Mutations in SARS-CoV-2 variants of concern link to increased spike cleavage and virus transmission

Abstract

SARS-CoV-2 lineages have diverged into highly prevalent variants termed "variants of concern" (VOCs). Here, we characterized emerging SARS-CoV-2 spike polymorphisms in vitro and in vivo to understand their impact on transmissibility and virus pathogenicity and fitness. We demonstrate that the substitution S:655Y, represented in the gamma and omicron VOCs, enhances viral replication and spike protein cleavage. The S:655Y substitution was transmitted more efficiently than its ancestor S:655H in the hamster infection model and was able to outcompete S:655H in the hamster model and in a human primary airway system. Finally, we analyzed a set of emerging SARS-CoV-2 variants to investigate how different sets of mutations may impact spike processing. All VOCs tested exhibited increased spike cleavage and fusogenic capacity. Taken together, our study demonstrates that the spike mutations present in VOCs that become epidemiologically prevalent in humans are linked to an increase in spike processing and virus transmission.

Keywords: H655Y mutation; SARS-CoV-2; fusion; gamma; omicron; spike cleavage; syncytia formation; variants of concern.

Graphical abstract

Figure 1

Characterization of spike protein processing of human SARS-CoV-2 isolates from New York (NY) (A) Time-calibrated phylogenetic analysis of the global distribution of H655Y substitution during the early SARS-CoV-2 outbreak. The phylogenetic tree was generated with Nextstrain with 7,059 genomes sampled from worldwide data deposited in the GISAID database from December 2019 to September 2020 for representation of the H655Y substitution over time. (B) Western blotting of spike protein cleavage of the 12 human SARS-CoV-2 viruses isolated from nasal swabs of COVID-19-infected patients and collected during the first pandemic wave in NY. Infections were performed in Vero E6 cells at an MOI of 0.01, and supernatants were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), and nucleocapsid (N; 50 kDa) were detected using specific antibodies. Levels of N protein were used as loading control. (C) Quantification of full-length and cleaved spike protein of the early human NY isolates in Vero E6 cells. Spike protein levels were normalized to nucleocapsid expression. (D) Western blotting of spike protein cleavage of the 12 human SARS-CoV-2 NY viruses in human pneumocyte-like cells. Cells were infected with 3000 pfu of the corresponding viral isolate per well, and cell extracts were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), nucleocapsid (N; 50 kDa), and β-actin (45 kDa) were detected using specific antibodies. Levels of N and β-actin protein were used as loading control. (E) Quantification of full-length and cleaved spike protein of the early human NY isolates in human pneumocyte-like cells. Spike protein levels were normalized to nucleocapsid expression.

Figure 2

The H655Y amino acid substitution enhances spike cleavage and viral growth (A) Spike polymorphisms present in the mink-adapted variants, early human SARS-CoV-2 New York (NY) isolates, WA1-655Y, and WA1 wild-type viruses. Wuhan1 is included as a reference. (B) Replication kinetics of early SARS-CoV-2 viruses in Vero E6 and Vero-TMPRSS2 cells. Infections were performed at an MOI of 0.01. Viral titers were determined by plaque assay at the indicated hour post-infection and expressed as PFU per milliliter. Shown are the means and SDs from 3 replicates. ANOVA test for multiple comparison was used to compare mean differences within different isolates and time points. Viral isolates were compared two by two using the Tukey’s correction. Statistical significance was considered when p ≤ 0.05 (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001; ns, not significant). Color codes relate to the isolates shown in (A). (C) Western blotting of S protein from supernatants of Vero E6- and Vero-TMPRSS2-infected cells. Infections were performed at an MOI of 0.01, and viral supernatants were collected at 48 h post-infection (p.i.). Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), and nucleocapsid (N; 50 kDa) were detected using specific antibodies. Levels of N protein were used as loading control. (D and E) Quantification of full-length and cleaved spike protein of the indicated viruses in Vero E6 and Vero-TMPRSS2 cells. Spike protein levels were normalized to nucleocapsid expression. (F) Replication kinetics of early SARS-CoV-2 viruses in human pneumocyte-like cells. Cells were infected with 3000 pfu of the corresponding viral isolate per well. Viral titers were determined by plaque assay at the indicated hour p.i. and expressed as PFU per milliliter. Shown are the means and SDs from 3 replicates. ANOVA test for multiple comparison was used to compare mean differences within different isolates and time points. Viral isolates compared two by two using the Tukey’s correction. Statistical significance was considered when p ≤ 0.05 (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001; ns, not significant). (G) Western blotting of S protein in human pneumocyte-like cells infected with 3000 pfu of the corresponding early SARS-CoV-2 virus per well. Cell extracts were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), nucleocapsid (N; 50 kDa), and β-actin (45 kDa) were detected using specific antibodies. Levels of N and β-actin protein were used as loading control. (H) Quantification of full-length and cleaved spike protein of the indicated viruses in human pneumocyte-like cells. Spike protein levels were normalized to nucleocapsid expression.

