Activation and evasion of type I interferon responses by SARS-CoV-2

Abstract

The pandemic of COVID-19 has posed an unprecedented threat to global public health. However, the interplay between the viral pathogen of COVID-19, SARS-CoV-2, and host innate immunity is poorly understood. Here we show that SARS-CoV-2 induces overt but delayed type-I interferon (IFN) responses. By screening 23 viral proteins, we find that SARS-CoV-2 NSP1, NSP3, NSP12, NSP13, NSP14, ORF3, ORF6 and M protein inhibit Sendai virus-induced IFN-β promoter activation, whereas NSP2 and S protein exert opposite effects. Further analyses suggest that ORF6 inhibits both type I IFN production and downstream signaling, and that the C-terminus region of ORF6 is critical for its antagonistic effect. Finally, we find that IFN-β treatment effectively blocks SARS-CoV-2 replication. In summary, our study shows that SARS-CoV-2 perturbs host innate immune response via both its structural and nonstructural proteins, and thus provides insights into the pathogenesis of SARS-CoV-2.

Fig. 1. SARS-CoV-2 infection induces the expression of antiviral genes.

a Calu-3 cells were mock-infected or infected with SARS-CoV-2 at an MOI of 0.5. At 4, 8, 12, and 24 h after infection, total RNA extracted from cells was evaluated by quantitative real-time PCR (qRT-PCR) using SYBR green method. The data are expressed as fold changes of the RNA levels of the viral N gene relative to the GAPDH control. b Calu-3 cells were mock-infected or infected with SARS-CoV-2 at an MOI of 0.5. At 4, 8, 12, and 24 h after infection, supernatants were collected, and viral titers were detected by using TCID₅₀ assay. c, d Cells from a were collected, and total RNA extracted from the cells was evaluated by qRT-PCR using SYBR green method. The data are expressed as fold change of the IFNB mRNA c and ISG56 mRNA d levels relative to the GAPDH control. e–g Calu-3 cells were mock-infected or infected with Sendai virus. Cells were harvested and analyzed as described in c and d. h, i Calu-3 cells were transfected with high molecular weight poly(I:C) (poly(I:C)-HMW). Cells were harvested at indicated times and analyzed by qRT-PCR. All experiments were done at least twice, and one representative is shown. Error bars indicate SD of technical triplicates. *P < 0.05, **P < 0.01, and ***P < 0.001, two-tailed Student’s t-test. Source data are provided as a Source Data file.

Fig. 2. Identification of viral proteins perturbing IFN-β production.

a Schematic diagrams of the SARS-CoV-2 genome. The genome includes 5′UTR-ORF1a-ORF1b-S-ORF3-E-M-ORF6-ORF7 (7a and 7b)-ORF8-N-3′UTR in order. Fifteen nonstructural proteins, four structural proteins, and four accessory proteins were delineated. b Protein expressions of SARS-CoV-2 genes. HEK293T cells were transfected with 500 ng plasmid in 24-well plates. Protein expressions were detected by Western blot. β-actin was used as a loading control. c Effect of SARS-CoV-2 proteins on SeV-induced IFN-β promoter activation. HEK293T cells were transfected with an IFN-β reporter plasmid, along with a control plasmid or with plasmids expressing the indicated SARS-CoV-2 proteins. At 24 h post-transfection, cells were infected with SeV for 12 h, and luciferase activity was measured. d, e Effects of SARS-CoV-2 proteins on RIG-IN and MDA5-induced IFN-β promoter activation. HEK293T cells were transfected with IFN-β promoter plasmid, along with a control plasmid or with plasmids expressing the indicated SARS-CoV-2 proteins, together with a plasmid expressing RIG-IN d or MDA5 e. At 24 h post-transfection, cells were harvested and luciferase activity was measured. f–h Expressions levels of SARS-CoV-2 protein, RIG-IN, and MDA5. Lysates of cells from c–e were subjected to Western blot analysis. Arrows indicate remnants of blots for SARS-CoV-2 proteins. All experiments were done at least twice, and one representative is shown. Error bars indicate SD of technical triplicates. *P < 0.05, **P < 0.01, and ***P < 0.001, two-tailed Student’s t-test. Source data are provided as a Source Data file.

