Capture of fusion-intermediate conformations of SARS-CoV-2 spike requires receptor binding and cleavage at either the S1/S2 or S2’ site

Figures

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  Fig 1. The inhibitory potency of the 36-HR2 peptide depends on the virus entry pathway. (A) Schematic representation of SARS-CoV-2 S2 subunit and the HR1 (green) and HR2 (magenta) derived peptides. Functional domains of the S2 protein: FP, fusion peptide; HR1, heptad repeat 1; CH, central helix; CD, connector domain; HR2, heptad repeat 2; TM, transmembrane domain. The S1/S2 and S2’ cleavage sites are marked. (B) Cartoon representation of the 6-helix bundle region of the SARS-CoV-2 spike in the post-fusion conformation (PDB 8FDW). The 42-HR1 and 36-HR2 are highlighted in green and magenta, respectively. (C) Dose-response curves of 36-HR2 peptide inhibition of D614G pseudovirus infection of 293T cells expressing ACE2 (red filled circles) or ACE2/TMPRSS2 (blue open circles). (D) Interaction of 42-HR1 and 36-HR2 peptides at different pHs. Bands corresponding to 36-HR2 and the 42-HR1+ 36-HR2 complexes are indicated. (E) 36-HR2 peptide inhibition of spike-mediated, cell-cell fusion between 293T effector cells expressing D614G spike and 293T targets cells expressing ACE2 (filled red circles) or ACE2/TMPRSS2 (open blue circles). (F) Time-dependence of 36-HR2 inhibition of cell-cell fusion…

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  Fig 2. ACE2 binding is sufficient to allow peptide capture of an S2 fusion intermediate, while TMPRSS2 allows binding to an S2’ fusion intermediate. (A) Experimental setup to assess trapping of fusion-intermediate conformations of spike. In the absence of peptide, spike and ACE2 interact, favoring the conformational changes in spike that facilitate membrane fusion and syncytia formation. In the presence of peptide, the spike cannot transition to the post-fusion conformation and syncytia are not formed. Formation of the trapped intermediate conformations can be analyzed by flow cytometry on the cell surface or by co-immunoprecipitation on cell lysates. (B) Flow cytometry histograms showing the interaction of 36-HR2-HA with effector cells (E), expressing D614G and target cells (T), expressing ACE2 (red trace) or ACE2/TMPRSS2 (blue trace). Control effector cells lacking spike (black trace) and target cells lacking receptors (gray trace) are shown. After incubation of HA-tagged peptide with cells, cells were washed and then incubated first with anti-HA, followed by Alexa 680 anti-mouse antibodies. One representative out of three experiments with similar results is shown. Capture of the…

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  Fig 3. At least one spike cleavage is needed for unwinding S2 to expose the HR1 in the fusion-intermediate conformation of spike. (A) Amino acid sequence alignment of the S1/S2 junction region with the furin cleavage site (FCS) of spikes used in this study. (B) 36-HR2 peptide inhibition of infection of ACE2/TMPRSS2 target cells by pseudoviruses with or without FCS spike mutations. Each datapoint corresponds to the average of three independent experiments. Mean IC50 and the standard deviation of three independent experiments is shown. (C) Co-immunoprecipitation of the D614G, D614G ∆FCS and D614G(SKPSK) spikes with 36-HR2-HA in the absence or the presence of ACE2 or ACE2/TMPRSS2 target cells. The S2, S2’, and S0 bands are indicated. One representative out of two experiments with similar results is shown.

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  Fig 4. Binding of the CB6 antibody to the RBD triggers spike conformational changes to fusion intermediates. (A) Superimposition of the structures of the SARS-CoV-2 spike trimer with 1-RBD-up conformation (PDB 7KDJ, gray) and structures of the RBD complexed with ACE2 (PDB 7A94, blue) and CB6 (PDB 7C01, green). Superposition of the 1-RBD-up and 3-RBD-down conformations (PDBs 7KDJ and KDG, respectively, gray) and the structure of the RBD complexed with Beb (PDB 7MMO, red). (B) Inhibition of CB6 and Beb against D614G spike-mediated cell fusion with 293T cells expressing ACE2 (red filled circles) or ACE2/TMPRSS2 (blue open circles). One representative out of three experiments with similar results is shown. Mean IC50 and the standard deviation of three independent experiments is shown. (C) Antibody bound spikes were co-immunoprecipitated with the 36-HR2-HA peptide in the absence or presence of ACE2 or ACE2/TMPRSS2. Effector cells were pre-incubated with CB6 or Beb antibodies before performing the co-immunoprecipitation. The S2, S2’, and S0 bands are indicated. One representative out of two experiments with similar results is shown.

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  Fig 5. The HR1 peptides preferentially trap S2 rather than S2’ fusion-intermediate spikes of Omicron variants in the presence of TMPRSS2. (A) Amino acid sequence alignment of the HR1 region in different mutants used in this study. Mutations are color coded. The sequence of the HR2 region is also presented. 42-HR1 and 36-HR2 peptides are underlined. (B) Cartoon representation of the 6-helix bundle region of the SARS-CoV-2 post-fusion conformation (PDB 8FDW). The 36-HR2 is highlighted in magenta. Residues mutated in Delta and Omicron variant are highlighted. (C) Spikes corresponding to the different variants were co-immunoprecipitated with 36-HR2-HA in the absence of any receptor, or in the presence of ACE2 or ACE2/TMPRSS2. Samples from the co-immunoprecipitation were analyzed by western blot using and anti-S2 antibody. The S2, S2’, and S0 bands are indicated in each panel. S2/S2’ densitometry ratio is presented below each lane. (D) 36-HR2 binding to different variants against 293T cells expressing ACE2 (red circles) or ACE2/TMPRSS2 (open blue circles) as measured by flow cytometry. One representative out of three experiments with similar results is shown. Characterization of the D614G…

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  Fig 6. Model of spike-mediated membrane fusion showing peptide capture of fusion intermediates. Schematic of spike-mediated membrane fusion during virus infection. Full-length model of the spike glycoprotein from refs [6,25,64] were used to highlight the HR1 (green) or HR2 (magenta) regions in the spike pre-fusion conformation. In the pre-fusion conformation (I), the RBD continuously samples the up and down conformations until it binds to ACE2 to form an ACE2-spike complex (II). ACE2 binding facilitates an open conformation (III, PDB ID 8Z7P) that exposes the HR1 and allows 3 trapping of the S2 intermediate by the 36-HR peptide. Formation of this intermediate occurs with displacement of the S1 subunit. The S2’ cleavage site is subsequently exposed to proteolysis by TMPRSS2 or other metalloproteases that allows trapping of a second S2’ fusion intermediate. The 36-HR peptide can trap either S2’ or S2’ fusion intermediate. The binding of the 36-HR2 peptide to the endogenous (viral) HR1 during transition to the fusion intermediate conformations interferes with formation of the endogenous six-helix bundle (IV, PDB ID 8FDW) preventing membrane fusion.