Introduction

The evolutionarily conserved Hippo-YAP signaling pathway integrates various extracellular signals, including mechanical force, cell adhesion, and nutrient availability, through the protein kinases LATS1/2 (1). YAP is the primary substrate of LATS kinases, and following phosphorylation, is inactivated via cytoplasmic retention or degradation. Unphosphorylated YAP translocates to the nucleus and complexes with transcription factor partners, notably TEAD, to activate target genes (1, 2) involved in tissue development, homeostasis, and regeneration across multiple organs (2–7). YAP/TEAD is regulated by intricate molecular mechanisms that govern its activity, localization, and interaction. Dysregulation of these processes has been implicated in various diseases, including cancer (8–11).

Recent studies reveal that YAP is an endogenous brake of innate antiviral immunity (12, 13). Genetic loss of YAP enhances antiviral responses, whereas expression of a transactivation deficient mutant (YAP 6SA) suppresses these responses. Conversely, multiple canonical YAP target genes, such as IL-6, Ccl2, and Csf1, modulate innate immune signaling and cytokine production (14–16). Notably, different types of viral infection distinctly influence YAP expression, degradation, and nuclear localization (17). Although these observations shed light on the complex dynamics of YAP during viral responses, the mechanisms underlying YAP/TEAD regulation after SARS-CoV-2 infection are poorly understood. SARS-CoV-2, the center of the COVID-19 pandemic, has well-documented effects on the respiratory, digestive, central nervous, and cardiovascular systems (18–20). Investigating SARS-CoV-2 and Hippo-YAP signaling provides molecular insight into virus–induced pathophysiology. Two groups recently reported opposite effects on YAP activity after SARS-CoV-2 infection (21, 22). However, the lack of YAP/TEAD target gene expression data in these studies prevents a full understanding of these disparate results. Thus, the precise understanding of YAP/TEAD regulation after SARS-CoV-2 infection remains elusive.

Here, we found that SARS-CoV-2 infection reduces YAP target gene expression in human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) and lung epithelial cells of COVID-19 patients. By comprehensively screening SARS-CoV-2 nonstructural proteins, we identified NSP13 as a key factor in inhibiting YAP transcriptional activity and suppressing active YAP5SA activity in vivo. Moreover, NSP13 suppression of YAP is dependent on its helicase activity, where both DNA binding and unwinding are required. Mechanistically, NSP13 is directly bound to TEAD4 and inhibits the transcriptional activity of the YAP/TEAD4 complex by remodeling chromatin and recruiting transcriptional suppressors such as TTF2. These findings reveal a novel function of NSP13 in regulating YAP/TEAD activity and provide key insights into how the SARS-CoV-2 genome modulates transcriptional activity of the YAP-TEAD complex.

Results

SARS-CoV-2 infection suppresses YAP activity in host cells

To assess YAP activity following SARS-CoV-2 infection in vitro, we evaluated a bulk RNA-sequencing dataset derived from hiPSC-CMs exposed to varying SARS-CoV-2 concentrations (23) (Figure 1A). Cardiomyocyte-specific YAP target gene (24) expression revealed that YAP targets were decreased in a dose-dependent manner after SARS-CoV-2 infection (Figure 1B). YAP is known to directly regulate several innate immunity genes, including Ccl2, Thbs1, and Csf1 (15, 16). We observed that these genes are down-regulated in SARS-CoV-2 infected cells, mirroring the decrease in canonical YAP targets such as Vgll3, Amotl2, and Ccn1 (Figure 1C).

Figure 1.

