Capture of fusion-intermediate conformations of SARS-CoV-2 spike requires receptor binding and cleavage at either the S1/S2 or S2' site
Although the 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 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 trigger conformational changes that allow the peptide to capture a fusion-intermediate conformation of S2 and inhibit membrane fusion. When TMPRSS2 is also present, an S2 fusion intermediate is captured 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; however, co-expression of ACE2 and TMPRSS2 allows 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-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. Author summaryThe SARS-CoV-2 spike protein undergoes two proteolytic cleavages and major conformational changes that facilitate fusion between viral and host membranes during virus infection. Spike is cleaved to S1 and S2 subunits during biogenesis, and S2 is subsequently cleaved to S2 as the virus enters host cells. While structures of pre-fusion and post-fusion spike conformations have been extensively studied, transient fusion-intermediate conformations during the fusion process are less well understood. Here, we use a peptide fusion inhibitor corresponding to a heptad repeat domain in the S2 subunit to investigate fusion-inducing conformational changes. During spike-mediated cell-cell fusion, we show that the peptide binds to spike only after spike engages ACE2 and is cleaved at the S1/S2, S2, or both sites. Thus, S2 needs at least one cleavage to refold to a peptide-sensitive fusion intermediate. SARS-CoV-2 variants differed in the proportion of S2 and S2 fusion intermediates captured after receptor binding, indicating that the virus has evolved not only to alter its entry pathway but also to modulate S2 unfolding. This work informs the development of antiviral strategies targeting conserved sites in fusion-intermediate conformations of spike and contributes more broadly to the understanding of the entry mechanisms of viral fusion proteins.