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. 2023 Mar 21;12:e83710. doi: 10.7554/eLife.83710

Figure 3. SARS-2 spike residues 985–988 are recognized by 3A3 and impair spike function upon substitution.

(a) Residues important for 3A3 binding were identified by single residue changes in HexaPro that increased or decreased binding to 3A3 relative to HexaPro. Each variant was tested with duplicate technical replicates in 2–6 independent ELISA assays. Significance relative to unaltered HexaPro was determined by ANOVA with post-hoc Tukey–Kramer test with α = 0.01; data meeting this criterion indicated by *. (b) Location of the residue changes altering binding to 3A3 shown in the HexaPro spike (6XKL) monomer and in (c) intact spike (side view) and the S2 domain (top-down view). All epitope residues (980–1006) are shown in space-fill, with residues colored according to their effect on 3A3 binding: improved binding (orange), reduced binding (teal), no effect (black), and those not altered (gray). The 2P stabilizing mutations within the hinge epitope are displayed in purple. (d) The infectivity of lentivirus pseudotyped with unmodified D614G SARS-2 spike or variants with D985L (green), E988A (light blue), and E988Q (dark blue) substitutions was compared by luciferase activity. Data shown are the mean luminescence with standard deviation of three technical replicates.

Figure 3—source data 1. ELISA binding data and relative luminescence data for pseudovirus infection assays.

Figure 3.

Figure 3—figure supplement 1. HexaPro variants with reduced 3A3 binding retain trimer SEC profile.

Figure 3—figure supplement 1.

The single-point mutants of HexaPro, D985L, E988I, D994A, and L1001A, had reduced binding to 3A3 relative to HexaPro (Figure 3a), but retained the overall size of unmodified HexaPro by SEC with elution of the main peak at ~13.75 mL on a Superose 6 Increase 30/100 column, indicating the spike was intact. Molecular weight markers (black triangles) on the x-axis are peak elution volumes from the following standards in order from left to right: thyroglobulin (669 kDa, 13.49 mL), ferritin (440 kDa, 15.40 mL), β-amylase (200 kDa, 16.75 mL), and aldolase (158 kDa, 17.91 mL).
Figure 3—figure supplement 2. The hinge epitope is nonlinear and inaccessible in aggregated or misfolded protein.

Figure 3—figure supplement 2.

(a) 3E11 binds reduced, denatured SARS-2 HP, SARS-2, and MERS spike proteins by Western blot, but 3A3 does not. The ladder molecular weight is labeled in kDa on the left side. (b) SDS-PAGE analysis of fresh and stressed SARS-2 and SARS-2 HexaPro spikes shows a substantial aggregation of the stressed SARS-2 spike. For each spike, 8 μg of protein was analyzed by SDS-PAGE under nonreducing conditions. Reduced band intensity of stressed SARS-2 spike (bottom arrow) and appearance of a band just under the loading wells (top arrow) indicates aggregation products not apparent in the other samples. Densitometry analysis (ImageJ) of these bands indicates that the stressed SARS-2 spike’s main peak is diminished by ~20% relative to the fresh SARS-2 spike intensity. Ladder molecular weights in kDa are indicated to the left of the gel image. Binding of 3A3 Fab to HexaPro S2 measured (c) ELISA capture of fresh (red circles) or stressed (pink diamonds) SARS-2 spike on 3A3 coated plates. (d) Antibodies coated on ELISA plates captured fresh (dark blue or red) or stressed (pink or light blue) SARS-2 HP (blue) or SARS-2 (red) spike proteins. Duplicate dilutions of spike over ~5 log in concentration were used to calculate EC50 values. For dilution series in which no binding was observed, EC50 was assumed to be >1000 nM. Open symbols are replicate data and filled rectangles are average data.
Figure 3—figure supplement 3. Original blot images for Figure 3—figure supplement 2a.

Figure 3—figure supplement 3.

Figure 3—figure supplement 4. Adding a disulfide bond slightly improves 3A3 binding by ELISA to spike with P986 and P987 reverted to the native sequence.

Figure 3—figure supplement 4.

Antibody 3A3 was coated on high binding plates and allowed to bind dilutions of SARS-2 HexaPro spike, 4P spike (HexaPro with P986K and P987V reversions), or 4P-DS spike (4P with an additional stabilizing disulfide). For detection, streptactin-HRP was allowed to bind the twin strep tag sequence at the C-terminus of any captured spike proteins.