We then assessed YAP activity with SARS-CoV-2 infection in vivo by integrating and reanalyzing single-nuclei RNA (snRNA) sequencing data derived from human lung samples (25, 26). We identified 10 major cell types (Figure 1D, Figure Supplement 1A). TMPRSS2 and ACE2, two key entry factors for SARS-CoV-2 infection (27) were more highly expressed in lung epithelial cells compared to other cell types (Figure 1E, Figure Supplement 1B). Lung epithelial cells were categorized into alveolar type 1 (AT1) and alveolar type 2 (AT2) (Figure Supplement 1C, D). SARS-CoV-2 is more likely to infect AT1 cells as they cover >95% of the alveolar surface. We performed an unbiased GO analysis of the differentially expressed genes (DEGs) and observed that genes associated with or regulating the Hippo pathway were downregulated in AT1 cells from COVID-19 patients (Figure 1F). Consistent with previous data in vitro, the YAP score, evaluated via YAP target gene expression, was lower in AT1 cells from COVID-19 patients than in controls (Figure 1G). Signature genes associated with AT1 cells, including the reported YAP target genes AGER and CLIC5 (28, 29) were downregulated in COVID-19 patient lung samples (Figure 1H). These data support the notion that SARS-CoV-2 infection suppresses YAP activity in host cells. However, further analysis revealed that multiple YAP target genes involved in innate immunity and cytokine signaling were paradoxically elevated (Figure Supplement 1E). This discrepancy likely reflects a combination of factors, including cell type specificity, activation of parallel signaling pathways, and alterations in mechanical cues or tissue architecture, that can independently drive expression of these genes.

NSP13 inhibits YAP5SA transactivation

To identify which SARS-CoV-2 protein suppresses YAP activity, we screened 11 NSP proteins using a dual-luciferase reporter assay. This assay measures firefly and Renilla luciferase activities sequentially from the same sample. Normalization to Renilla luciferase serves as an internal control, ensuring accuracy and reproducibility of the results. Compared with other NSPs, NSP13 strongly inhibited YAP transcription activity (Figure 2A). By using a constitutively active form of YAP (YAP5SA) that is resistant to phosphorylation and inactivation by LATS1/2 (30), we observed that NSP13 suppresses YAP transactivation independent of its upstream kinase LATS2 (Figure Supplement 2A). Moreover, NSP13 attenuated YAP5SA activity dose-dependently (Figure 2B). A recent study reported that NSP13 suppresses episomal DNA transcription, as evidenced by reduced Renilla luciferase activity and decreased GFP expression upon co-expression with NSP13 (31). To evaluate this in our system, we examined our raw Renilla luciferase data and found that while 100 ng of NSP13 had no effect, 400 ng of NSP13 reduced Renilla luciferase levels by approximately 50% compared to the YAP5SA-only group (Figure Supplement 2B and 3C-D). Notably, the firefly luciferase signal, driven by YAP/TEAD interaction (HOP-Flash), exhibited an even greater reduction. To assess the specificity of this suppression, we also conducted a Notch reporter assay and observed that co-expression of NSP13 with NICD (Notch intracellular domain) did not inhibit Notch signaling (Figure Supplement 2C). These results reveal a specific suppressive effect of NSP13 on YAP-mediated transcriptional activity.

Figure 2.

To investigate NSP13 function in vivo, we used YAP5SA transgenic mice (aMyHC-MerCreMer;YAP5SA), which express YAP5SA in cardiomyocytes following tamoxifen administration. YAP5SA induction produces cardiomyocyte hyperplasia and increased ejection fraction (EF) and fractional shortening (FS), ultimately leading to mortality (24). We used the adenovirus expressing NSP13 (AAV9-NSP13), which specifically infects cardiomyocytes (Figure Supplement 2D and Figure 2C). NSP13-induced expression in YAP5SA mouse cardiomyocytes increased survival rates (Figure 2D) and restored cardiac function (Figure 2E and Figure Supplement 2E). Furthermore, NSP13 expression reversed the smaller left ventricle (LV) chamber in YAP5SA mouse hearts (24) (Figure 2F, G). To investigate NSP13 induction in the mouse heart further, we collected heart tissue from surviving mice 21 days after tamoxifen injection. Notably, NSP13 expression in YAP5SA mice reversed heart overgrowth (Figure 2H, I, and Figure Supplement 2F). Together, these data indicate that NSP13 suppresses YAP activity in vitro and in vivo.

