Hippo Signaling Pathway Activation during SARS-CoV-2 Infection Contributes to Host Antiviral Response

Abstract

SARS-CoV-2, responsible for the COVID-19 pandemic, causes respiratory failure and damage to multiple organ systems. The emergence of viral variants poses a risk of vaccine failures and prolongation of the pandemic. However, our understanding of the molecular basis of SARS-CoV-2 infection and subsequent COVID-19 pathophysiology is limited. In this study, we have uncovered a critical role for the evolutionarily conserved Hippo signaling pathway in COVID-19 pathogenesis. Given the complexity of COVID-19 associated cell injury and immunopathogenesis processes, we investigated Hippo pathway dynamics in SARS-CoV-2 infection by utilizing COVID-19 lung samples, and human cell models based on pluripotent stem cell-derived cardiomyocytes (PSC-CMs) and human primary lung air-liquid interface (ALI) cultures. SARS-CoV-2 infection caused activation of the Hippo signaling pathway in COVID-19 lung and in vitro cultures. Both parental and Delta variant of concern (VOC) strains induced Hippo pathway. The chemical inhibition and gene knockdown of upstream kinases MST1/2 and LATS1 resulted in significantly enhanced SARS-CoV-2 replication, indicating antiviral roles. Verteporfin a pharmacological inhibitor of the Hippo pathway downstream transactivator, YAP, significantly reduced virus replication. These results delineate a direct antiviral role for Hippo signaling in SARS-CoV-2 infection and the potential for this pathway to be pharmacologically targeted to treat COVID-19.

Figure 1.. Hippo signaling pathway is activated in lung tissues of COVID-19 patients and infected human lung in vitro cell cultures.

(A) Transcriptome analysis of five negative control and autopsy lung samples from patients deceased due to SARS-CoV-2 infection are depicted. KEGG pathway database was used to examine the Hippo signaling pathway genes in the differentially expressed genes (DEGs). Heatmap shows the expression levels of the 45 DEGs (p <0.01) involved in Hippo signaling pathway. Blue and red colors represent downregulated and upregulated genes, respectively. (B) Immunohistochemistry (IHC) of COVID-19 lung autopsy tissue shows high level of phospho-YAP Ser127 protein (dark brown). SARS-CoV-2 RNA (red) presence in COVID-19 lung was confirmed by RNA-FISH (RNAscope). Images are obtained at 20x magnification. (C) Western blot analyses of lung ALI cells show activation of Hippo-TBK1 pathways during SARS-CoV-2 infection. Proximal lung air-interface cultures are susceptible to SARS-CoV-2 infection. Antibody targeting Spike protein was used for probing infection by immunohistochemistry. Cell-specific markers, such as Ac-tubulin (ciliated cells) and Mucin5 (mucus cells) were detected by antibody probes to define infected cell types. Scale bar 10 μm. (D) Human airway epithelial cells (Calu-3) were analyzed with IHC for pYAP127 and SARS-CoV-2 Spike protein in uninfected (Mock) and infected cells. Parental and Delta strains were used for infection studies. Scale bar: 25 μm. Representative data from three independent studies are provided.

Figure 2.. SARS-CoV-2 infection activates Hippo and antiviral STAT pathways in hPSC-CMs.

(A) Confocal image analysis of SARS-CoV-2 (red) infected cardiomyocytes shows extensive damage to cTNT positive (green) cells, which undergo apoptotic cell death (green; cleaved caspase 3). Scale bar 5 μm. n=6 independent experiments. (B) Western blot analyzes show activation of Hippo and STAT1 pathways. Phospho-YAP127 level is increased at 2 and 24 hpi upon SARS-CoV-2 infection. N=2 independent experiments. (C) Immunohistochemistry analysis of SARS-CoV-2 infected PSC-CMs at 24 hpi reveals cytoplasmic localization of YAP/TAZ and (D) increase in pYAP127 level. Scale bar 25 μm.

Figure 3.. ShRNA-mediated knockdown and pharmacological modulation of SARS-CoV-2 replication.

(A) IHC analysis of shRNA-mediated knockdown of YAP1 and LATS1 specific shRNAs showed efficiently reduced or increased SARS-CoV-2 replication (green) relative to shRNA control, respectively in hPSC-CMs. Scale bar 50 μm. (B) Western blot analysis of shRNA-mediated knockdown of YAP1 and LATS1 respective protein expression. (Con: Control shRNA). (C) IHC images of XMU-MP-1 (MST1/2 inhibitor) and vehicle treated hPSC-CMs are shown. Note: XMU-MP-1 increased SARS-CoV-2 replication (green) in hPSC-CM. (D) Graphs depict quantification of SARS-CoV-2 positive cells in infected hPSC-CM respective to panels A and C. Student T-test. **P >0.001. n=2 independent experiments. (E) IHC Images show YAP/TAZ protein (green) and SARS-CoV-2 Spike (red) in Calu-3cells. Note, MST1/2 inhibitor treated Calu-3 cells have higher number of infected cells. Inset and white asterisk hovers infected cells showing depletion of YAP/TAZ. Scale bar: 25 μm. Inset scale bar 10 μm. (F) Western blot analysis of Calu-3 cells treated with Verteporfin (1μM) and SARS-CoV-2 infection. Drug treatment resulted in reduction in SARS-CoV-2 infection. Graph shows the viral titer (TCID50/ml) measurement of infected as well as treated Calu-3 culture supernatant(representative data from two independent experiments) (G) Schematic diagram of our hypothetical model integrating Hippo and TBK1 signaling pathways during preinfection (Hippo off) and SARS-CoV-2 infection states (Hippo on). c-GAS, cyclic GMP-AMP synthase; IKKe, inhibitor of nuclear factor kappa B kinase subunit epsilon; MAVS, mitochondrial antiviral-signaling protein; RIG-I, retinoic acid inducible gene I protein; STING, stimulator of interferon response cGAMP interactor 1; TEAD, TEA domain transcription factors.