Figure 6.
Glu-290 and Gln-349 in the CTD of the SARS-CoV-2 N protein are crucial for inhibiting the NF-κB pathway. (A) Domain organization of SARS-CoV-2 N and its truncated mutants. HEK293T cells were transfected with Myc-N full-length (FL), Myc-N′, Myc-C′, Myc-NTD, and Myc-CTD. Lysates were collected for immunoblot analysis. (B) Domain organization of SARS-CoV N and its truncated mutants. (C and D) HEK293T cells were transfected with Myc-SARS-CoV-2 N-C′, Myc-SARS-CoV-2 N-N′, Myc-empty vector, HA-empty vector, HA-TAB2 (C), or HA-TAB3 (D). Lysates were collected for immunoprecipitation (IP) (anti-HA) and immunoblot (IB) analysis. (E) HEK293T cells were transfected with Myc-SARS-CoV-2 N-C′, Myc-SARS-CoV N-C′, Myc-empty vector, HA-TAB2, HA-TAB3, and HA-empty vector. Lysates were collected for immunoprecipitation (anti-HA) and immunoblot analysis. (F and G) HEK293 cells were transfected with NF-κB-Luc, along with plasmids encoding Myc-SARS-CoV-2 N full-length (FL), Myc-SARS-CoV N FL, Myc-SARS-CoV-2 N-N′, Myc-SARS-CoV-2 N-C′, Myc-SARS-CoV-2 N-NTD, Myc-SARS-CoV-2 N-CTD, or Myc-SARS-CoV N-CTD. At 24 h after transfection, cells were infected with SeV for 10 h (F) or IL-1β for 4 h (G), and luciferase activity was measured. (H) Domain organization of SARS-CoV-2 N and its amino acid point mutations. (I) HEK293 cells were transfected with NF-κB-Luc, along with plasmids encoding Myc-SARS-CoV-2 N wild type (WT), Myc-SARS-CoV N WT, or SARS-CoV-2 N single-point mutants (Myc-A267Q, Myc-E290D, Myc-T334H, Myc-N345Q, and Myc-Q349N). At 24 h after transfection, cells were stimulated by IL-1β for 4 h, and luciferase activity was measured. (J) The structure of N (SARS-CoV N, SARS-CoV-2 N, and SARS-CoV-2 N mutations) was predicted by Alphafold3. Blue box, SARS-CoV N and SARS-CoV-2 N mutations CTD terminal common structure; red box, SARS-CoV-2 CTD terminal structure. (K) Caco-2 cells stably expressing SARS-CoV-2 N, SARS-CoV N, or SARS-CoV-2 N mutations (E290D and Q349N) via lentiviral transduction were lysed for immunoblot analysis. (L and M) Caco-2 cells stably expressing SARS-CoV-2 N, SARS-CoV N, or SARS-CoV-2 N mutations (E290D and Q349N) were infected with RDPs for 24 or 48 h. Total RNA extracted from the cells was evaluated by RT-qPCR. Horizontal lines in figures represent the average value of the positive control group. Graphs show mean ± SEM (n = 3 in F, G, I, L, and M) from one representative experiment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (unpaired, two-tailed Student’s t test). Data in C–E and K are done at least twice, and one representative is shown. Data in F, G, I, L, and M are done in at least three independent experiments. Source data are available for this figure: SourceData F6.

Glu-290 and Gln-349 in the CTD of the SARS-CoV-2 N protein are crucial for inhibiting the NF-κB pathway. (A) Domain organization of SARS-CoV-2 N and its truncated mutants. HEK293T cells were transfected with Myc-N full-length (FL), Myc-N′, Myc-C′, Myc-NTD, and Myc-CTD. Lysates were collected for immunoblot analysis. (B) Domain organization of SARS-CoV N and its truncated mutants. (C and D) HEK293T cells were transfected with Myc-SARS-CoV-2 N-C′, Myc-SARS-CoV-2 N-N′, Myc-empty vector, HA-empty vector, HA-TAB2 (C), or HA-TAB3 (D). Lysates were collected for immunoprecipitation (IP) (anti-HA) and immunoblot (IB) analysis. (E) HEK293T cells were transfected with Myc-SARS-CoV-2 N-C′, Myc-SARS-CoV N-C′, Myc-empty vector, HA-TAB2, HA-TAB3, and HA-empty vector. Lysates were collected for immunoprecipitation (anti-HA) and immunoblot analysis. (F and G) HEK293 cells were transfected with NF-κB-Luc, along with plasmids encoding Myc-SARS-CoV-2 N full-length (FL), Myc-SARS-CoV N FL, Myc-SARS-CoV-2 N-N′, Myc-SARS-CoV-2 N-C′, Myc-SARS-CoV-2 N-NTD, Myc-SARS-CoV-2 N-CTD, or Myc-SARS-CoV N-CTD. At 24 h after transfection, cells were infected with SeV for 10 h (F) or IL-1β for 4 h (G), and luciferase activity was measured. (H) Domain organization of SARS-CoV-2 N and its amino acid point mutations. (I) HEK293 cells were transfected with NF-κB-Luc, along with plasmids encoding Myc-SARS-CoV-2 N wild type (WT), Myc-SARS-CoV N WT, or SARS-CoV-2 N single-point mutants (Myc-A267Q, Myc-E290D, Myc-T334H, Myc-N345Q, and Myc-Q349N). At 24 h after transfection, cells were stimulated by IL-1β for 4 h, and luciferase activity was measured. (J) The structure of N (SARS-CoV N, SARS-CoV-2 N, and SARS-CoV-2 N mutations) was predicted by Alphafold3. Blue box, SARS-CoV N and SARS-CoV-2 N mutations CTD terminal common structure; red box, SARS-CoV-2 CTD terminal structure. (K) Caco-2 cells stably expressing SARS-CoV-2 N, SARS-CoV N, or SARS-CoV-2 N mutations (E290D and Q349N) via lentiviral transduction were lysed for immunoblot analysis. (L and M) Caco-2 cells stably expressing SARS-CoV-2 N, SARS-CoV N, or SARS-CoV-2 N mutations (E290D and Q349N) were infected with RDPs for 24 or 48 h. Total RNA extracted from the cells was evaluated by RT-qPCR. Horizontal lines in figures represent the average value of the positive control group. Graphs show mean ± SEM (n = 3 in F, G, I, L, and M) from one representative experiment. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant (unpaired, two-tailed Student’s t test). Data in C–E and K are done at least twice, and one representative is shown. Data in F, G, I, L, and M are done in at least three independent experiments. Source data are available for this figure: SourceData F6.

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