Although structures of pre- and post-fusion conformations of SARS-CoV-2 spikes have been solved by cryo-electron microscopy, the transient spike conformations that mediate virus fusion with host cell membranes remain poorly understood. In this study, we used a peptide fusion inhibitor corresponding to the heptad repeat 2 (HR2) in the S2 transmembrane subunit of the spike to investigate fusion-intermediate conformations that involve exposure of the highly conserved heptad repeat 1 (HR1). The HR2 peptide disrupts the assembly of the HR1 and HR2 regions of the spike, which form a six-helix bundle during the transition to the post-fusion conformation. We show that binding of the spike S1 subunit to ACE2 is sufficient to induce conformational changes that allow S1 shedding and enable the HR2 peptide to bind to fusion-intermediate conformations of S2 and inhibit membrane fusion. When TMPRSS2 is also present, the peptide captures an S2' fusion intermediate though the proportion of the S2' intermediate relative to the S2 intermediate is lower in Omicron variants than pre-Omicron variants. In spikes lacking the natural S1/S2 furin cleavage site, ACE2 binding alone is not sufficient for trapping fusion intermediates, but the presence of ACE2 and TMPRSS2 allows peptide trapping of an S2' intermediate. These results indicate that, in addition to ACE2 engagement, at least one spike cleavage is needed for unwinding S2 into an HR2 peptide-sensitive, fusion-intermediate conformation. Our findings elucidate fusion-intermediate conformations of SARS-CoV-2 spike variants that expose conserved sites on spike that could be targeted by inhibitors or antibodies.
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