SARS-CoV-2 Alpha, Beta, and Delta variants display enhanced Spike-mediated syncytia formation

Abstract

Severe COVID-19 is characterized by lung abnormalities, including the presence of syncytial pneumocytes. Syncytia form when SARS-CoV-2 spike protein expressed on the surface of infected cells interacts with the ACE2 receptor on neighboring cells. The syncytia forming potential of spike variant proteins remain poorly characterized. Here, we first assessed Alpha (B.1.1.7) and Beta (B.1.351) spread and fusion in cell cultures, compared with the ancestral D614G strain. Alpha and Beta replicated similarly to D614G strain in Vero, Caco-2, Calu-3, and primary airway cells. However, Alpha and Beta formed larger and more numerous syncytia. Variant spike proteins displayed higher ACE2 affinity compared with D614G. Alpha, Beta, and D614G fusion was similarly inhibited by interferon-induced transmembrane proteins (IFITMs). Individual mutations present in Alpha and Beta spikes modified fusogenicity, binding to ACE2 or recognition by monoclonal antibodies. We further show that Delta spike also triggers faster fusion relative to D614G. Thus, SARS-CoV-2 emerging variants display enhanced syncytia formation.

Keywords: SARS-CoV-2; coronavirus; fusion; spike; syncytia.

Figure 1. Replication kinetics of D614G, Alpha, and Beta variants in cell culture

1. A–D

   Cells were infected at the indicated MOI. Viral replication (left) and release (right) were assessed by flow cytometry and RT‐qPCR. (A) Caco2/TC7 cells (MOI 0.01), (B) Calu‐3 cells (MOI 0.001), (C) Vero cells (MOI 0.01), (D) primary human airway epithelial cells (HAEC) virus release (Right) and infectious virus release (Left) (MOI 0.01). Data are mean ± SD of at least three independent experiments. Statistical analysis: mixed‐effect analysis or two‐way ANOVA compared with D614G reference, ns: non‐significant, *P < 0. 05, ****P < 0.0001.

Figure 2. SARS‐CoV‐2 variant infection increases formation of syncytia in U2OS‐ACE2 and Vero GFP‐split cells

1. U2OS‐ACE2 or Vero cells expressing either GFP 1–10 or GFP 11 (1:1 ratio) were infected 24 h after plating and imaged 20 h (U2OS‐ACE2) or 48 h (Vero) post‐infection.

2. Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for U2OS‐ACE2 20 h post‐infection at MOI 0.001. Right Panel: Representative images of U2OS‐ACE2 20 h post‐infection, GFP‐Split (green), and Hoechst (blue). Top and bottom are the same images with and without Hoechst channel.

3. Left Panel: Quantified fusion of Vero cells infected at MOI 0.01. Right Panel: Representative images of Vero cells 48 h post‐infection, GFP‐Split (green), and Hoechst (blue).

Data information: Scale bars: 200 µm. Data are mean ± SD of eight independent experiments. Statistical analysis: one‐way ANOVA compared with D614G reference, ns: non‐significant, **P < 0.01, ****P < 0.0001.

Figure EV1. Qualitative and quantitative assessment of syncytia formation

1. U2OS‐ACE2 GFP‐split cells were infected at MOI 0.01 with the Wuhan, D614G, Alpha, and Beta strains for 20 h. Cells were stained for S protein with the human pan‐SARS‐CoV‐2 102 mAb and Alex647 fluorescent secondary antibody. Representative confocal images of the variant induced syncytia formation: GFP‐Split (green), Spike (red), and Hoechst (blue). Scale bars: 50 µm.

2. Quantification method for syncytia formation using the Opera Phenix high content imager and harmony software: Total syncytia area (GFP area) is normalized for cell number upon quantifying the number of nuclei (Hoechst).

Figure 3. Alpha and Beta SARS‐CoV‐2 S proteins induce more robust syncytia formation than D614G

1. Vero GFP‐split cells were transfected with variant S proteins and imaged 18 h post‐transfection.

2. Left Panel: Fusion was quantified by GFP area/number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Vero GFP‐split cells 18 h post‐transfection, GFP (green), and Hoechst (blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm.

3. Left Panel: Quantification of variant S protein‐mediated fusion in Vero GFP‐split cells by video microscopy. Results are mean ± SD from three fields per condition from one representative experiment. Right Panel: Fusion quantification of at least three independent video microscopy experiments, 20 h post‐transfection, normalized to D614G.

