In SARS-CoV-2 delta variants, Spike-P681R and D950N promote membrane fusion, Spike-P681R enhances spike cleavage, but neither substitution affects pathogenicity in hamsters

Abstract

Background: The SARS-CoV-2 delta (B.1.617.2 lineage) variant was first identified at the end of 2020 and possessed two unique amino acid substitutions in its spike protein: S-P681R, at the S1/S2 cleavage site, and S-D950N, in the HR1 of the S2 subunit. However, the roles of these substitutions in virus phenotypes have not been fully characterized.

Methods: We used reverse genetics to generate Wuhan-D614G viruses with these substitutions and delta viruses lacking these substitutions and explored how these changes affected their viral characteristics in vitro and in vivo.

Findings: S-P681R enhanced spike cleavage and membrane fusion, whereas S-D950N slightly promoted membrane fusion. Although S-681R reduced the virus replicative ability especially in VeroE6 cells, neither substitution affected virus replication in Calu-3 cells and hamsters. The pathogenicity of all recombinant viruses tested in hamsters was slightly but not significantly affected.

Interpretation: Our observations suggest that the S-P681R and S-D950N substitutions alone do not increase virus pathogenicity, despite of their enhancement of spike cleavage or fusogenicity.

Funding: A full list of funding bodies that contributed to this study can be found under Acknowledgments.

Keywords: COVID-19; Hamster; Reverse genetics; SARS-CoV-2.

Fig. 1

Characterization of amino acid substitutions at positions 681 and 950 in vitro. a and b, VeroE6/TMPRSS2 cells were infected with each virus at an MOI of 1. (Left) At 12 h post-infection, cells were lysed, and spike and TUBA (internal control) expression was analyzed by western blotting. (Right) Ratios of S2 to full-length S were determined based on the band intensity of the western blots (n = 6, mean ± s.e.m). c and d, spike-expressing BHK cells and human ACE2-expressing VeroE6 (c) or VeroE6/TMPRSS2 (d) cells were co-cultured and fusion activity was determined by using the dual-luciferase assay (n = 3, mean ± s.e.m.). a–d, Data were analyzed by using a one-way ANOVA with Tukey's multiple comparisons test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig. 2

Growth kinetics of mutant viruses. VereE6 (a and b), VeroE6/TMPRSS2 (c and d), or Calu-3 cells (e and f) were infected with each virus at an MOI of 0.001. Virus titers at the indicated timepoints were determined by using plaque assays (n = 3, mean ± s.e.m.). Data were analyzed by using a two-way ANOVA with Dunnet's multiple comparisons test. Statistical significance was calculated against the values in Wuhan-D614G- or delta-infected cells. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. The lower limit of detection is indicated by the horizontal dashed line.

Fig. 3

The effect of amino acid substitutions at 681 and/or 950 on viral in vivo characteristics. Syrian hamsters were intranasally inoculated with 10⁵ PFU (in 30 μL) of the indicated virus. a, Body weights of virus-infected and mock-infected hamsters (n = 5) were monitored daily for 10 days. Data are presented as the mean percentages of the starting weight (±s.e.m.). b, Pulmonary function analyses in infected hamsters. Penh and Rpef were measured by using whole-body plethysmography. (n = 5, mean ± s.e.m.). c, Virus titers in infected Syrian hamsters. Hamsters (n = 5) were euthanized at 3 and 7 days post-infection for virus titration. Virus titers in the nasal turbinates and lungs were determined by using plaque assays. Vertical bars show the mean ± s.e.m. Points indicate data from individual hamsters. The lower limit of detection is indicated by the horizontal dashed line.

Fig. 4

Pathological findings in the lungs of infected Syrian hamsters. Representative histopathological images of the lungs of hamsters infected with the indicated virus on days 3 and 7 post-infection. (Top) Hematoxylin/eosin (H&E) staining. (Middle) in situ hybridization for SARS-CoV-2 viral RNA. (Bottom) Immunohistochemistry (IHC) for SARS-CoV-2 nucleocapsid protein. Scale bars, 200 μm.