Delta spike P681R mutation enhances SARS-CoV-2 fitness over Alpha variant

Abstract

SARS-CoV-2 Delta variant has rapidly replaced the Alpha variant around the world. The mechanism that drives this global replacement has not been defined. Here we report that Delta spike mutation P681R plays a key role in the Alpha-to-Delta variant replacement. In a replication competition assay, Delta SARS-CoV-2 efficiently outcompeted the Alpha variant in human lung epithelial cells and primary human airway tissues. Delta SARS-CoV-2 bearing the Alpha-spike glycoprotein replicated less efficiently than the wild-type Delta variant, suggesting the importance of Delta spike in enhancing viral replication. The Delta spike has accumulated mutation P681R located at a furin cleavage site that separates the spike 1 (S1) and S2 subunits. Reverting the P681R mutation to wild-type P681 significantly reduced the replication of Delta variant, to a level lower than the Alpha variant. Mechanistically, the Delta P681R mutation enhanced the cleavage of the full-length spike to S1 and S2, leading to increased infection via cell surface entry. In contrast, the Alpha spike also has a mutation at the same amino acid (P681H), but the spike cleavage from purified Alpha virions was reduced compared to the Delta spike. Collectively, our results indicate P681R as a key mutation in enhancing Delta variant replication via increased S1/S2 cleavage. Spike mutations that potentially affect furin cleavage efficiency must be closely monitored for future variant surveillance.

Extended Data Figure 1.. Construction of infectious cDNA clones for Alpha and Delta variants.

a, Construction of infectious cDNA clones for Alpha and Delta variants. A three-fragment in vitro ligation was performed to construct the full-length cDNA clones of Alpha and Delta SARS-CoV-2. The construction method was detailed previously.^, ORFs, the Open reading frames; L, leader sequence; S, spike gene; E, envelope glycoprotein gene; M, membrane glycoprotein gene; N, nucleocapsid gene; UTR, untranslated region. b,c, Mutations from Alpha and Delta variants. The whole genome sequences of Alpha (EPI_ISL_999340) (b) and Delta (EPI_ISL_2100646) (c) were compared to USA/WA1–2020 strain. Nucleotide and amino acid mutations are presented.

Extended Data Figure 2.. Construction of Delta-P681 SARS-CoV-2 and plaque morphologies of different recombinant SARS-CoV-2s.

a, Construction of revertant Delta-P681 SARS-CoV-2. Single nucleotide G-to-C substitution was engineered into the Delta variant to construct Delta-P681 SARS-CoV-2. The nucleotide positions of viral genome are annotated. b, Plaque morphologies of Alpha, Alpha-spike/Delta-backbone, Delta, Delta-P681, and USA-WA1/2020 viruses. The plaque images were taken on day 2.5 post infection of Vero E6 cells.

Extended Data Figure 3.. Viral replication competition between Delta and Alpha variants on Calu-3 cells.

Recombinant Delta and Alpha SARS-CoV-2s were mixed in equal PFUs to infect Calu-3 cells at a total MOI of 0.1. At 2 h post infection, the cells were washed thrice with DPBS to remove free viruses. Culture medium were sampled for Sanger sequencing at 24 h, 36 h, and 48 h post infection. Red dots represent individual cell cultures (n=6); horizontal lines in each catseye represent the mean; shaded regions represent standard error of the mean; y-axes use a log₁₀ scale. Black numbers above each set of values (catseye) indicate the ratios of two viral RNA species. P values are calculated for group coefficient using linear regression model. **p<0.01, ***p<0.001.

Extended Data Figure 4.. Validation of viral competition results by next generation sequencing.

a-c, RNA samples from competition assays between Delta and Alpha (a), Delta and Alpha-spike/Delta-backbone (b), Alpha-spike/Delta-backbone and Alpha (c) were initially assessed using Sanger sequencing (Fig. 1b–d). The same RNA samples were retested here using next generation sequencing (NGS). Red dots represent individual cell cultures (n=6); the horizontal lines in each catseye represent the mean; shaded regions represent standard error of the mean; y-axes use a log₁₀ scale. Black numbers above each set of values (catseye) indicate the relative fitness estimates. P values are calculated for group coefficient using linear regression model. ***p<0.001.

Extended Data Figure 5.. Binding affinities of Alpha and Delta RBDs to human ACE2 receptor.

a-b, Alpha RBD (a) and Delta RBD (b) proteins were captured onto protein A biosensors. The biosensors were then dipped into serially diluted human ACE2 protein and buffers to measure the association and dissociation kinetics. The binding affinity-related parameters, including association (K_on), dissociation (K_off), and affinity (K_D) are shown. The affinity of ACE2 to Alpha RBD (N501Y) is below the detection limit and is presented as <1.0×10⁻¹². The result for Alpha RBD and ACE2 binding was adopted from our previous study for comparison.

Figure 1.. Delta P681R mutation enhances SARS-CoV-2 fitness over Alpha variant through improving spike protein processing.

a, Schemes of Alpha variant, Delta variant, Delta variant bearing Alpha-spike, and Delta variant with a P681R-to-P681 reversion. The spike gene of Delta variant was swapped with Alpha variant, resulting in chimeric SARS-CoV-2 “Alpha-spike/Delta-backbone.” The Delta P681R mutation was reverted wild-type P681, resulting in “Delta-P681” virus. Blue and red colors indicate Alpha and Delta variants, respectively. b-f, Viral replication competitions among Alpha, Delta, Alpha-spike/Delta-backbone, and Delta-P681 viruses on primary human airway epithelial (HAE) cells. Equal PFU of two viruses were mixed and inoculated onto HAE cells at an MOI of 5. Five pairs of viral competition are presented: Delta and Alpha (b), Delta and Alpha-spike/Delta-backbone (c), Alpha-spike/Delta-backbone and Alpha (d), Delta variant and Delta-P618 (e), and Delta-P681 and Alpha (f). After 2 h incubation, the cells were washed thrice with DPBS and maintained for 5 days. The secreted viruses were collected daily in DPBS after incubation at 37°C for 30 min. Red dots represent individual cell cultures (n=6), the horizontal lines in each catseye represent the mean, shaded regions represent standard error of the mean; y-axes use a log₁₀ scale. Black numbers above each set of values (catseye) indicate the ratios of two viral RNA species. P values are calculated for group coefficient using linear regression model. **p<0.01, ***p<0.001. g, Spike cleavages of purified virions. USA/WA1–2020, Alpha, Delta, and Delta-P681 viruses were purified and analyzed by Western blot using polyclonal antibodies against spike and anti-nucleocapsid antibodies. Full-length spike (FL), cleaved S1/S2, and S2’ proteins were annotated. One representative image of two experiments is shown. h, Quantification of spike processing of different variants from g. Densitometry was performed to quantify the cleavage efficiency of FL spike to S1/S2 subunits using ImageLab 6.0.1. The ratios of S1/S2 over FL were calculated to indicate spike processing efficiencies. The average results of two experiments were presented as mean ± standard deviation.