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

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.

The deadly coronaviruses: The 2003 SARS pandemic and the 2020 novel coronavirus epidemic in China

“…For β-CoVs, the RBD of the spike protein mediates the interaction with host receptor. Upon binding the receptor, the spike protein is cleaved by nearby host proteases and releases the signal peptide to facilitate virus entry into host cells [112][113][114][115].…”

Discovery of Critical Residues for Viral Entry and Inhibition through Structural Insight of HIV-1 Fusion Inhibitor CP621–652

“…Cell-Cell Fusion Assays-To evaluate the effect of mutations on HIV-1 Env-driven cell-cell fusion, a sensitive assay was adapted from our previous studies (23,24). Briefly, 293T effector cells seeded in 6-well plates at 4 ϫ 10 5 cells per well were transfected with the plasmid encoding HIV-1 NL4-3 Env or its mutants (M626A or T627A) in combination with plasmid pGAL4-VP16, which encodes the herpes simplex virus VP16 transactivator fused to the DNA-binding domain of the Saccharomyces cerevisiae transcription factor GAL4.…”

Bat-to-human: spike features determining ‘host jump’ of coronaviruses SARS-CoV, MERS-CoV, and beyond

“…In addition, the viral S can also be activated by extracellular enzymes such as trypsin, thermolysin, and elastase, which are shown to induce syncytia formation and virus entry, possibly at the plasma surface [42]. Other proteases that are of potential biological relevance in potentiating SARS-CoV S include TMPRSS2, TMPRSS11a, and HAT [43][44][45], which are localized on the cell surface and are highly expressed in the human airway [46]. It is also noteworthy that TMPRSS2 can associate with ACE2 to form a receptorprotease complex, enabling efficient virus entry directly at the cell surface [47].…”