Fig 1.
SARS-CoV-2 infection dynamically regulates viral defence pathways in infected host cells.
a: PCA (principal component analysis) of the transcriptomes of Calu-3 cells infected with SARS-CoV-2 at an MOI of 2. Infected cells were collected at 6, 24, 48 and 72 hours post-infection (hpi). b: Gene clustering based on temporal expression patterns during infection was performed using the Mfuzz R package. The color scale reflects the membership values of the genes; blue, white and pink indicate low membership values, whereas red indicates high membership values. The numbers in brackets indicate the number of genes in each cluster (C) with a membership value ≥ 0.3. c: Gene Ontology enrichment analysis revealed that ‘Defense response to virus’ is significantly enriched only in genes of C1 (red bar) but not in those of other clusters (gray bars). The dashed line represents the threshold of the significant enrichment P value with Benjamini-Hochberg (B-H) correction (adjusted p = 0.05). d: Heatmap of the activity scores of immune-related pathways at different time points post infection calculated using PROGENy. The color scale indicates the Z score of pathway activity scores, with blue indicating lower activity and red indicating higher activity. e: The level of activation (red) or inhibition (blue) of immune-related pathways at 24 and 48 hpi. The regulation level of the pathways is presented as a t-statistic based on PROGENy with linear modeling. The color intensity indicates the -log10(B-H adjusted p values). f: Pearson correlation network of immune-related pathways in SARS-CoV-2-infected Calu-3 cells. Nodes represent individual pathways, and the edges between the nodes indicate the correlation of their activity scores. The color scale represents the Pearson correlation coefficient, in which red indicates a positive correlation, and blue indicates a negative correlation. Only edges with an absolute correlation greater than 0.3 are shown. g: Western blot analysis showing the dynamic regulation of key members of the canonical NF-κB pathway in N protein-expressing Caco-2 cells infected with SARS-CoV-2 replicon delivery particles (RDPs) lacking the N gene at an MOI of 0.001. The mock infection group was cultured with an equal volume of DMEM. The phosphorylated and total protein levels of p65 and IκBα were detected with the corresponding antibodies, and the signal intensity was quantified using ImageJ. The color scale represents the Z scores of the relative ratios of phosphorylated and total proteins, with red indicating upregulation and blue indicating downregulation. The results are representative of two independent experiments.
Fig 2.
A pretrained protein language model identifies viral protein interactions with the canonical NF-κB pathway.
a: Schematic overview of the geometric weighted pathway interaction score (GWPIS) for quantifying the interactions between viral proteins and immune-related pathways in infected host cells. Step 1: Data collection of the sequences of SARS-CoV-2 proteins and host proteins in the immune pathways shown in Fig 1d. Step 2: V-H protein interaction score: The D-SCRIPT protein language model was used to compute the interaction scores between SARS-CoV-2 proteins and host proteins in immune-related pathways. Step 3: Pathway-level interaction scores were calculated by integrating the pairwise interaction scores from Step 2 and the gene weights obtained from the PROGENy gene-pathway weight matrix. Created in BioRender. Yang, Q. (2026) https://BioRender.com/41xkykd. b: Interactions between SARS-CoV-2 proteins and the canonical NF-κB pathway, as calculated by GWPIS. Each circle represents a SARS-CoV-2 protein, and the color intensity and thickness of the lines reflect the interaction scores. Green, structural proteins (S, E, M, and N); blue, ORF proteins; red/pink, nonstructural proteins. c: Western blot analysis showing the regulation of key proteins in the canonical NF-κB pathway in HEK293T cells expressing SARS-CoV-2 Nsps or GFP as a control (Ctrl). The phosphorylated and total protein levels of p65 and IκBα were probed with the corresponding antibodies, and the relative intensity signals were quantified using ImageJ. d: Bar chart showing the ratios of phosphorylated and total protein levels in (c). The red dashed line represents the ratio in the GFP-expressing control cells (black). e: Activities of the promoter containing NF-κB response elements (κB sites) were measured by a dual luciferase assay. HEK293T cells were transfected with a plasmid expressing a Renilla luciferase reporter driven by a promoter containing 2 κB sites and a control Firefly luciferase 24 hr before transfection of Nsp1 or control GFP-expressing plasmids. The cells were collected 24 h later for the dual luciferase assay. Each dot represents a replicate, and the mean and SEM are shown. P values, Mann-Whitney test. f: Relative mRNA levels of IL-8, IP-10 and TNF-α in HEK293T cells transfected with plasmids expressing cMYC-tagged Nsp1 or the GFP control (Ctrl). GAPDH was used as an internal control. Each dot represents a replicate, and the mean and SEM are shown. P values, Mann-Whitney test. The results in e-f are representative of two independent experiments.
Fig 3.
