SARS-CoV-2 NSP13 interacts with TEAD to suppress Hippo-YAP signaling
- McGill Gene Editing Lab, The Texas Heart Institute, United States
- Cardiomyocyte Renewal Laboratory, The Texas Heart Institute, United States
- Department of Integrative Physiology, Baylor College of Medicine, United States
- The Heart Institute, Division of Molecular Cardiovascular Biology, Cincinnati Children's Hospital Medical Center, United States
- Department of Pediatrics, University of Cincinnati College of Medicine, United States
Figures
Figure 1 with 4 supplements
SARS-CoV-2 infection suppresses YAP activity.
(A) Overview of SARS-CoV-2 infection in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). (B) Box plot showing the mean expression scores of known YAP target genes in hiPSC-CM bulk RNA-sequencing data. Each dot represents a biological replicate. Student’s t-test; **p<0.01, ****p<0.0001. (C) Heatmap displaying the expression Z-scores of example YAP targets in the iPSC-CM bulk RNA-sequencing data. Each column corresponds to a single biological sample. (D) Overview of integrated single-nucleus RNA sequencing and uniform manifold approximation and projection (UMAP) of cell types in lung samples from controls and patients with COVID-19. EC, endothelial cells; NK, natural killer cells; SMC, smooth muscle cells. (E) UMAP visualization of TMPRESS2 expression in the 10 cell types. (F) GO analysis of downregulated genes in AT1 cells from COVID patients. (G) Yap scores in alveolar type 1 (AT1) and alveolar type 2 (AT2) epithelial cells from lung samples in controls and patients with COVID-19. Wilcoxon test, ****p<0.0001. (H) Expression of Yap targets in AT1 and AT2 cells of control and COVID-positive lung samples.
Figure 1—figure supplement 1
Heatmap showing the most highly expressed markers for each cell type in human lung samples.
Figure 1—figure supplement 2
Uniform manifold approximation and projection (UMAP) visualization showing ACE2 expression in all cell types.
Figure 1—figure supplement 3
Epithelial cell subtypes AT1 and AT2 with marker genes.
(A) UMAP of epithelial cell subclusters. (B) Dot plot showing the relative expression of alveolar type 1 (AT1) and alveolar type 2 (AT2) markers in human lung samples.
Figure 1—figure supplement 4
Expression of Yap targets genes involved in innate immune response or regulation in AT1 and AT2 cells from control and COVID-positive lung samples.
Figure 2 with 6 supplements
NSP13 inhibits YAP transactivation.
(A) Screening of 11 NSPs for YAP activation by using a dual-luciferase reporter assay (HOP-flash). Compared with other NSPs, NSP13 strongly inhibited YAP transactivation at low protein expression levels. (n=3 independent experiments; data are reported as mean ± SD). ****p<0.0001, one-way ANOVA. (B) Reporter assay (8xGTIIC) results showing that NSP13 but not YAP upstream kinase LATS2 inhibited YAP5SA transactivation in a dose-dependent manner. (n=3 independent experiments; data are presented as the mean ± SD). ***p<0.001, ****p<0.0001, one-way ANOVA. (C) Experimental design of NSP13 study in mice. Control (aMyHC-MerCreMer;WT) and YAP5SA (aMyHC-MerCreMer;YAP 5SA) mice were injected with AAV9-GFP or AAV9-NSP13. At 12 days after virus injection, the mice received two low doses of TAM (10 ug/g). Cardiac function was recorded by echocardiography at days 4 and 8 after the second shot of tamoxifen. Hearts of all surviving mice were collected at day 21 post-tamoxifen injection. (D) NSP13 expression in cardiomyocytes improved the survival rate of YAP5SA mice after TAM injection compared to YAP5SA mice with AAV9-GFP infection. **p=0.0099, log-rank (Mantel–Cox) test. (E) Ejection fraction in YAP5SA mice was increased on day 8 after tamoxifen injections (10 ug/g x2). NSP13 expression reversed the increase of EF in YAP5SA mice. (n=6 in MCM + GFP, MCM + NSP13, and YAP5SA + GFP group; n=8 in YAP5SA + NSP13 group). ****p<0.0001, three-way ANOVA. (F) Representative B-mode and M-mode echocardiographic images of mouse hearts in four groups, 8 days after tamoxifen (TAM) induction. (G) A reduction in the left ventricle size was seen in YAP5SA mice at day 8 after tamoxifen injection. NSP13 introduction reversed this trend as evidenced by an increase in the diameter of the left ventricle (n=6 in MCM + GFP, MCM + NSP13, and YAP5SA + GFP group; n=8 in YAP5SA + NSP13 group). ***p<0.001, three-way ANOVA. (H, I) Representative whole mount and H&E images of mouse hearts at 21 days after tamoxifen induction. Scale bar, 2 mm.