Fig. 3. SARS-CoV-2 ORF6 inhibits RIG-I-like signaling pathways.

a Effects of SARS-CoV-2 ORF6 on SeV-induced IFN-β promoter activation. HEK293T cells were transfected with an IFN-β reported plasmid, along with a control plasmid or with increasing amounts plasmids expressing ORF6. Cells were infected with SeV for 12 h and assayed for luciferase activity. b Effects of SARS-CoV-2 ORF6 on poly(I:C)-induced IFN-β promoter activation. HEK293T cells were transfected as described in a. At 24 h post-infected, cells were transfected with high molecular weight poly(I:C) (poly(I:C)-HMW) for 12 h and assayed for luciferase activity. c–f Effects of ORF6 on RIG-IN, MDA5, MAVS, or IRF3-induced IFN-β promoter activation. HEK293T cells were transfected with an IFN-β reporter plasmid, along with a control plasmid or with increasing amount plasmids expressing ORF6, together with plasmids expressing RIG-IN (c), MDA5 (d), MAVS (e), or IRF3-5D (f). At 24 h post-transfection, luciferase activity was measured. g, h Effect of SARS-CoV ORF6 and SARS-CoV-2 ORF6 on MAVS and IRF3-5D-induced IFN-β promoter activation. 293T cells were transfected with IFN-β reporter plasmid, along with a control plasmid or plasmids expressing SARS-CoV ORF6 or SARS-CoV-2 ORF6, together with plasmids expressing MAVS (g) or IRF3-5D (h). At 24 h post-transfection, luciferase activity was measured. i Confocal immunofluorescence imaging of IRF3 and SARS-CoV-2 ORF6. HEK293 cells were transfected with a control plasmid or a plasmid expressing SARS-CoV-2 ORF6. At 24 h of post-infection, cells were infected with SeV. At 4 h of post-infection, cells were stained with indicated antibodies and subjected to immunofluorescence analyses. Red: IRF3 antibody signal; Green: ORF6 signal; Blue: DAPI (nuclei staining). Merge 1 and Merge 2 indicate the merged red and green channels and the merged red, green, and blue channels, respectively. Scale bar, 10 μm. All experiments were done at least twice, and one representative is shown. Error bars indicate SD of technical triplicates. *P < 0.05, **P < 0.01, and ***P < 0.001, two-tailed Student’s t-test. Source data are provided as a Source Data file.

Fig. 4. ORF6 antagonizes innate immune response via its C terminus.

a Schematic diagram of SARS-CoV-2 ORF6 variants. ORF6-WT: wildtype, ORF6-M1: amino acids 49–52 were substituted with alanines, ORF6-M2: amino acids 53–55 were substituted with alanines; ORF6-M3: amino acids from 56 to 61 were substituted with alanines. b–e The effect of ORF6 mutants on IFN-β promoter activation. HEK293T cells were transfected with an IFN-β reporter plasmid, along with a control plasmid or plasmids expressing wildtype ORF6 or indicated ORF6 variants, together with plasmids expressing RIG-IN (b), MDA5 (c), MAVS (d), or IRF3-5D (e). At 24 h after transfection, luciferase activity was measured. Protein expression levels were detected by Western blot. All experiments were done at least twice, and one representative is shown. Error bars indicate SD of technical triplicates. *P < 0.05, **P < 0.01, and ***P < 0.001, ns not significant, two-tailed Student’s t-test. Source data are provided as a Source Data file.

Fig. 5. ORF6 inhibits ISRE promoter activation.

a The effects of SARS-CoV-2 proteins on ISRE-promoter activation. HEK293T cells were transfected with an ISRE reporter plasmid, along with a control plasmid or with plasmids expressing the indicated SARS-CoV-2 proteins. At 24 h post-transfection, cells were treated with 800 U/ml of human IFN-β for 12 h, and luciferase activity was measured. b, c The effects of SARS-CoV-2 ORF6 on ISRE and ISG56 promoter activity. HEK293T cells were transfected with am ISRE reporter plasmid (b) and an ISG56 reporter plasmid (c), along with a control plasmid or the increasing amounts of plasmids expressing SARS-CoV-2 ORF6. At 24 h post-transfection, cells were treated with 800 U/ml of human IFN-β for 12 h, and luciferase activity was measured. d, e Effect of SARS-CoV ORF6 and SARS-CoV-2 ORF6 on ISRE and ISG56 promoters. HEK293T cells were transfected with an ISRE reporter plasmid (d) or an ISG56 reporter plasmid (e), along with a control plasmid or plasmids expressing SARS-CoV ORF6 or SARS-CoV-2 ORF6. At 24 h post-transfection, cells were treated with 800 U/ml of human IFN-β for 12 h, and luciferase activity was measured. All experiments were done at least twice, and one representative is shown. Error bars indicate SD of technical triplicates. *P < 0.05, **P < 0.01, and ***P < 0.001, two-tailed Student’s t-test. Source data are provided as a Source Data file.