SARS-CoV-2 Nsp1 interacts with the TAK1/TAB1 complex.
a-b: Co-IP and reciprocal co-IP analyses confirm the interaction between Nsp1 and TAK1. HEK293T cells were transfected with plasmids expressing FLAG-tagged TAK1 and cMYC-tagged Nsp1 or GFP as a control (Ctrl). Immunoprecipitation (IP) was performed using anti-cMYC (a) and anti-FLAG (b) magnetic beads. Nsp1 (red arrowhead) and GFP (green arrowhead) were detected by anti-cMYC, and TAK1 was detected by anti-FLAG antibodies in a, b and f. c: N-Caco-2 cells were infected with ΔN SARS-CoV-2 RDPs and harvested at 48 hpi. IP was performed using anti-Nsp1 antibody or anti-IgG as a control. TAK1 was detected by an anti-TAK1 antibody. d: Schematics of the TAK1 truncation proteins (upper panel) and co-IP analysis showing that the N-terminus of TAK1 interacts with Nsp1 (lower panel). HEK293T cells were transfected with plasmids expressing FLAG-tagged N (1-303 aa)- or C (304-606 aa)-terminal truncation of TAK1 24 hr prior to the transfection of cMYC-tagged Nsp1- or GFP (Ctrl)-expressing plasmids. Immunoprecipitation (IP) was performed using anti-cMYC magnetic beads. e: SPR (surface plasmon resonance) analysis. Recombinant TAK1 protein (1-303 aa) was diluted and immobilized onto the sensor chip CM5. The recombinant Nsp1 protein was diluted in PBS at different concentrations. The KD value of the Nsp1-TAK1 interaction was 2.3 × 10-7 M. f-g: HEK293T cells were cotransfected with plasmids expressing FLAG-tagged TAK1 and cMYC-tagged Nsp1 at different concentrations (0.5, 1, 2 or 4 μg) (f) or with only the Nsp1-expressing plasmid (g). TAK1 was detected by anti-FLAG in (f) or by anti-TAK1 antibody in (g). The relative signals were quantified using ImageJ. The results in a-g are representative of two independent experiments.
Fig 4.
SARS-CoV-2 Nsp1 inhibits TAK1-mediated NF-κB and MAPK signaling.
a, d: Western blot analysis showing the phosphorylated and total protein levels of p65 and IκBα (a) or p38 and Erk (d) in Nsp1-expressing cells. HEK293T cells were transfected with a plasmid expressing FLAG-tagged GFP (Ctrl), TAK1 or TAB1 24 hr prior to transfection of a plasmid expressing cMYC-tagged Nsp1 (Nsp1-cMYC) or GFP (GFP-cMYC, Ctrl), and the cell lysates were collected 24 hr later. FLAG-tagged GFP (green arrowhead), TAK1 (red arrowhead) and TAB1 (blue arrowhead) were detected by anti-FLAG antibody. GFP was detected by anti-cMYC and anti-Nsp1 antibodies in a, b, d & e. The relative intensity signals quantified using ImageJ are shown in the right panel of a-e. b, e: Phosphorylated and total protein levels of p65 and IκBα (b) and p38 and Erk (e) in Nsp1-expressing cells. HEK293T cells were transfected with the Nsp1-cMYC or GFP-cMYC plasmid for 24 h, after which the culture medium was replaced with DMEM supplemented with 1% FBS for 12 h and then stimulated with IL-1β (20 ng/mL, +) or PBS (-) for 10 min. c: HEK293T cells were (co-)transfected with plasmids expressing Strep-tagged Nsp2, Nsp1 (Nsp1-cMYC) and/or GFP (GFP-cMYC, Ctrl). f: Activities of the promoters of AP-1 family transcription factors measured by a dual luciferase assay. HEK293T cells were transfected with a plasmid expressing a Renilla luciferase reporter driven by the promoter of GJA1, CDH3 or NECTIN2 and a control firefly luciferase; 24 h later, the cells were transfected again with an Nsp1- or GFP (Ctrl)-expressing plasmid and collected for the dual luciferase assay 24 h after the second transfection. Each dot represents a replicate, and the mean and SEM are shown. P values, Mann-Whitney test. The results in a-f are representative of two independent experiments.
Fig 5.
SARS-CoV-2 Nsp1 promotes TAK1 K48-ubiquitin degradation.