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Figure 2—source data 1
Original files for western blot analysis displayed in Figure 2A.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig2-data1-v1.zip
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Figure 2—source data 2
Original western blots for Figure 2A, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig2-data2-v1.zip
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Figure 2—source data 3
Original files for western blot analysis displayed in Figure 2B.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig2-data3-v1.zip
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Figure 2—source data 4
Original western blots for Figure 2B, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig2-data4-v1.zip
Figure 2—figure supplement 1
Reporter assay (HOP-flash) results indicating that NSP13 inhibited YAP5SA transactivation at low protein expression levels when compared with other NSPs.
n=3 independent experiments; data are presented as mean ± SD. ****p<0.0001, one-way ANOVA.
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Figure 2—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
Original western blots for Figure 2—figure supplement 1, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig2-figsupp1-data2-v1.zip
Figure 2—figure supplement 2
Raw reads of Renilla luciferase in Figures 2A and 3B and D.
Figure 2—figure supplement 3
Reporter assay (Notch reporter) results showing that NSP13 cannot suppress NICD activation.
n=3 independent experiments; data are presented as mean ± SD.
Figure 2—figure supplement 4
NSP13 expression at different time points in mouse hearts after AAV9-HA-NSP13 virus injection.
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Figure 2—figure supplement 4—source data 1
Original files for western blot analysis displayed in Figure 2—figure supplement 4.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig2-figsupp4-data1-v1.zip
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Figure 2—figure supplement 4—source data 2
Original western blots for Figure 2—figure supplement 4, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig2-figsupp4-data2-v1.zip
Figure 2—figure supplement 5
Fractional shortening was increased in YAP5SA mice at day 8 after tamoxifen injection (10 ug/g x2).
This increase was reversed by NSP13 expression. (n=6 in MCM + GFP, MCM + NSP13, and YAP5SA + GFP group; n=8 in YAP5SA + NSP13 group). ****p<0.0001, three-way ANOVA.
Figure 2—figure supplement 6
The overgrowth of the heart in YAP5SA mice, as evidenced by heart/body ratio, was reversed by NSP13 expression.
n=6 in MCM + GFP and MCM + NSP13 group; n=4 in YAP5SA + GFP group; n=8 in YAP5SA + NSP13 group. *p=0.0175, one-way ANOVA.
Figure 3 with 1 supplement
NSP13 helicase activity is required for suppressing YAP activity.
(A) Conserved amino acid sequences of NSP13 among coronaviruses. (B) SARS-CoV-2 NSP13 mutant plasmids were constructed to examine YAP suppression mechanisms. NSP13-R567A, which loses its ATP consumption ability, did not inhibit YAP5SA transactivation, whereas NSP13 K345A/K347A, which loses its nucleic acid binding activity, mildly promoted YAP5SA transactivation (n=3 independent experiments; data are reported as the mean ± SD). **p<0.01, ****p<0.0001, one-way ANOVA. (C) Six NSP13 truncations were constructed based on the NSP13 domain map. (D) Reporter assay: none of the truncations led to a reduction in YAP transactivation, and the NSP13 DNA binding domains 1A and 2A slightly increased YAP5SA activation, suggesting that the full-length NSP13 with helicase activity may be required for suppression of YAP transactivation (n=3 independent experiments; data are reported as the mean ± SD). *p<0.05, ****p<0.0001, one-way ANOVA. (E) Summary of NSP13 mutants from SARS-CoV-2 variants. (F) HOP-flash reporter assay: NSP13 mutations did not affect suppression of YAP5SA transactivation (n=3 independent experiments; data are reported as mean ± SD). ****p<0.0001, one-way ANOVA.
