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. 2022 Jun 16;13(4):e00519-22. doi: 10.1128/mbio.00519-22

FIG 4.

FIG 4

The metalloproteinase-dependent entry pathway requires both the furin cleavage site and S2 region of the SARS-CoV-2 S protein. The effects of drugs on the entry of S protein-bearing vesicular stomatitis virus (VSV) pseudotype virus produced by 293T cells are shown. The relative pseudovirus entry was calculated by normalizing the FL activity for each condition to the FL activity of cells infected with pseudovirus in the presence of DMSO alone, which was set to 100%. Values are means ± SD (n = 3/group). **, P < 0.01. Cont, control (cells infected with pseudovirus without S protein); E-64d, 25 μM E-64d; marima, 1 μM marimastat; nafamo, 10 μM nafamostat. (a) Effects of E-64d and marimastat on the entry of pseudoviruses bearing SARS-CoV S, SARS-CoV-2 S, MERS-CoV S, or VSV G in HEC50B cells. (b) Effects of E-64d and marimastat on the entry of HCoV-NL63 S and WIV1-CoV S pseudovirus in HEC50B cells. (c) Schematic illustration of C-terminally Flag-tagged chimeric S proteins in which the S1, S1/S2 boundary, and S2 domain from SARS-CoV S (red) and SARS-CoV-2 S (yellow) are indicated (top). Amino acid sequences of the residues around the S1/S2 boundary of the coronaviruses (bottom). Numbers refer to the amino acid residues. F, Flag tag. Arginine residues in the S1/S2 cleavage site and furin cleavage motif are highlighted in red. (d) Expression of chimeric S protein in pseudoviruses. S proteins were detected using an anti-Flag tag antibody that binds to a Flag tag on the C terminus of the S proteins (top). Detection of the vesicular stomatitis virus matrix protein (VSV M) served as the control (bottom). Culture supernatants of 293T cells containing the pseudoviruses were centrifuged at 109,000 × g for 35 min at 4°C using a TLA100.3 rotor with an Optima TLX ultracentrifuge (Beckman Coulter, CA, USA), and the pellet was then lysed for Western blotting. S0, uncleaved S protein; S2, cleaved S2 domain of the S protein. (e and f) Effects of E-64d and marimastat on the entry of pseudoviruses bearing chimeric S proteins in HEC50B cells. (g) Effects of E-64d and nafamostat on the entry of pseudoviruses bearing SARS-CoV S, SARS-CoV-2 S, MERS-CoV S, or VSV G in HEC50B-TMPRSS2 cells in the presence of marimastat. (h and i) Effects of E-64d and nafamostat on the entry of pseudoviruses bearing chimeric S proteins in HEC50B-TMPRSS2 cells in the presence of marimastat.