells transfected with control or NSP9 vectors and infected with VSV for 12 hours. (H) Immunoassay of cell lysates from HEK293T cells transfected with various vectors. (I) Immunoassay of cell lysates from HEK293T cells transfected with various vectors and stimulated with DMSO or MG132. (J) Immunoassay of cell lysates from L929 cells transfected with various vectors. (K) Immunoassay of cell lysates from HEK293T cells transfected with various vectors and stimulated with DMSO or MG132. Data are representative of at least three independent experiments (mean ± SEM in A, F). ***P < 0.001, two-tailed unpaired Student’s t-test.