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Figure 3—source data 1
Original files for western blot analysis displayed in Figure 3B.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig3-data1-v1.zip
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Figure 3—source data 2
Original western blots for Figure 3B, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig3-data2-v1.zip
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Figure 3—source data 3
Original files for western blot analysis displayed in Figure 3D.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig3-data3-v1.zip
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Figure 3—source data 4
Original western blots for Figure 3D, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig3-data4-v1.zip
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Figure 3—source data 5
Original files for western blot analysis displayed in Figure 3F.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig3-data5-v1.zip
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Figure 3—source data 6
Original western blots for Figure 3F, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig3-data6-v1.zip
Figure 3—figure supplement 1
SARS-CoV-2 NSP13 mutations: structural locations and functional effect on YAP5SA transactivation.
(A) The location of the NSP13 mutations from SARS-CoV2 variants on the NSP13 protein structure model (PDB: 7NN0). (B) Reporter assay (8xGTIIC) results indicating that NSP13 mutations did not affect its suppression on YAP5SA transactivation (n=3 independent experiments; data are presented as mean ± SD). ****p<0.0001, one-way ANOVA.
Figure 4 with 10 supplements
NSP13 inactivates the YAP/TEAD4 complex by recruiting YAP repressors.
(A) Immunofluorescence imaging showing that NSP13 colocalized with YAP5SA in cardiomyocytes of YAP5SA transgenic mice 3 days after tamoxifen injection. Scale bar, 20 μm. (B) Co-IP: NSP13 interacts with TEAD4, a major binding partner of YAP, in the nucleus. (C) Co-IP of HEK293T nuclei: NSP13 does not disrupt the interaction between YAP and TEAD4, whereas TEAD4 promotes the interaction between YAP and NSP13. (D, E) Immunofluorescence imaging and western blot analysis reveal that NSP13 protein levels increase after YAP5SA expression is induced in YAP5SA mouse cardiomyocytes. Scale bar, 50 μm. (F) Workflow of IP-MS. (G) IP-MS in nuclei (IP: NSP13), suggesting NSP13 interacts with proteins with or without YAP co-expression. Significance Analysis of INTeractome (SAINT), AvgP >0.6 are labeled in red. (H) GO analysis in subclusters of NSP13 interacting proteins (SAINT, AvgP >0.6, labeled with red in Figure S4C). (I) HOP-flash reporter assay: endogenous YAP activity is increased after the siRNA knockdown of CCT3 and TTF2 in HeLa cells (n=3 independent experiments; data are reported as mean ± SD). ***p<0.001, ****p<0.0001, one-way ANOVA. (J) Working model for NSP13 regulation of YAP/TEAD.
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Figure 4—source data 1
Original files for western blot analysis displayed in Figure 4B.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig4-data1-v1.zip
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Figure 4—source data 2
Original western blots for Figure 4B, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig4-data2-v1.zip
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Figure 4—source data 3
Original files for western blot analysis displayed in Figure 4C.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig4-data3-v1.zip
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Figure 4—source data 4
Original western blots for Figure 4C, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig4-data4-v1.zip
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Figure 4—source data 5
Original files for western blot analysis displayed in Figure 4E.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig4-data5-v1.zip
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Figure 4—source data 6
Original western blots for Figure 4E, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig4-data6-v1.zip
Figure 4—figure supplement 1
Western blot analysis showed that neither wild-type nor mutant NSP13 directly interacted with YAP5SA in HEK 293T cells.