Fig. 6. ORF6 inhibits STAT1 nuclear translocation but not phosphorylation.

a Effect of SARS-CoV ORF6 and SARS-CoV-2 ORF6 on IFN-β-induced phosphorylation of STAT1. HEK293T cells were transfected with a control plasmid or with plasmids expressing SARS-CoV ORF6, SARS-CoV-2 ORF6, or SOCS1. At 24 h after transfection, cells were left untreated or treated with 1000 U/ml IFN-β for 30 min. The phosphorylation of STAT1 was detected by Western blot analyses. b Effect of SARS-CoV ORF6 and SARS-CoV-2 ORF6 on IFN-β-induced nuclear translocation of STAT1. Vero cells were transfected with plasmids expressing SARS-CoV ORF6 and SARS-CoV-2 ORF6. At 24 h after transfection, cells were treated with 1000 U/ml IFN-β for 30 min and stained with indicated antibodies. Merge 1 and Merge 2 indicate the merged red and green channels and the merged red, green, and blue channels, respectively. Scale bar, 10 μm. c Quantitation of the nuclear translocation of STAT1. All experiments were done at least twice, and one representative is shown. Error bars indicate SD of technical triplicates. ***P < 0.001, two-tailed Student’s t-test. Source data are provided as a Source Data file.

Fig. 7. ORF6 inhibits STAT1 nuclear translocation via its C-terminus.

a, b Effect of SARS-CoV-2 ORF6 and its mutants on IFN-β-induced ISRE- and ISG56-promoter activation. HEK293T cells were transfected with an ISRE reporter plasmid (a) or an ISG56 reporter plasmid (b), along with a control plasmid or plasmids expressing wildtype or indicated SARS-CoV-2 ORF6 variants. At 24 h post-transfection, cells were treated with 800 U/ml IFN-β for 12 h, and luciferase activity was measured. Protein expression levels were detected by Western blot analyses. c Effect of SARS-CoV-2 ORF6 and its variants on IFN-β-induced STAT1 nuclear translocation. Vero cells were transfected with plasmids expressing wildtype ORF6 or indicated ORF6 variants. At 24 h post-transfection, cells were treated with 1000 U/ml of human IFN-β for 30 min and stained with indicated antibodies. Merge 1 and Merge 2 indicate the merged red and green channels and the merged red, green, and blue channels, respectively. Scale bar, 10 μm. All experiments were done at least twice, and one representative is shown. Error bars indicate SD of technical triplicates, **P < 0.01, and ***P < 0.001, ns not significant, two-tailed Student’s t-test. Source data are provided as a Source Data file.

Fig. 8. IFN-β inhibits SARS-CoV-2 replication.

a, b Expressions of ISG54 and ISG56 upon IFN-β treatment. Calu-3 cells were left untreated or treated with 100 or 500 U/ml of recombinant human IFN-β for 18 h. Total RNA was extracted, and the ISG54 and ISG56 mRNA were detected by qRT-PCR. c Susceptibility of SARS-CoV-2 to IFN-β treatment. Calu-3 cells were left untreated or pretreated with 100 or 500 U/ml human IFN-β for 18 h, and then cells were mock-infected or infected with SARS-CoV-2 at an MOI of 0.5. After 24 h post-infection, SARS-CoV-2 RNA was detected by qRT-PCR using SYBR green. d Viral titer assessment. Supernatants from c were harvested and subjected to TCID₅₀ analyses for measuring viral titers. All experiments were done at least twice, and one representative is shown. Error bars indicate SD of technical triplicates. ***P < 0.001(***), two-tailed Student’s t-test. Source data are provided as a Source Data file.