explain please: Spike-Driven Syncytia Formation Is Coupled to S20 Fragment Generation in the Presence of ACE2. To investigate the functional and biochemical signatures of spike (S) protein upon engaging its receptor ACE2 in live cells, we first utilized a cell–cell fusion assay to obtain potentially cleaved spike protein products from syncytia . In this system, HEK293T cells transfected with plasmids encoding WT spike readily formed syncytia after coculturing with HEK293T cells expressing human and Vero E6-ACE2, as well as colorectal adenocarcinoma Caco-2 and lung adenocarcinoma Calu-3 cells expressing endogenous ACE2. We then collected these adherent syncytia and immunoblotted for cleaved spike species using a rabbit polyclonal antibody specifically detecting the S2 ectodomain through standard reducing Tris-glycine sodium dodecyl sulfate polyacrylamide gel electrophoresis. This experiment revealed that spike expression in HEK293T cells displayed full-length S and autocleaved S2 fragments, respectively, as reported earlier. Upon addition of HEK293T-ACE2 cells onto the spike-expressing HEK293T cells, an additional band S2' was detected, which was absent from spike-expressing HEK293T cells cocultured with control HEK293Tcells. The abundance of S2' band was dependent on the number of ACE2-expressing cells being added, suggesting that the increasing amount of ACE2 promoted the proteolytic processing of spike. To validate the peptide sequence of the S2' , we performed immunoprecipitation of spike protein products from HEK293T syncytia lysates. Purified S2 protein fragments were separated and examined on polyacrylamide gels using silver staining; the S2' fragment was then extracted for mass spectrometry analysis. Within the S2 subunit, 12 unique peptides were detected, listed, and mapped between the spike 816 and 1266 amino acid positions, confirming that the S2' is a further cleaved spike species downstream of S2. To go further, we tested three SARS-CoV-2 variants of concern, namely, Alpha, Beta, and Delta, using our cell–cell fusion system. These variants carry multiple forms of mutations spanning different regions of the spike S1 and S2 subunits , notably, the D614G substitution that promotes the S1 incorporation and infectivity, and the P681H and P681R substitutions that are adjacent to the S1/S2 furin cleavage site in the Alpha and Delta variants, respectively . HEK293T cells expressing these spike variants exhibited slightly increased autocleaved S2 in Beta and Delta variants when cocultured with control HEK293T cells without ACE2; after adding HEK293T cells expressing ACE2, generation of the cleaved S20 band was robustly detected in all spike variants, and was increased in Beta and Delta variants when compared with the Alpha variant. These data indicate that SARS-CoV-2 spike S2 can be autocleaved in the absence of ACE2, whereas S20 cleavage occurs exclusively in the presence of ACE2- expressing cells. To examine whether generation of a SARS-CoV-2 S20 fragment is associated with membrane, we physically homogenized spike-expressing cells and syncytia to separate soluble cytosol and insoluble membranes. S20 band was only detected from the membrane fractions of the fused HEK293T cells, but not in the cytosolic fraction, suggesting that the S2' remained as a membrane-associating fragment. To confirm whether cleaved S2' functionally formed a homotrimeric coiled-coil six-helix bundle, we measured its resistance to limited proteolysis using proteinase K. This experiment revealed that proteinase K digested the full-length S, autocleaved S2, and ACE2 in both nonfused and fused HEK293T cell lysates, whereas the S2' fragment remained the only species resistant to the proteinase K digestion in the syncytia lysates detected using the anti-S2 antibody . Hence, the cleaved S20 fragment functionally formed a rigid spike fusion core, but the autoprocessed S2 and full-length spike expressed in HEK293Tcells did not. To quantitatively validate spike-driven membrane fusion and subsequent blending of the cytosolic contents of donor and acceptor cells, we utilized a bioluminescence assay system wherein a firefly luciferase (Stop-Luc) reporter gene is only expressed when Cre excises the Stop cassette inside the fused syncytia . When spike + Cre-expressing HEK293T cells were cocultured with increasing ratio of HEK293T-ACE2 cells carrying Stop-Luc, an ACE2-dependent increase in luciferase signal, quantified as the relative luminescence units (RLU) in postfusion HEK293T syncytia, was detected . Coculturing spike + Cre-expressing cells with control Stop-Luc cells without ACE2 expression did not produce any luminescence signal, suggesting that ACE2 is required for cell–cell fusion.
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