NSP13 helicase activity is required for YAP suppression

NSP13, a helicase with conserved sequence across all coronaviruses (Figure 3A), plays a critical role in viral replication and is a promising target for antiviral treatment (32–39). K131, K345/K347, and R567 were identified as important amino acid sites for NSP13 helicase activity in SARS-CoV (40). Given the 99.8% sequence identity of NSP13 between SARS-CoV-2 and SARS-CoV, we speculated that these sites were similarly crucial for SARS-CoV-2 NSP13. Reporter assays revealed that NSP13-K131A, which retains partial helicase activity, can suppress YAP, whereas NSP13-R567A (no ATP consumption) and NSP13-K345A/K347A (obstructed nucleic acid binding channel) failed to inhibit YAP transcriptional activity (Figure 3B). To assess the contribution of each domain to YAP regulation, we constructed six NSP13 truncations (Figure 3C), yet none of these truncations reduced YAP transactivation (Figure 3D). These findings suggest that full-length NSP13 with helicase activity (DNA binding and ATP dependent unwinding) is required to suppress YAP transactivation, and that partial helicase function is sufficient to fully inhibit YAP.

Figure 3.

SARS-CoV-2 has consistently mutated over time, resulting in variants that differ from the original virus. We evaluated NSP13 mutations (35) (Figure 3E and Figure Supplement 3A) and observed that all mutants can suppress YAP transactivation (Figure 3F and Figure Supplement 3B).

NSP13 interacts with TEAD4 and recruits YAP repressors

To determine the molecular mechanism(s) underlying NSP13 suppression of YAP, we performed immunofluorescence (IF) analyses. Our data revealed that NSP13 localizes to the cytoplasm and nucleus, whereas most YAP5SA colocalizes with NSP13 in the nucleus three days after tamoxifen injection (Figure 4A). However, co-immunoprecipitation (co-IP) assays revealed no direct interaction between NSP13 and YAP5SA (Figure Supplement 4A). Additional assays indicated that NSP13 is associated with the transcription factor TEAD4 (Figure 4B), and both the N– and C-terminal ends of TEAD4 interact with NSP13 (Figure Supplement 4B), suggesting that NSP13 prevents YAP transactivation by competitively interacting with TEAD4. Unexpectedly, introduction of NSP13 had no effect on the YAP/TEAD4 association (Figure Supplement 4C). However, the YAP/NSP13 interaction was much stronger in the presence of TEAD4, indicating that TEAD4 is a platform to recruit YAP and NSP13 (Figure 4C). Consistent with this, IF analysis of HeLa cells and heart sections revealed that YAP5SA is restricted to the nucleus when co-expressed with either NSP13 WT or R567A, in contrast to the vector control (Figure 4D, Figure Supplement 4D). Moreover, NSP13 protein levels in cardiomyocytes accumulated following YAP5SA induction (Figure 4D, E), suggesting that nuclear NSP13/YAP/TEAD4 prevents NSP13 degradation. Given that NSP13 preferentially interacts with TEAD4 in the nucleus (Figure 4B-C, Figure Supplement 4C), we next determined whether this association was DNA dependent using multiple nucleases: Universal Nuclease (which degrades all forms of DNA and RNA), DNase I (which cleaves both single and double stranded DNA), and RNase H (which selectively cleaves the RNA strand in RNA/DNA hybrids). These treatments did not disrupt the NSP13/TEAD4 interaction (Figure Supplement 4E), revealing that their binding is not dependent on nucleic acids.

Figure 4.

Given that NSP13 forms a complex with YAP and TEAD4, we hypothesized that it recruits nuclear repressors to inhibit YAP transactivation. To investigate this, we performed immunoprecipitation followed by mass spectrometry (IP-MS) to identify NSP13-interacting proteins in the presence or absence of YAP in HEK293T cell nuclei (Figure 4F). Our analyses identified hundreds of candidate proteins that interact with NSP13 in the nucleus (Figure 4G), which were further analyzed by STRING-based network analysis (Figure Supplement 5A). Gene Ontology (GO) analysis indicated that the largest clusters are involved in RNA polymerase II transcription termination, chromatin remodeling, and protein folding (Figure 4H). To validate these candidate interactors, we evaluated several proteins—CCT3, SMARCD1, EIF4A1, LMNA, TTF2, and YY2—and conducted co-IP assays. Whereas TEAD4 strongly interacts with NSP13 (Figure Supplement 5B), the candidate repressors exhibited relatively weak binding to NSP13, suggesting that NSP13 associates with these proteins indirectly, potentially through a larger multiprotein complex or via chromatin-dependent interactions (Figure 4J).