Figure 3

The 655Y spike polymorphism increases cell-cell fusion (A) Immunofluorescence of SARS-CoV-2 S and N protein localization in Vero-TMPRSS2-infected cells at an MOI of 0.01 and 24 h p.i. Spike protein was detected using a specific monoclonal antibody 3AD7 (green), N protein was detected using a polyclonal antiserum (red), and 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus. (B) Schematic representation of the split-GFP fusion assay. (C) Quantification of cell-cell fusion represented as GFP expression produced by each spike variant over WA1 wild-type spike. GFP signals were normalized to spike expression and DAPI counts. Shown are the means and SDs of 3 independent experiments. (D) Images showing GFP-positive syncytia formation obtained by Celigo image cytometer. Cell nuclei were stained using DAPI.

Figure 4

The 655Y polymorphism prevails over the 655H in the transmission in vivo model (A) Ten 3-week-old female Syrian hamsters were placed in pairs. Only 1 hamster per cage was infected intranasally with a total of 10⁵ pfu of SARS-CoV-2 WA1 and WA1-655Y isolates in a one-to-one ratio. Nasal washes were collected at day 2, 4, and 6 post-infection (p.i.). Lungs and nasal turbinates were harvested from direct infected (DI) and direct contact (DC) hamsters at day 5 and 7 p.i., respectively. (B) Body weight change of individual hamsters over time. (C) Viral titers of nasal washes expressed as PFU per milliliter. Shown are the medians with 95% confidence intervals (CI). Mann-Whitney t test was performed to compare differences within each group. Statistical significance was considered when p ≤ 0.05 (ns, not significant). (D) Relative abundance of 655Y mutation in the RNA from nasal washes in the DI and DC hamsters. The y axis shows the percentage of 655Y polymorphism in the total good quality sequencing reads from each biological RNA sample, and the x axis indicates the day p.i. samples were collected. (E) Viral titers of lungs and nasal turbinates expressed as PFU per gram of tissue. Shown are the medians with 95% CI. Mann-Whitney t test was performed to compare differences within each group. Statistical significance was considered when p ≤ 0.05 (ns, not significant). Titers of DI and DC hamsters are shown at day 5 and 7 p.i., respectively. (F and G) Proportion of hamsters with 655Y (blue) and H (green) in the nasal turbinates and lungs from DI and DC as confirmed by next generation sequencing. (H) Competition experiments between SARS-CoV-2 WA1 and WA1-655Y isolates in human pneumocyte-like cells at different ratios. Proportion of 655Y (blue) and H (green) expressed as percentage are shown for the input and after 24 and 48 h p.i. Shown are the medians of 3 independent experiments.

Figure 5

Global epidemiology of SARS-CoV-2 variants of concern (VOCs) The amino acid substitution frequencies around the cleavage site region (655 to 701) from globally available data (2,072,987 sequences deposited in GISAID database as of 28 June 2021) were estimated. (A) The high prevalent mutations identified mapped onto the structure of the S glycoprotein. The model was generated by superposition of PDB: 6M0J and 7C2L (Chi et al., 2020; Lan et al., 2020). One RBD in the up conformation (red) is bound with ACE2 receptor (pink). The N-terminal domain (NTD) is colored blue, the amino-acid substitutions are shown as gold spheres, and the furin cleavage loop (disordered and therefore missing in most atomic models) is flanked with cyan spheres. One spike protomer is shown in bold colors while the other two are colored white. A zoomed-in image of the region of interest and the sequence of the furin site loop is also shown. Amino acid residues of interest are highlighted in gold. (B) Time-calibrated phylogenetic tree of SARS-CoV-2 circulating variants illustrating the temporal distribution and phylogenetic relationships of the most prevalent S mutations along the S1/S2 region (highlighted in color). The phylogenetic tree was generated using NextStrain, and analysis was performed using a sample of 13,847 genomes focused on the most prevalent substitutions between S:655 and S:701 between February 2020 and June 2021 from GISAID database. (C–E) Frequency per clade of H655Y, P681H/R, and A701V spike polymorphisms.

