SARS-CoV-2 Spike Protein-Priming Cysteine Cathepsin L Acting at Microvilli of Intestine Epithelial Cells

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SARS-CoV-2, responsible for causing COVID-19, enters via epithelial cells including enterocytes of the small intestine. While the proteases ACE2 and TMPRSS2 are recognized as entry co-receptor/s of SARS-CoV-2 target cells, it is not sufficiently understood how the cysteine peptidase cathepsin L involves, which can act as a viral spike protein-activating enzyme in principle. Thus, the proposed research shall test a novel and non-conventional mechanism of SARS-CoV-2 spike protein activation, namely, by cathepsin L acting at microvilli of small intestine epithelial cells. We base this proposal on our previous work demonstrating that cathepsin L is secreted from intestine epithelial and goblet cells into the gut lumen from where is re-associates with the surface of the mucosal cells. Hence, cathepsin L is present at well-exposed microvilli of the small intestine epithelium, where it might be involved in priming of the coronaviral spike protein, thereby promoting viral entry and spreading, despite and in addition to the prominent tasks that ACE2 and TMPRSS2 play in this regard. Therefore, this project will address expression and sub-cellular co-localization of cathepsin L, ACE2 and TMPRSS2 in the small intestine in situ as well as the enzymatic activity of cathepsin L at microvilli of intestine epithelial cells using our established organoid-typical co-culture model consisting of enterocytes and goblet cells. In addition, we will investigate the possibility of cathepsin L-mediated spike protein cleavage in vitro by simulating conditions expected for the gut lumen. Eventually, the significance of cathepsin L activity for maintenance of microvilli at cells of the small intestine will be analyzed. We expect to clarify whether cathepsin L can play an important role in SARS-CoV-2 infection of the small intestine. The results of this basic science project will help to more comprehensively approach therapeutic prevention of coronavirus entry into target cells.