Next, we evaluated the functional consequence of siRNA knockdown of selected candidates in YAP transactivation (Figure Supplement 5C). Reporter assays revealed that knockdown of CCT3 and TTF2 increased endogenous YAP transactivation in HeLa cells (Figure 4I and Figure Supplement 5D). To confirm the function of CCT3, we performed quantitative PCR analysis of classical YAP target genes and observed increased expression of Ctgf and Cyr61 following Cct3 knockdown. These findings support a model where NSP13 suppresses YAP activity by recruiting suppressors to the YAP/TEAD4 complex.

Discussion

In this study, we observed that SARS-CoV-2 infection in human lung and hiPSC-CMs reduces YAP transcriptional activity, wherein the SARS-CoV-2 helicase NSP13 significantly inhibited YAP activity. Mechanistically, NSP13 directly interacts with TEAD4 to form a YAP/TEAD4/NSP13 complex in the nucleus, which recruits suppressors such as TTF2 and CCT3 to repress YAP-TEAD transcriptional activity (Figure 4J).

Our data reveal a helicase dependent inhibition of YAP by NSP13, where the K131, K345/K347, and R567 NSP13 residues are required. Based on published structural and biochemical studies, each of these residues uniquely supports helicase function: Substituting K131 with alanine (K131A) severely reduces helicase efficiency; K345/K347 are key DNA binding residues as mutating both (K345A/K347A) abolishes DNA binding; mutation of the ATP hydrolysis residue R567 (R567A) disables DNA unwinding. As illustrated in Fig. 4J, NSP13 must bind DNA and hydrolyze ATP to unwind nucleic acids. This helicase dependent process likely enables NSP13 to remodel chromatin by binding TEAD and organizing YAP repressors at the YAP/TEAD complex to prevent YAP/TEAD transactivation. In support of this mechanism, the K345A/K347A mutant, unable to anchor to DNA, fails to repress YAP as YAP driven transcription is slightly increased in this mutant (Figure 3B). Likewise, the ATPase dead R567A can bind DNA but does not unwind and remodel chromatin to recruit YAP repressors, resulting in a loss of YAP suppression (Figure 3B and 3F). Our model demonstrates that both DNA binding and ATP dependent unwinding are essential for NSP13 to suppress YAP transcriptional activity.

TEAD transcription factors, the major partners of YAP, are the primary nuclear effectors of Hippo-YAP signaling and play critical roles in cancer development (11, 41). We observed that NSP13 interacts with TEAD4, but does not disrupt the YAP/TEAD4 interaction, suggesting a novel regulatory mechanism for TEAD. IP-MS revealed that NSP13-interacting proteins such as TTF2 and CCT3 suppress the YAP-TEAD4 complex. TTF2, a SWI2/SNF2 family member, facilitates the removal of RNA polymerase II from the DNA template via ATP hydrolysis (42, 43). Importantly, termination of transcription complex elongation by TTF2 appears minimally affected by template position (43). CCT3 is a component of the chaperonin-containing T-complex, a molecular chaperone complex that promotes protein folding following ATP hydrolysis. Previous reports revealed that the liver cancer biomarker CCT3 (44) positively regulates YAP protein stability. We found that CCT3 interacts with NSP13 and inhibits YAP transactivation. Moreover, CCT3 knockdown increases YAP target gene expression, suggesting a context-dependent role of CCT3 in YAP regulation. While Co-IP assays detected weak interactions between NSP13 and CCT3 or TTF2, a strong NSP13/TEAD4 interaction was evident. These data suggest that NSP13 associates with CCT3 and TTF2 indirectly as part of a multiprotein complex in a chromatin structure-dependent manner, although further study is required to elucidate these mechanisms.

