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 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 cell of control and COVID-positive lung samples.

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 day 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. ****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. ***p< 0.001, three-way ANOVA. (H-I) Representative whole mount and hematoxylin & eosin images of mouse hearts at 21 days after tamoxifen induction. Scale bar, 2 mm.

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) 6 NSP13 truncations were contructed 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. ****p < 0.0001, one-way ANOVA.

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. (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. (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, labelled 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. ***p< 0.001, ****p< 0.0001, one-way ANOVA. (J) Working model for NSP13 regulation of YAP/TEAD.

SARS-CoV-2 infection suppresses YAP activity in vivo and in vitro.

(A) Heatmap showing the most highly expressed markers for each cell type in human lung samples. (B) Uniform manifold approximation and projection (UMAP) visualization showing ACE2 expression in all cell types. (C) UMAP of epithelial cell subclusters. (D) Dot plot showing the relative expression of alveolar type 1 (AT1) and alveolar type 2 (AT2) markers in human lung samples. (E) Expression of Yap targets genes involved in innate immune response or regulation in AT1 and AT2 cells from control and COVID-positive lung samples.

NSP13 inhibits YAP transactivation in vitro and in vivo.

(A) 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. (B) Raw reads of Renilla luciferase in Figure 2A. (C) Reporter assay (Notch reporter) results showing that NSP13 can not suppress NICD activation. (n = 3 independent experiments; data are presented as mean ± SD). (D) NSP13 expression at different time points in mouse hearts after AAV9-HA-NSP13 virus injection. (E) Fractional shortening was increased in YAP5SA mice at day 8 after tamoxifen injection (10 ug/g x2). This increase was reversed by NSP13 expression. ****p<0.0001, three-way ANOVA. (F) The overgrowth of the heart in YAP5SA mice, as evidenced by heart/body ratio, was reversed by NSP13 expression. *p=0.0175, one-way ANOVA.

NSP13 helicase activity is required for suppressing YAP activity.

(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. ****p<0.0001, one-way ANOVA. (C) Raw reads of Renilla luciferase in Figure 3B. (D) Raw reads of Renilla luciferase in Figure 3D.

NSP13 interacts with TEAD4.

(A) Western blot analysis showed that neither wild-type nor mutant NSP13 directly interacted with YAP5SA in HEK 293T cells. (B) TEAD4 domain mapping experiments showing that both the N-terminus and C-terminus are required for the interaction with NSP13. (C) Co-immunoprecipitation in nucleus indicated that NSP13 wild-type and R567A did not disrupt YAP and TEAD4 interaction. (D) Immunofluorescence imaging showing that both NSP13 WT and R567A restricted YAP5SA in cell nucleus. (E) Western blot analysis of co-IP showed that nucleic acid removal did not disrupt the NSP13–TEAD4 interaction.

NSP13 inactivates the YAP/TEAD4 complex by recruiting YAP repressors.

(A) Immunoprecipitation mass spectrometry in nuclei (IP: NSP13) suggesting NSP13 interacting proteins with or without YAP co-expression. Significance Analysis of INTeractome (SAINT), AvgP >0.6 were labeled with red. (B) co-IP assays indicated that the NSP13 interactors from mass spectrometry had weaker binding with NSP13 compared to TEAD4. (C) siRNA knockdown efficiency for genes in HeLa cells, determined by using quantitative polymerase chain reaction. (D) Reporter assay (8xGTIIC) results in HeLa cells revealed that endogenous YAP activity was increased after the siRNA-mediated knockdown of CCT3 and TTF2. ****p<0.0001, one-way ANOVA. (E) Quantitative PCR analysis indicated increased expression of Ctgf and Cyr61 following Cct3 knockdown. *p<0.05, **p<0.01, ***p<0.001, two-way ANOVA.