Cleavage and Activation of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein by Human Airway Trypsin-Like Protease

TLDR

SARS‑CoV remains a major public‑health threat, with its spike protein mediating host cell entry and requiring proteolytic activation that is not yet fully understood. The study investigates the role of the type II transmembrane serine proteases HAT and TMPRSS2 in activating the SARS‑CoV spike protein. The authors examined HAT and TMPRSS2 activity using surrogate systems and authentic SARS‑CoV infection assays. HAT cleaves SARS‑S at R667, activates it for cell‑cell fusion in both cis and trans, and is co‑expressed with ACE2 in airway cells, whereas TMPRSS2 cleaves at multiple sites, activates fusion only in trans, and renders SARS‑S‑driven fusion cathepsin‑independent; thus HAT likely facilitates viral spread.

Abstract

The highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) poses a constant threat to human health. The viral spike protein (SARS-S) mediates host cell entry and is a potential target for antiviral intervention. Activation of SARS-S by host cell proteases is essential for SARS-CoV infectivity but remains incompletely understood. Here, we analyzed the role of the type II transmembrane serine proteases (TTSPs) human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2), in SARS-S activation. We found that HAT activates SARS-S in the context of surrogate systems and authentic SARS-CoV infection and is coexpressed with the viral receptor angiotensin-converting enzyme 2 (ACE2) in bronchial epithelial cells and pneumocytes. HAT cleaved SARS-S at R667, as determined by mutagenesis and mass spectrometry, and activated SARS-S for cell-cell fusion in cis and trans, while the related pulmonary protease TMPRSS2 cleaved SARS-S at multiple sites and activated SARS-S only in trans. However, TMPRSS2 but not HAT expression rendered SARS-S-driven virus-cell fusion independent of cathepsin activity, indicating that HAT and TMPRSS2 activate SARS-S differentially. Collectively, our results show that HAT cleaves and activates SARS-S and might support viral spread in patients.