In conclusion, our study reveals a novel function of NSP13 in suppressing the YAP/TEAD transcriptional complex. These findings advance our understanding of the pathological effects of SARS-CoV-2 on host cells. Given that NSP13 directly interacts with TEAD4 and strongly suppresses YAP transactivation, this work provides a new avenue for modulating YAP activity as a potential treatment for YAP-driven diseases.

Materials and methods

Mice

We used αMyHC-MerCreMer mice, wild-type (WT) mice, and αMyHC-MerCreMer; YAP5SA mice (24). Mice had a mixed genetic background of C57BL/6 and 129SV. The AAV9 virus (a total of 5 × 10¹¹ viral genomes, 120 μl total volume) was delivered by retro-orbital injection two weeks before tamoxifen injection. For the survival experiment in Figure 2, two low doses of tamoxifen (10 ug/g) were administered to 6-week-old mice by intraperitoneal injection. The mouse cardiac function was evaluated by echocardiography at day 4 and day 8 post-tamoxifen injection, and all the surviving mice were sacrificed at day 21. For the experiments in Figure 4, two tamoxifen doses were injected (Figure 4A, 10 ug/g; Figure 4D-E, 50 ug/g), and mouse hearts were collected at day three post-tamoxifen injection.

RNA-seq analysis

To analyze lung samples from COVID-19 patients, snRNA sequencing data of 7 control lungs and 35 COVID-19 lungs from GSE171668 (25) and GSE171524 (26) were downloaded and analyzed using the Seurat v4 software suite (45). A total of 223,106 nuclei were used in the analysis. Each sample was normalized and batch corrected using SCTransformation. Mitochondrial percentage was used to regress out technical variability between batches, and we used harmony (46) integration to remove technical variation among samples. The YAP score was evaluated using 38 canonical YAP target genes. For analyzing iPSC-CMs infected with SARS-CoV-2, bulk RNA-seq data were obtained from Perez-Bermejo et al. (23) With this dataset, we re-analyzed and evaluated the average expression of 302 cardiomyocyte-specific YAP target genes (24).

Expression plasmids

Expression plasmids encoding HA-tagged WT, mutant, or truncated NSP13 were generated by polymerase chain reaction (PCR) and subcloned into pXF4H (N-terminal HA tag) derived from pRK5 (Genetech). Myc-tagged WT or truncated TEAD4, flag-tagged WT YAP, flag-tagged YAP5SA, and Myc-tagged NSP13 interacting proteins, including CCT3, SMARCD1, EIF4A1, LMNA, TTF2, and YY2, were generated by PCR and subcloned into a pcDNA3 backbone. HA-tagged WT LATS2, LATS2 KR, and pRL-TK_Luc were gifts from Dr. Pinglong Xu’s lab. Reporters of HOP-flash (#83467) and 8xGTIIC-luciferase (#34615) were purchased from Addgene. The NSP plasmids used in YAP transactivation screening experiments were gifts from Dr. Nevan J. Krogan’s lab. All plasmids were confirmed by performing DNA sequencing.

Cell culture and transfection

HEK293T and HeLa cells were cultured in Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum. Lipofectamine™ 2000 (ThermoFisher) or Lipofectamine™ 3000 (ThermoFisher) reagents were used for plasmid transfection. LipofectAmine RNAiMAX (ThermoFisher) was used for siRNA transfection.