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Figure 4—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Original western blots for Figure 4—figure supplement 1, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig4-figsupp1-data2-v1.zip
Figure 4—figure supplement 2
TEAD4 domain mapping experiments showing that both the N-terminus and C-terminus are required for the interaction with NSP13.
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Figure 4—figure supplement 2—source data 1
Original files for western blot analysis displayed in Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig4-figsupp2-data1-v1.zip
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Figure 4—figure supplement 2—source data 2
Original western blots for Figure 4—figure supplement 2, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig4-figsupp2-data2-v1.zip
Figure 4—figure supplement 3
Co-immunoprecipitation in nucleus indicated that NSP13 wild-type and R567A did not disrupt YAP and TEAD4 interaction.
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Figure 4—figure supplement 3—source data 1
Original files for western blot analysis displayed in Figure 4—figure supplement 3.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig4-figsupp3-data1-v1.zip
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Figure 4—figure supplement 3—source data 2
Original western blots for Figure 4—figure supplement 3, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig4-figsupp3-data2-v1.zip
Figure 4—figure supplement 4
Immunofluorescence imaging showing that both NSP13 WT and R567A restricted YAP5SA in cell nucleus.
Scale bar, 50 μm.
Figure 4—figure supplement 5
Western blot analysis of co-IP showed that nucleic acid removal did not disrupt the NSP13–TEAD4 interaction.
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Figure 4—figure supplement 5—source data 1
Original files for western blot analysis displayed in Figure 4—figure supplement 5.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig4-figsupp5-data1-v1.zip
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Figure 4—figure supplement 5—source data 2
Original western blots for Figure 4—figure supplement 5, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig4-figsupp5-data2-v1.zip
Figure 4—figure supplement 6
STRING analysis of NSP13 interacting proteins.
Figure 4—figure supplement 7
Co-IP assays indicated that the NSP13 interactors from mass spectrometry had weaker binding with NSP13 compared to TEAD4.
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Figure 4—figure supplement 7—source data 1
Original files for western blot analysis displayed in Figure 4—figure supplement 7.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig4-figsupp7-data1-v1.zip
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Figure 4—figure supplement 7—source data 2
Original western blots for Figure 4—figure supplement 7, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/100248/elife-100248-fig4-figsupp7-data2-v1.zip
Figure 4—figure supplement 8
siRNA knockdown efficiency for genes in HeLa cells, determined by using quantitative polymerase chain reaction (n=3 independent experiments; data are presented as mean ± SD).
Figure 4—figure supplement 9
Reporter assay (8xGTIIC) results in HeLa cells revealed that endogenous YAP activity was increased after the siRNA-mediated knockdown of CCT3 and TTF2.
n=3 independent experiments; data are presented as mean ± SD. ****p<0.0001, one-way ANOVA.
Figure 4—figure supplement 10
Quantitative PCR analysis indicated increased expression of CTGF and CYR61 following CCT3 knockdown.
Data are presented as mean ± SD (n=3). *p<0.05, **p<0.01, ***p<0.001, two-way ANOVA.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/100248/elife-100248-mdarchecklist1-v1.pdf
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Source data 1
IP-MS raw data.
- https://cdn.elifesciences.org/articles/100248/elife-100248-data1-v1.xlsx
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Source data 2
IP-MS analysis.
- https://cdn.elifesciences.org/articles/100248/elife-100248-data2-v1.xlsx
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Source data 3
STRING analysis.
- https://cdn.elifesciences.org/articles/100248/elife-100248-data3-v1.xlsx
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Source data 4
Supporting data values in figures.
- https://cdn.elifesciences.org/articles/100248/elife-100248-data4-v1.xlsx
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SARS-CoV-2 NSP13 interacts with TEAD to suppress Hippo-YAP signaling
eLife 13:RP100248.
https://doi.org/10.7554/eLife.100248.3
