Figure S5.
SARS-CoV-2 N protein suppresses LPS-induced pulmonary inflammation in mice. (A) The lungs of K18-hACE2 KI mice infected with lentivirus expressing SARS-CoV-2 N, SARS-CoV N, E290D, or Q349N. Nuclear markers DAPI (blue), SARS-CoV-2 N, SARS-CoV N, E290D, and Q349N (red), and hACE2 (green) were visualized by microscopy. Scale bars, 100 μm. (B) K18-hACE2 KI mice were infected with lentivirus expressing SARS-CoV-2 N WT, SARS-CoV N WT, E290D, or Q349N. Lung lysates were collected for immunoblotting, n = 3. (C) Establishment of lung models of K18-hACE2 KI mice infected with lentivirus expressing SARS-CoV-2 N, SARS-CoV N, E290D, or Q349N, and activation of the NF-κB pathway by LPS. (D) The lungs of K18-hACE2 KI mice were infected with lentivirus expressing SARS-CoV-2 N, SARS-CoV N, E290D, or Q349N. SARS-CoV-2 N, SARS-CoV N, E290D, and Q349N (green) were visualized by microscopy. Scale bars, 50 μm. (E) K18-hACE2 KI mice were infected with lentivirus expressing SARS-CoV-2 N WT, SARS-CoV N WT, E290D, or Q349N, and lung lysates were collected for immunoblotting. (F and G) Levels of N (F), Tnfα, Il6, and Il1β (G) were measured by RT-qPCR in the lungs of mice infected with either empty lentivirus or lentivirus expressing SARS-CoV-2 N, SARS-CoV N, E290D, or Q349N. LPS activated inflammatory pathways, PBS as control, n = 5. Horizontal lines in F represents the average value of the Lenti-SARS-CoV-2 N+LPS group. Horizontal lines in G represents the average value of the positive control group. (H) Heatmap of inflammatory genes in the Lenti-Vector, Lenti-SARS-CoV-2 N, Lenti-SARS-CoV N, Lenti-E290D, and Lenti-Q349N. LPS activated inflammatory pathways, PBS as control, n = 3. (I) GSEA plot depicting the enrichment of genes in cytokine-cytokine receptor interaction, NF-κB signaling pathway, and Toll-like receptor signaling pathway in the lungs of mice infected with empty lentivirus and lentivirus expressing SARS-CoV-2 N protein. LPS activated inflammatory pathways, PBS as control, n = 3. A positive normalized enrichment score (NES) indicates that the gene set is enriched in upregulated genes, while a negative NES suggests enrichment in downregulated genes. Graphs show mean ± SEM (n = 5 in F and G). *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant (unpaired, two-tailed Student’s t test). Source data are available for this figure: SourceData FS5.

SARS-CoV-2 N protein suppresses LPS-induced pulmonary inflammation in mice. (A) The lungs of K18-hACE2 KI mice infected with lentivirus expressing SARS-CoV-2 N, SARS-CoV N, E290D, or Q349N. Nuclear markers DAPI (blue), SARS-CoV-2 N, SARS-CoV N, E290D, and Q349N (red), and hACE2 (green) were visualized by microscopy. Scale bars, 100 μm. (B) K18-hACE2 KI mice were infected with lentivirus expressing SARS-CoV-2 N WT, SARS-CoV N WT, E290D, or Q349N. Lung lysates were collected for immunoblotting, n = 3. (C) Establishment of lung models of K18-hACE2 KI mice infected with lentivirus expressing SARS-CoV-2 N, SARS-CoV N, E290D, or Q349N, and activation of the NF-κB pathway by LPS. (D) The lungs of K18-hACE2 KI mice were infected with lentivirus expressing SARS-CoV-2 N, SARS-CoV N, E290D, or Q349N. SARS-CoV-2 N, SARS-CoV N, E290D, and Q349N (green) were visualized by microscopy. Scale bars, 50 μm. (E) K18-hACE2 KI mice were infected with lentivirus expressing SARS-CoV-2 N WT, SARS-CoV N WT, E290D, or Q349N, and lung lysates were collected for immunoblotting. (F and G) Levels of N (F), Tnfα, Il6, and Il1β (G) were measured by RT-qPCR in the lungs of mice infected with either empty lentivirus or lentivirus expressing SARS-CoV-2 N, SARS-CoV N, E290D, or Q349N. LPS activated inflammatory pathways, PBS as control, n = 5. Horizontal lines in F represents the average value of the Lenti-SARS-CoV-2 N+LPS group. Horizontal lines in G represents the average value of the positive control group. (H) Heatmap of inflammatory genes in the Lenti-Vector, Lenti-SARS-CoV-2 N, Lenti-SARS-CoV N, Lenti-E290D, and Lenti-Q349N. LPS activated inflammatory pathways, PBS as control, n = 3. (I) GSEA plot depicting the enrichment of genes in cytokine-cytokine receptor interaction, NF-κB signaling pathway, and Toll-like receptor signaling pathway in the lungs of mice infected with empty lentivirus and lentivirus expressing SARS-CoV-2 N protein. LPS activated inflammatory pathways, PBS as control, n = 3. A positive normalized enrichment score (NES) indicates that the gene set is enriched in upregulated genes, while a negative NES suggests enrichment in downregulated genes. Graphs show mean ± SEM (n = 5 in F and G). *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant (unpaired, two-tailed Student’s t test). Source data are available for this figure: SourceData FS5.

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