Data information: Data are mean ± SD of at least three independent experiments. Statistical analysis: one‐way ANOVA compared with D614G reference, ns: non‐significant, *P < 0. 05, ***P < 0.001, ****P < 0.0001.

Figure EV2. SARS‐CoV‐2 variant S proteins are expressed equally at the cell surface

293T cells were transfected with variant S proteins for 20 h and stained with human pan‐coronavirus mAb10 without permeabilization. 293T cells were chosen because they lack ACE2 and do not fuse upon S transfection; this makes them suitable for single‐cell flow cytometry.

1. Left Panel: Quantification of percent of cells expressing each S protein at the surface. Right Panel: Representative FACs plots.

2. Quantification of median florescent intensity (MFI) of variant S protein at the cell surface and representative histograms of MFI of the Wuhan, D614G, Alpha, Beta, and Alpha + E484K variants S protein using mAb10.

3. Quantification of median florescent intensity (MFI) of variant S protein at the cell surface and representative histograms of MFI of the Wuhan, D614G, Alpha, Beta, and Alpha + E484K variants S protein using mAb129.

4. Quantification of median florescent intensity (MFI) of variant S protein at the cell surface and representative histograms of MFI of the Delta variant compared with the Alpha and D614G using mAb129.

5. Controlled acceptor/donor experiment 293T GFP1‐10 donor cells transfected with S protein and verified to have equal S protein expression on the surface (top), were then added to Vero GFP11 acceptor cells (bottom) to assess fusion.

Data information: Flow cytometry data are mean ± SD of at least three independent experiments. Statistical analysis: one‐way ANOVA compared with D614G reference, ns: non‐significant, *P < 0. 05, ****P < 0.0001.

Figure EV3. Impact of IFN‐β1 and IFITMs on SARS‐CoV‐2 variant replication and S protein‐mediated cell–cell fusion

1. A

   Vero cells were pre‐treated for 2 h with a serial dilution of IFN‐β1 prior to infection with the SARS‐CoV‐2 variants. Infected cells were maintained in media containing IFN‐β1 and analyzed by flow cytometry 48 h post‐infection to determine relative infection change.

2. B

   U20S‐ACE2 GFP‐split cells were pre‐treated for 2 h with a serial dilution of IFN‐β1 prior to infection with the SARS‐CoV‐2 variants. Infected cells were maintained in media containing IFN‐β1 and relative inhibition of syncytia formation 20 h post‐infection was determined via GFP signal.

3. C–G

   A co‐culture of 293T GFP‐Split cells were transfected with combination of S, control, ACE2, TMPRSS2, and IFITM plasmids and then imaged 18 h post‐transfection. Effect of IFITMs and TMPRSS2 on the cell–cell fusion induced by different S proteins, (D) Wuhan, (E) D614G, (F) Alpha, and (G) Beta.

Data information: Data are mean ± SD of at least three independent experiments. Statistical analysis: one‐way ANOVA compared with D614G reference or control plasmid transfection, ns: non‐significant, *P < 0. 05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 4. Mutations associated with Alpha and Beta S proteins differentially affect cell–cell fusion

1. Top Panel: Schematic representation of the S protein color‐coded for the functional regions: N‐terminal domain (NTD), receptor‐binding domain (RBD), fusion peptide (FP), heptad repeat 1,2 (HR1, HR2), transmembrane anchor (TA), C‐terminal domain (CTD). Bottom left Panel: Vero GFP‐split cells were transfected with S plasmids containing each of the individual mutations associated with Alpha variant in the D614G background. The amount of fusion was quantified at 20 h and normalized to D614G reference plasmid. Bottom right Panel: Quantified fusion for each of the individual S protein mutations associated with the Beta variant. Color code of each mutation corresponds to S protein functional regions represented in the schematic on the Top Panel. Data set for N501Y and D614G reference mutations are duplicated between bottom left and bottom right panels for presentation as these mutations are common to both variants.

2. Left Panel: Quantified fusion of the Alpha + E484K variant S protein normalized to D614G S. Right Panel: Representative images of fusion at 20 h. Scale bar: 200 µm. Top and bottom are the same images with and without Hoechst channel.

Data information: Data are mean ± SD of at least four independent experiments. Top and bottom are the same images with and without Hoechst channel. Statistical analysis: statistics for both left and right panels of A were conducted together. One‐way ANOVA compared with D614G reference, ns: non‐significant, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure EV4. SARS‐CoV‐2 variant S protein‐associated mutations are expressed equally at the cell surface

293T cells were transfected with S proteins with each of the variant‐associated mutations for 18 h and stained with human pan‐coronavirus mAb10 without permeabilization.

