The role of furin cleavage site in SARS-CoV-2 spike protein-mediated membrane fusion in the presence or absence of trypsin

摘要

The function of furin cleavage site in SARS-CoV-2-S mediated fusion. Schematic representation of SARS-CoV-2 S protein and the location of S1/S2 and S2′ cleavage site. SP, signal peptide; FP, fusion peptide; HR, heptad repeat domain; TM, transmembrane domain; CP, cytoplasmic domain. Mutated SARS-CoV-2 S proteins with mutation in S1/S2 region, including SARS-CoV-2-m1 (mutating “RRAR” into “SSAR”), SARS-CoV-2-m2 (deleting four amino acids, “PRRA”), SARS-CoV-2-m3 (replacing “QTQTNSPRRARSVASQSII” in SARS-CoV-2 with “HTVSLLRSTSQKSIV” derived from SARS-CoV), SARS-CoV-m1 (replacing “HTVSLLRSTSQKSIV” in SARS-CoV with “QTQTNSPRRARSVASQSII” derived from SARS-CoV-2), and SARS-CoV-m2 (mutating “RRAR” in SARS-CoV-m1 into “SSAR”). Prediction scores for the S1/S2 furin cleavage site in S protein were analyzed by using the ProP 1.0 server ( ). Western blot analysis of S protein expression in 293T cells. Representative images of cell–cell fusion between 293T/SARS-CoV-2/EGFP, 293T/SARS-CoV-2-m1/EGFP, 293T/SARS-CoV-2-m2/EGFP, 293T/SARS-CoV-2-m3/EGFP, 293T/SARS-CoV/EGFP, 293T/SARS-CoV-m1/EGFP, and 293T/SARS-CoV-m2/EGFP effector cells and target cells (Huh-7), using the fusion between target cells and 293T/EGFP effector cells without S-expression as negative control. Representative fused cells are indicated by white arrows. Scale bar = 400 µm. Statistical analysis of fusion rates mediated by wild-type or mutated S protein after co-culture for 4 h (left) or 24 h (right), taking the fusion rate of SARS-CoV-2 group as 100%. Representative images of cell–cell fusion between target and effector cells with indicated S protein in the presence of trypsin (200 ng/ml). Scale bar = 400 µm. Statistical analysis for ( ), taking the fusion rate of the SARS-CoV-2 group as 100%. , Fusion rates of cell–cell fusion between target cells and series of effector cells in the presence of indicated concentration of trypsin ( ) or HAT ( ), taking the fusion rate of SARS-CoV-2 group treated by trypsin (200 ng/ml) or HAT (500 ng/ml) as 100%. Experiments were repeated twice, and the data are expressed as means ± SD (error bar). Asterisks indicate significant differences (***