SARS-CoV-2 infection suppresses YAP activity in vivo and in vitro. (A) 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. (B) UMAP visualization of TMPRESS2 expression in the 10 cell types. (C) 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. (D) Overview of SARS-CoV-2 infection in human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs). (E) 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. (F) 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.

NSP13 inhibits YAP transactivation in vitro and in vivo. (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 levels of protein expression. (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. Both 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-dose of TAM (10 ug/g). Cardiac function was recorded by echocardiography at day 4 and 8 after the second shot of tamoxifen. The mouse hearts of all the surviving mice were collected at day 21 post tamoxifen injection. (D) NSP13 expression in cardiomyocytes improved 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 size of the left ventricle 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) We constructed SARS-CoV-2 NSP13 mutant plasmids to examine the mechanisms underlying YAP suppression. 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) We constructed 6 NSP13 truncations on the basis of the NSP13 domain map. (D) Reporter assay results indicated that 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-CoV2 variants. (F) Reporter assay (HOP-flash) results indicated that NSP13 mutations did not affect its 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-immunoprecipitation results suggesting that NSP13 interacts with TEAD4, a major binding partner of YAP, in the nucleus. (C) Results of co-immunoprecipitation experiments in nucleus of HEK293T cells showing that NSP13 did not disrupt the interaction between YAP and TEAD4, whereas TEAD4 promoted the interaction between YAP and NSP13. A working model for the YAP/TEAD4/NSP13 complex is that TEAD4 acts as a platform for recruiting YAP and NSP13. (D-E) Immunofluorescence imaging and western blot analysis showing that NSP13 protein levels increased after YAP5SA expression in cardiomyocytes of YAP5SA transgenic mice. (F) GO analysis in subclusters of NSP13 interacting proteins (SAINT, AvgP >0.6, labelled with red in Figure S4C). (G) Reporter assay (HOP-flash) results showed that endogenous YAP activity was increased after the siRNA-mediated knockdown of CCT3 and TTF2 in HeLa cells. ***p< 0.001, ****p< 0.0001, one-way ANOVA. (H) Working model: NSP13, together with its interacting proteins (YAP-TEAD repressors), are recruited to the YAP/TEAD complex by interacting with TEAD4, which results in YAP-TEAD inactivation.