Figure 6

SARS-CoV-2 VOCs evolve to a convergent phenotype associated to an increase on S cleavage (A) Multiple alignments of the S protein of the indicated SARS-CoV-2 VOCs. Diagram shows the corresponding S amino acid substitutions mapped to the S gene. (B) Viral growth of SARS-CoV-2 variants in Vero E6 and Vero-TMPRSS2 cells. Infections were performed at an MOI of 0.01. Viral titers were determined by plaque assay at the indicated hour post-infection (p.i.) and expressed as PFU per milliliter Shown are the means and SDs from 3 replicates. ANOVA test for multiple comparison was used to compare mean differences within different isolates and time points. Viral isolates were compared two by two using the Tukey’s correction. Statistical significance was considered when p ≤ 0.05 (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001; ns, not significant). Color codes relate to the isolates shown in (A). (C) Western blotting of spike cleavage in supernatants from Vero E6- and Vero-TMPRSS2-infected cells at an MOI of 0.01. Viral supernatants were collected at 48 h p.i. Full-length (FL) S protein (180 kDa), S2 cleaved spike (95 kDa), and nucleocapsid (N; 50 kDa) were detected using specific antibodies. Levels of N protein were used as loading control. (D) Quantification of full-length and cleaved spike protein of the VOCs in Vero E6 and Vero-TMPRSS2 cells. Spike protein levels were normalized to nucleocapsid expression. Asterisk (^∗) indicates under limit of detection. (E) Replication kinetics of SARS-CoV-2 VOCs in human pneumocyte-like cells. Cells were infected with 3000 pfu of the corresponding viral isolate per well. Viral titers were determined by plaque assay at the indicated hour p.i. and expressed as PFU per milliliter. Shown are the means and SDs from 3 replicates. ANOVA test for multiple comparison was used to compare mean differences within different isolates and time points. Viral isolates were compared two by two using the Tukey’s correction. Statistical significance was considered when p ≤ 0.05 (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001; ns, not significant). (F) Western blotting of S protein in human pneumocyte-like cells infected with 3000 pfu of the corresponding VOC per well. Cell extracts were collected at 48 h p.i. Full-length (FL) spike protein (180 kDa), S2 cleaved spike (95 kDa), nucleocapsid (N; 50 kDa), and β-actin (45 kDa) were detected using specific antibodies. Levels of N and β-actin protein were used as loading control. (G) Quantification of full-length and cleaved spike protein of the VOCs in human pneumocyte-like cells. Spike protein levels were normalized to nucleocapsid expression. (H) Quantification of the cleavage efficiency by mass spectrometry. Vero-TMPRSS2 cells were infected at an MOI of 0.1 with the indicated VOCs and NY7 (S:H655Y) and WA1-655Y isolates. WA1 and NY6 were used as controls. Cells extracts were collected after 24 h p.i. Cleavage efficiency was determined by measuring the abundance of the resulting peptide (SVASQSIIAYTMSLGAE) after cleavage at the terminal arginine of the furin cleavage site. Total spike, ORF3a, and N protein were used as internal standard to normalize across variants. The y axis shows the log₂ of fold change between cleaved peptide abundance for each variant normalized by WA1 control.

Figure 7

SARS-CoV-2 VOCs exhibit enhanced cell-cell fusion (A and B) Immunofluorescence of SARS-CoV-2 S and N protein in Vero-TMPRSS2-infected cells at an MOI of 0.01 and 24 h p.i. for the indicated VOCs. Spike protein was detected using a specific monoclonal antibody 3AD7 (green), N protein was detected using a polyclonal antiserum (red), and 4′,6-diamidino-2-phenylindole (DAPI) was used to stain the nucleus. (C) Quantification of cell-cell fusion represented as GFP expression showed by each spike VOC over WA1 wild-type spike. GFP signals were normalized to spike expression and DAPI counts. Shown are the means and SD of 3 independent experiments. Two-tailed t test was performed to compare mean differences between each VOC S and corresponding reverse mutant. Statistical significance was considered when p ≤ 0.05 (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001; ns, not significant). (D) Images showing GFP-positive syncytia formation obtained by Celigo image cytometer. Cell nuclei was stained using DAPI.