Wqpprari

Heat-inactivated SARS-CoV-2 (strain: 2019-nCoV/USA-WA1/2020) was purchased from ATCC (Manassas, VA, USA; cat. no. ATCC VR-1986HK), the N-terminally His-tagged recombinant SARS-CoV-2 spike protein S1 subunit (region Val16–Gln690) from RayBiotech (Peachtree Corners, GA, USA) recombinant human syndecan-4 from R&D Systems (Minneapolis, MN, USA; cat. no. 2918-SD) and recombinant human ACE2 from Abcam (Cambridge, UK; ab151852). Peptide inhibitors utilized for the studies were purchased from either Bachem (Bubendorf, Switzerland; DX600) or Genscript (Leiden, The Netherlands; SPRRAR and WQPPRARI). SARS-CoV-2 pseudovirus-RFP (SARS-CoV-2 PSV-RFP), recombinant pseudotyped lentiviral particles containing SARS-CoV-2 spike protein and encoding red fluorescent protein (RFP) in their lentiviral genome was purchased from GeneMedi (Shanghai, China; cat. no. GM-2019nCoV-PSV01).

To reveal the involvement of various endocytosis pathways, A549 cells were preincubated with the following inhibitors 30 min before SARS-CoV-2 admission: amiloride hydrochloride (at a concentration of 100 µM; Sigma, St. Louis, MO, USA); DX600 (10 µM; Bachem, Bubendorf, Switzerland); Gö 6983 (10 µM; Sigma, St. Louis, MO, USA); heparin (200 ug/mL; Sigma, St. Louis, MO, USA); a heparin-binding peptide (WQPPRARI, 100 µM; Genscript, Leiden, The Netherlands); and SPRRAR derived from spikeS1 (100 µM; Genscript, Leiden, The Netherlands). After incubation with these inhibitors, the cells were then treated with SARS-CoV-2 and processed for the flow cytometric analyses as described above.