Immunoprecipitation-mass spectrometry, database search, and data analysis

HEK293T cells were plated on a 150 mm tissue culture dish using 10% FBS DMEM 24 hours post-plating. Cells were transfected with Flag-YAP1, HA-NSP13, or Flag-YAP1+HA-NSP13 by Lipofectamine 3000 using standard manufacturer’s protocols. Each experimental group was performed in duplicate to ensure reproducibility and reliability. 24 hours post-transfection, cells were collected in a hypotonic lysis buffer (5 mM NaCl, 20 mM HEPES, pH 7.5, 0.4% NP-40) supplemented with protease inhibitor (Roche) and phosphatase inhibitor (Roche). Cell suspensions were drawn into a 3 ml syringe using a 25G needle and expelled in one rapid stroke into a new tube. After three rounds of lysis, the suspension was centrifuged at 600 x g 4 °C for 10 minutes to pellet nuclei. The supernatant was collected in a different tube, and the nuclear fraction was washed in the buffer twice. After the final wash, nuclei were resuspended in 20 mM HEPS, 150 mM NaCl, 2.5 mM MgCl2, 0.1% NP 40, and nuclear fractions were sonicated using Bioruptor® Pico for 10 cycles of 30 sec on and 30 sec off. Subsequently, lysate was centrifuged for 20 mins at 2 x 1000 g at 4°C. The supernatant was incubated with HA magnetic beads (ThermoFisher) for two hours at 4°C with continuous rotation. Antibody and protein-bound beads were washed thrice for 15 minutes in lysis buffer at 4 °C with constant rotation. Washed beads were boiled in 40 μL of 1X NUPAGE® LDS sample buffer (Invitrogen) and subjected to SDS-PAGE (NuPAGE 10% Bis-Tris Gel, Invitrogen). Eluted proteins were visualized with Coomassie Brilliant blue stain and excised into gel pieces according to molecular size. Individual gel pieces were destained and subject to in-gel digestion using trypsin (GenDepot T9600). Tryptic peptides were resuspended in 10 μl of loading solution (5% methanol containing 0.1% formic acid) and subjected to nanoflow LC-MS/MS analysis with a nano-LC 1000 system (Thermo Scientific) coupled to Orbitrap Elite (Thermo Scientific) mass spectrometer. Peptides were loaded onto a Reprosil-Pur Basic C18 (1.9 µm, Dr.Maisch GmbH, Germany) precolumn of 2 cm X 100 µm size. The precolumn was switched in-line with an in-house 50 mm x 150 µm analytical column packed with Reprosil-Pur Basic C18 equilibrated in 0.1% formic acid/water. Peptides were eluted using a 75 min discontinuous gradient of 4-26% acetonitrile/0.1% formic acid at a 700 nl/min flow rate. Eluted peptides were directly electro-sprayed into Orbitrap Elite mass spectrometer operated in the data-dependent acquisition mode acquiring fragmentation spectra of the top 25 strongest ions and under the direct control of Xcalibur software (Thermo Scietific).

MS/MS spectra were analyzed using the target-decoy mouse RefSeq database in the Proteome Discoverer 1.4 interface (Thermo Fisher), employing the Mascot algorithm (Mascot 2.4, Matrix Science). The precursor mass tolerance was set to 20 ppm, with a fragment mass tolerance of 0.5 daltons and a maximum of two allowed missed cleavages. Dynamic modifications included oxidation, protein N-terminal acetylation, and destreaking. Peptides identified from the Mascot results were validated at a 5% false discovery rate (FDR), and the iBAQ algorithm was applied to assess protein abundance, enabling comparisons of relative quantities among different proteins in the sample. The iBAQ value was determined by normalizing the summed peptide intensity by the number of theoretically observable tryptic peptides for each protein. PSM values from Proteome Discover were uploaded to the SAINT user interface on the CRAPOME website for SAINT analysis. SAINT probability and fold change were calculated using YAP IP as a user-defined control.

Luciferase reporter assay

HEK293T or HeLa cells were transfected with WT YAP-or YAP5SA-responsive HOP-flash plasmid or 8xGTIIC-luciferase reporter, which has an open reading frame encoding firefly luciferase, along with the pRL-Luc with Renilla luciferase coding as the internal control for transfection and other expression vectors (NSPs), as specified. After 24 h of transfection with the indicated treatments, cells were lysed with passive lysis buffer (Promega). Luciferase assays were performed by using a dual-luciferase assay kit (Promega), and data were quantified with POLARstar Omega (BMG Labtech) and normalized to the internal Renilla luciferase control.

