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

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.

Production of novel Spike truncations in Chinese hamster ovary cells (preprint)

SARS-CoV-2 Spike is a key protein that mediates viral entry into cells and elicits antibody responses. Its importance in infection, diagnostics, and vaccinations has created a large demand for purified Spike for clinical and research applications. Spike is difficult to express, prompting modifications to the protein and expression platforms to improve yields. Alternatively, Spike receptor binding domain (RBD) is commonly expressed with higher titers, though it has lower sensitivity in serological assays. Here, we improve transient Spike expression in Chinese hamster ovary (CHO) cells. We demonstrate that Spike titers increase significantly over the expression period, maximizing at 14 mg/L at day 7. In comparison, RBD titers peak at 54 mg/L at day 3. Next, we develop 8 Spike truncations (T1-T8) in pursuit of a truncation with high expression and antibody binding. The truncations T1 and T4 express at 130 mg/L and 73 mg/L, respectively, which are higher than our RBD titers. Purified proteins were evaluated for binding to antibodies raised against full-length Spike. T1 has similar sensitivity as Spike against a monoclonal antibody and even outperforms Spike for a polyclonal antibody. These results suggest T1 is a promising Spike alternative for use in various applications.