1. Representative FAC plots of percent of cells expressing each mutant S protein at the surface.

2. Quantification of percent of cells expressing each S protein at the surface.

3. Quantification of median florescent intensity (MFI) of the mutant S protein at the cell surface.

4. Representative histograms of MFI of each mutant S protein.

5. Representative images of Vero GFP‐split cells 20 h after transfection with each Alpha variant‐associated mutant S protein, GFP‐Split (Green). Scale bars: 200 µm.

6. Representative images of Vero GFP‐split cells 20 h after transfection with each Beta variant‐associated mutant S protein. Scale bars: 200 µm.

Data information: Data are mean ± SD of at least three independent experiments. Statistical analysis: one‐way ANOVA compared with D614G reference, ns: non‐significant.

Figure 5. ACE2 and monoclonal antibody binding to S proteins with Alpha and Beta associated mutations

1. 293T cells were transfected S proteins with each variant‐associated mutation for 24 h and stained with biotinylated ACE2 and fluorescent streptavidin before analysis by flow cytometry.

2. Left Panel: EC50 values (concentration of ACE2 needed for 50% binding) for Alpha and associated mutations. Color code corresponds to location on S protein functional domains and lower EC50 values signify higher affinity to ACE2 binding. Right Panel: EC50 values for Beta and associated mutations. Data set for N501Y and D614G reference mutations are duplicated between left and right panels as mutations are common to both variants.

3. S protein transfected 293T cells were stained with human monoclonal antibodies targeting the S2 (mAb10), RBD (mAb48 and mAb98), and the NTD (mAb71). Cells were analyzed by flow cytometry. The percentage of positive cells is indicated.

Data information: Data are mean of at least three independent experiments. Statistical analysis: one‐way ANOVA compared with D614G reference, ns: non‐significant, *P < 0. 05, **P < 0.01, ****P < 0.0001.

Figure EV5. ACE2 binding curves to SARS‐CoV‐2 variant S proteins and associated mutations

293T cells were transfected with variant or mutant S proteins for 24 h and stained with a serial dilution of soluble biotinylated ACE2 and revealed by fluorescent streptavidin before analysis by flow cytometry.

1. A

   ACE2 binding dilution curves of each variant S protein.

2. B–E

   ACE2 binding dilution curves of each variant‐associated mutation located in (B) S protein n‐terminal domain (NTD), (C) receptor‐binding domain (RBD), (D) S1/S2 cleavage site, and (E) heptad repeat 1–2 site (HR1‐HR2).

Data information: Data are mean ± SD of three independent experiments.

Figure 6. Delta SARS‐CoV‐2 S protein induces more syncytia formation and binds more to ACE2 than D614G

1. Vero GFP‐split cells were transfected with variant S proteins and imaged 18 h post‐transfection. Left Panel: Fusion was quantified by GFP area/number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Vero GFP‐split cells 18 h post‐transfection, GFP (green), and Hoechst (blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm.

2. Left Panel: Quantification of Delta S protein‐mediated fusion in Vero GFP‐split cells by video microscopy. Results are mean ± SD from three fields per condition from one representative experiment. Right Panel: Fusion quantification of three independent video microscopy experiments, 20 h post‐transfection, normalized to D614G.

3. 293T cells were transfected S proteins with each variant‐associated mutation for 24 h and stained with biotinylated ACE2 and fluorescent streptavidin before analysis by flow cytometry. Left Panel: Representative ACE2 binding dilution curves for the Delta variant in relation to Alpha and D614G. Right Panel: EC50 values (concentration of ACE2 needed for 50% binding) for Alpha for the Delta variant.

4. Caco2 GFP‐split cells were transfected with variant S proteins and imaged 18 h post‐transfection. Left Panel: Fusion was quantified by GFP area/ number of nuclei and normalized to D614G for each of the transfected variant S proteins. Right Panel: Representative images of Caco2 GFP‐split cells 18 h post‐transfection, GFP (green), and Hoechst (blue). Top and bottom are the same images with and without Hoechst channel. Scale bars: 200 µm.

Data information: Data are mean ± SD of at least three independent experiments. Statistical analysis: one‐way ANOVA compared with D614G reference, *P < 0. 05, **P < 0.01, ***P < 0.001, ****P < 0.0001.