a: The ubiquitination of TAK1 increased in N-Caco-2 cells infected with GFP-expressing ΔN SARS-CoV-2 RDPs at an MOI of 0.01. The cells were transfected with plasmids expressing HA-Ub and FLAG-tagged TAK1 (TAK1-FLAG). Twenty-four hours later, the cells were infected at an MOI of 0.01, and samples were collected at different time points. Ubiquitinated TAK1 was immunoprecipitated (IP) using anti-FLAG magnetic beads, and the ubiquitination level was analyzed with an anti-ubiquitin antibody. TAK1 was detected by anti-FLAG. The relative signals quantified by ImageJ are shown in the right panels of a-c and e-f. b: HEK293T cells were transfected with plasmids expressing HA-Ub and TAK1-FLAG 24 h prior to transfection with different quantities of plasmids expressing cMYC-tagged Nsp1 (Nsp1-cMYC; 0, 0.5, 1, 2 or 4 μg). Nsp1 was detected by an anti-cMYC antibody in b-d. c: HEK293T cells were transfected with plasmids containing TAK1-FLAG and wild-type (WT) or mutant HA-Ub in which the corresponding ubiquitin-accepting lysine (K) residue was replaced with arginine (R), and 24 h later, the cells were transfected again with the cMYC-tagged Nsp1 (Nsp1-cMYC) or GFP control (Ctrl, GFP-cMYC) plasmid. Green arrowhead, GFP-cMYC; red arrowhead, Nsp1-cMYC in c, e and f. d: HEK293T cells were transfected with the Nsp1-cMYC plasmid 24 hr prior to treatment with different concentrations of MG132 (0, 2, 5 or 10 μM) for 12 hr. The relative signals between TAK1 (by anti-TAK1) and β-ACTIN (by anti-β-ACTIN) were quantified by ImageJ and are shown in the right panel. e: HEK293T cells were treated with 10 μM MG132 or DMSO (Ctrl) prior to transfection with the TAK1-FLAG and HA-Ub plasmids, and 24 h later, they were transfected again with the Nsp1-cMYC or control GFP-cMYC (Ctrl) plasmid. f: Polyubiquitination levels of TAK1 mutants in which the lysine (K) residue at position 34, 72, 209 or 589 was mutated to alanine (A). HEK293T cells were first transfected with plasmids expressing HA-Ub and wild-type (WT) or mutant TAK1-FLAG; 24 h later, the cells were transfected again with Nsp1-cMYC or GFP-cMYC (Ctrl) plasmids. The results in a, c, d, e, and f are representative of two independent experiments.
Fig 6.
TRIM21 is involved in the Nsp1-mediated upregulation of TAK1 ubiquitination.
a: HEK293T cells were transfected with plasmids expressing FLAG-tagged TAK1 (TAK1-FLAG) and wild-type (WT) or mutant (K48R/K63R) HA-Ub, and 24 h later, were transfected again with a plasmid expressing cMYC-tagged TRIM21 (cMYC-TRIM21) or GFP (GFP-cMYC, Ctrl). Ubiquitinated TAK1 was immunoprecipitated (IP) by anti-FLAG magnetic beads, and the ubiquitination level was analyzed by an anti-HA antibody. GFP (green arrowhead) and TRIM21 (red arrowhead) were detected by anti-cMYC and anti-TAK1 with anti-FLAG antibodies in a-b. The relative signals of HA-Ub and TAK1-FLAG quantified by ImageJ are shown in the right panels in a & e. b: Plasmids expressing cMYC-tagged full-length (FL) or truncated TRIM21 mutants expressing only SPRY or lacking (Δ) the SPRY domain were constructed (left panel). SPRY, SPla and the RYanodine Receptor domain. HEK293T cells were cotransfected with plasmids expressing TAK1-FLAG and FL or truncated cMYC-TRIM21 or GFP. Immunoprecipitation (IP) was performed using anti-cMYC magnetic beads (right panel). c: HEK293T cells were cotransfected with plasmids expressing cMYC-TRIM21 and FLAG-tagged full-length (FL) or truncated N-terminal (1-303 aa, N) or C-terminal (304-606 aa, C) TAK1 mutants. FLAG-tagged GFP (green arrowhead) and TAK1 (blue arrowhead) were detected with an anti-FLAG antibody. d: HEK293T cells were transfected with plasmids expressing HA-tagged TAK1 and cMYC-TRIM21 24 h later and transfected with a plasmid expressing FLAG-tagged Nsp1 (red arrowheads) or a GFP control (Ctrl, green arrowhead). Immunoprecipitation (IP) was performed using anti-HA magnetic beads. The relative signals of cMYC-TRIM21 and TAK1-HA quantified using ImageJ are shown in the right panel. e: HEK293T cells were transfected with a lentiviral vector encoding shRNA targeting TRIM21 (shTRIM21) or luciferase (shLuc) as a control. After puromycin (10 μg/mL) selection for 72 hr, the cells were transfected with an HA-Ub plasmid, and 24 hr later, transfected with an Nsp-cMYC (red arrowheads) or a GFP-cMYC plasmid (Ctrl, green arrowheads). The results in a, d and e are representative of two independent experiments.
Fig 7.
SARS-CoV-2 Nsp1 inhibits TAK1-mediated immune pathways by promoting the TRIM21-dependent K48-linked polyubiquitination of TAK1.
In uninfected cells (left), TAB1 binds to TAK1, and the TAK1-TAB1 complex phosphorylates downstream regulators, including IκBα, p38, and Erk, which in turn activate the downstream NF-κB, MAPK and AP-1 pathways. In SARS-CoV-2-infected cells (right), Nsp1 binds to TAK1 at the N-terminal TAB1-binding domain, preventing the formation of the TAK1-TAB1 complex and promoting the binding of TRIM21 to the C-terminus of TAK1. As a result, Nsp1 promotes the proteasomal degradation of TAK1 by enhancing the TRIM21-dependent K48-linked polyubiquitination (Ub) of TAK1, thereby attenuating the activity of the NF-κB and AP-1 pathways. Created in BioRender. Yang, Q. (2026) https://BioRender.com/41xkykd.