AAV9 viral packaging

Viral vectors were used as previously described (47). The construct containing HA-tagged NSP13 was cloned into the pENN.AAV.cTNT, p1967-Q vector (AAV9-HA-NSP13). Empty vector–encoding green fluorescent protein was used as the control (AAV9-GFP). Both vectors were packaged into the muscle-trophic serotype AAV9 by the Intellectual and Developmental Disabilities Research Center Neuroconnectivity Core at Baylor College of Medicine. After being titered, viruses were aliquoted (1 × 10¹³ viral genome particles per tube), immediately frozen, and stored long-term at −80°C. Each aliquot was diluted in saline to make a 120-ul injection solution.

Echocardiography

For cardiac function analysis, echocardiography was performed on a VisualSonics Vevo 2100 system with a 550-s probe. B-mode images and M-mode images were captured on a short-axis projection. Ejection fraction, fractional shortening, diameter of diastolic left ventricle, diameter of systolic left ventricle, and end-diastolic volume were calculated by using a cardiac measurement package installed in the Vevo2100 system.

Histology and Immunofluorescence staining

Hearts were fixed in 4% paraformaldehyde overnight at 4 °C, dehydrated in serial ethanol and xylene solutions, and embedded in paraffin. For immunofluorescence staining, slides were sectioned at 7-μm intervals. For paraffin sections, samples were deparaffinized and rehydrated, treated with 3% H₂O₂ in EtOH and then with antigen retrieval solution (Vector Laboratories Inc., Burlingame, CA, USA), blocked with 10% donkey serum in phosphate-buffered saline, and incubated with primary antibodies. Antibodies used were rabbit anti-HA (#3724, Cell signaling) and rat anti-flag (NBP1-06712, Novus Bio-logicals). Immunofluorescence-stained images were captured on a Zeiss LSM 780 NLO Confocal/2-hoton microscope.

Statistical Analysis

For the data in our manuscript, the specific statistical test used is presented in the figure legend. In snRNA-seq analyses, Yap score differences between AT1 from Control and COVID-19 patients were identified via the Wilcoxon test. For analyzing iPSC-CMs infected with SARS-CoV-2, the expression score of cardiomyocyte YAP targets was evaluated by Student’s t-test. Mouse survival rates in Figure 2D were analyzed by the log-rank (Mantel-Cox) test. Statistical significances were evaluated using three-way ANOVA and Šídák’s multiple comparisons test for other cardiac data, including EF, FS, and systolic LV diameter. For reporter assays in cells and heart/body ratio in Figure S2E, the statistical significance of the observed differences in mean was evaluated using a one-way or two-way ANOVA and the post hoc Tukey’s multiple comparisons test. P values less than 0.05 were considered statistically significant (*p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001).

Supplemental files

Figure supplement 1.

Figure supplement 2.

Figure supplement 3.

Figure supplement 4.

Figure supplement 5.

Data and materials availability

All data are available in the main text or the supplementary materials.

Acknowledgements

We are grateful to Drs. Nevan J. Krogan and Pinglong Xu for the plasmid gifts. Dr. Hongxin Guan assisted with NSP13 mutant structure analysis. Rebecca Bartow, PhD, of the Department of Scientific Publications at The Texas Heart Institute, provided editorial support.

Additional information

Funding

National Institutes of Health grant HL 127717 (JFM).

National Institutes of Health grant HL 130804 (JFM).

National Institutes of Health grant HL 118761 (JFM).

Vivian L. Smith Foundation (JFM).

2020 COVID-19 RFA (JFM).

AHA postdoctoral fellowship 903411 (FM).

AHA postdoctoral fellowship 903651 (RL).

K99 HL169742 (JS)

Don McGill Gene Editing Laboratory of The Texas Heart Institute (XL).

Author contributions

Conceptualization: FM, JFM

Methodology: JK, JS, XL

Investigation: FM, C-RT, JW, YS, VD, BX, RL, SL

Supervision: JFM

Writing – original draft: FM

Writing – review & editing: FM, JFM

Funding

National Institutes of Health (HL 127717)

National Institutes of Health

National Institutes of Health (HL 118761)

American Heart Association

American Heart Association (903651)

National Institutes of Health (HL169742)

Additional files

Supporting data values

WB raw figure

sp1 IP-MS raw data

sp2 IP-MS analysis

sp3 STRING analysis