Human cathepsin l

SARS-CoV-2 S FKO-FLAG pseudotyped HIV viruses were generated in the presence or absence of SERINC5-HA as mentioned above (see Pseudovirus production section). As previously described^{35 (link)}, virus pellets were resuspended in 100 μl of 1× PBS with Ca²⁺ and Mg²⁺, pH 5.5 and either treated with PBS (mock) or human Cathepsin L (10 μg/ml, Millipore Sigma, 219402) for 1 h at room temperature (RT). We used 20 μM of Cathepsin L inhibitor SID 26681509 (MedChemExpress) as a control. Virus supernatants were then resolved on 8% SDS-PAGE gels. SARS-CoV-2 S FKO was detected using rabbit anti- FLAG as described above (see Immunoblotting section).

Enzymes included bovine trypsin, bovine chymotrypsin, human cathepsin B, human cathepsin L, and human cathepsin S (all Sigma-Aldrich) and human cathepsin K (Enzo Life Sciences). Purified F. hepatica cathepsin L1 (FhCL1), F. hepatica cathepsin L2 (FhCL2) and F. hepatica cathepsin L3 (FhCL3)^{66 (link)} and recombinant FhKT1 and FhKT1Leu¹⁵/Arg^{1510 (link)} were expressed as active recombinant proteins in P. pastoris. Reaction conditions and substrates employed for measuring the activity of each protease were as reported by Smith et al.^{10 (link)}. Additionally, rFhCL3 activity was measured using the fluorogenic substrate Z-Gly-Pro-Arg-NHMec (20 μM).
KT inhibitors (2 μM) was incubated with each protease in a 100 μl volume of reaction buffer for 15 min at 37 °C. Reaction were brought to 200 μl with the addition of fluorogenic substrate dissolved in reaction buffer and proteolytic activity measured as RFU (relative fluorescent units) using a PolarStar Omega spectrophotometer (BMG LabTech, UK). Inhibition constants were determined using the Morrison equation for tight-binding inhibition as previously described^{10 (link)}.