Figure S4.
Glu-290 and Gln-349 in the CTD of SARS-CoV-2 N protein are key sites for inhibiting NF-κB pathway. (A) 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 stimulated by TNF-α for 4 h, and luciferase activity was measured. (B) HEK293T cells were transfected with Myc-SARS-CoV-2 N wild type (WT) or single-point mutants (Myc-A267Q, Myc-E290D, Myc-T334H, Myc-N345Q, and Myc-Q349N). Lysates were collected for immunoblotting. (C) HEK293 cells were transfected with NF-κB-luc, along with plasmids encoding Myc-SARS-CoV-2 N 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 TNF-α for 4 h, and luciferase activity was measured. (D and E) HEK293 cells were transfected with NF-κB-luc, along with plasmids encoding Myc-SARS-CoV-2 N WT, Myc-SARS-CoV N WT, SARS-CoV-2 N single-point mutants (Myc-E290D or Myc-Q349N), or SARS-CoV-2 N double-point mutant (Myc-E290D+Q349N). At 24 h after transfection, cells were stimulated by IL-1β (D) or TNF-α (E) for 4 h, and luciferase activity was measured. (F and G) HEK293T cells were transfected with Flag-TAK1, Flag-empty vector, Myc-Q349N, Myc-empty vector, HA-empty vector, HA-TAB2 (F), or HA-TAB3 (G). Lysates were collected for immunoprecipitation (IP) (anti-HA) and immunoblotting (IB). The ratio of the gray value between IP TAK1-Flag and IP TAB2-HA or IP TAB3-HA was determined. (H) N protein sequence comparison between SARS-CoV-2, SARS-CoV, BA.1, BA.1.1, BA.2, BF.7, BF7.4, BQ.1, BQ.1.1, XBB.1, and XBB.1.16. The full-length N protein sequences of SARS-CoV-2 (YP_009724397.2), SARS-CoV (YP_009825061.1), BA.1 (EPI_ISL_10543999), BA.1.1 (EPI_ISL_10065996), BA.2 (EPI_ISL_10746692), BF.7 (EPI_ISL_16536280), BF7.4 (EPI_ISL_15800501), BQ.1 (EPI_ISL_16536390), BQ.1.1 (EPI_ISL_16549162), XBB.1 (EPI_ISL_16385568), and XBB.1.16 (EPI_ISL_17233345) were aligned using MUSCLE. The aligned sequences were further confirmed using a similarity plot implemented in Simplot 3.5.1. Horizontal lines in figures represent the average value of the positive control group. Graphs show mean ± SEM (n = 3 in A and C–E). ***P < 0.001, ****P < 0.0001, ns, not significant (unpaired, two-tailed Student’s t test). Data in B, F, and G are expressed at least twice in independent experiments, and one representative is shown. Data in A and C–E are expressed in at least three independent experiments. Source data are available for this figure: SourceData FS4.

Glu-290 and Gln-349 in the CTD of SARS-CoV-2 N protein are key sites for inhibiting NF-κB pathway. (A) 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 stimulated by TNF-α for 4 h, and luciferase activity was measured. (B) HEK293T cells were transfected with Myc-SARS-CoV-2 N wild type (WT) or single-point mutants (Myc-A267Q, Myc-E290D, Myc-T334H, Myc-N345Q, and Myc-Q349N). Lysates were collected for immunoblotting. (C) HEK293 cells were transfected with NF-κB-luc, along with plasmids encoding Myc-SARS-CoV-2 N 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 TNF-α for 4 h, and luciferase activity was measured. (D and E) HEK293 cells were transfected with NF-κB-luc, along with plasmids encoding Myc-SARS-CoV-2 N WT, Myc-SARS-CoV N WT, SARS-CoV-2 N single-point mutants (Myc-E290D or Myc-Q349N), or SARS-CoV-2 N double-point mutant (Myc-E290D+Q349N). At 24 h after transfection, cells were stimulated by IL-1β (D) or TNF-α (E) for 4 h, and luciferase activity was measured. (F and G) HEK293T cells were transfected with Flag-TAK1, Flag-empty vector, Myc-Q349N, Myc-empty vector, HA-empty vector, HA-TAB2 (F), or HA-TAB3 (G). Lysates were collected for immunoprecipitation (IP) (anti-HA) and immunoblotting (IB). The ratio of the gray value between IP TAK1-Flag and IP TAB2-HA or IP TAB3-HA was determined. (H) N protein sequence comparison between SARS-CoV-2, SARS-CoV, BA.1, BA.1.1, BA.2, BF.7, BF7.4, BQ.1, BQ.1.1, XBB.1, and XBB.1.16. The full-length N protein sequences of SARS-CoV-2 (YP_009724397.2), SARS-CoV (YP_009825061.1), BA.1 (EPI_ISL_10543999), BA.1.1 (EPI_ISL_10065996), BA.2 (EPI_ISL_10746692), BF.7 (EPI_ISL_16536280), BF7.4 (EPI_ISL_15800501), BQ.1 (EPI_ISL_16536390), BQ.1.1 (EPI_ISL_16549162), XBB.1 (EPI_ISL_16385568), and XBB.1.16 (EPI_ISL_17233345) were aligned using MUSCLE. The aligned sequences were further confirmed using a similarity plot implemented in Simplot 3.5.1. Horizontal lines in figures represent the average value of the positive control group. Graphs show mean ± SEM (n = 3 in A and C–E). ***P < 0.001, ****P < 0.0001, ns, not significant (unpaired, two-tailed Student’s t test). Data in B, F, and G are expressed at least twice in independent experiments, and one representative is shown. Data in A and C–E are expressed in at least three independent experiments. Source data are available for this figure: SourceData FS4.

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