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

Abstract

We report that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta spike mutation P681R plays a key role in the Alpha-to-Delta variant replacement during the coronavirus disease 2019 (COVID-19) pandemic. Delta SARS-CoV-2 efficiently outcompetes the Alpha variant in human lung epithelial cells and primary human airway tissues. The Delta spike mutation P681R is located at a furin cleavage site that separates the spike 1 (S1) and S2 subunits. Reverting the P681R mutation to wild-type P681 significantly reduces the replication of the Delta variant to a level lower than the Alpha variant. Mechanistically, the Delta P681R mutation enhances the cleavage of the full-length spike to S1 and S2, which could improve cell-surface-mediated virus entry. In contrast, the Alpha spike also has a mutation at the same amino acid (P681H), but the cleavage of the Alpha spike is reduced compared with the Delta spike. Our results suggest P681R as a key mutation in enhancing Delta-variant replication via increased S1/S2 cleavage.

Keywords: CP: Microbiology; Delta variant; S1/S2 cleavage; SARS-CoV-2; fitness; human airway culture; spike P681R; viral entry.

Graphical abstract

Figure 1

Delta variant has an improved fitness over Alpha variant on the Calu-3 and primary human airway epithelial cultures (A) Plaque morphologies of USA-WA1/2020, Alpha, and Delta viruses. The plaque images were taken on day 2.5 post infection of Vero E6 cells. The average diameters of the plaques are presented in the parentheses. (B) Viral kinetics on Vero E6 cells. Recombinant Alpha and Delta variants were inoculated onto Vero E6 cells at an MOI of 0.01. After 1 h infection, the cells were washed thrice with DPBS to remove unattached viruses. The viral titers in the culture supernatant were detected at 12, 24, 36, and 48 h post infection. Red dots represent individual cell cultures (n = 6) pooled from 2 independent biological repeats. Data are mean ± SEM. Statistical analysis was performed using nonparametric two-tailed Mann-Whitney test. (C) Replication competition between Delta and Alpha variants on Calu-3 cells. Culture medium was sampled for Sanger sequencing at 24, 36, and 48 h post infection. (D) Replication competitions between Alpha and Delta variants on primary human airway epithelial (HAE) cells. Secreted progeny viruses were collected by incubating the apical side of the HAE culture with 300 μL DPBS at 37°C for 30 min from day 1 to 5. For both (C) and (D), equal PFUs of Alpha and Delta SARS-CoV-2s were mixed and inoculated onto Calu-3 and HAE cells at an MOI of 5. At 2 h post infection, the cells were washed thrice with DPBS to remove unattached viruses. Red dots represent individual cell cultures (n = 6) pooled from 2 independent biological repeats. 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 were calculated for group coefficient using linear regression model. ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 2

The spike glycoprotein is responsible for the improved replication fitness of Delta variant in primary human epithelial airway cultures (A) Schemes of Alpha variant, Delta variant, and Delta variant bearing Alpha spike. The spike gene of Delta variant was swapped with Alpha variant, resulting in a chimeric SARS-CoV-2 of “Alpha-spike/Delta-backbone.” (B) Plaques of Alpha-spike/Delta-backbone chimeric virus. The plaque images were taken on day 2.5 post infection of Vero E6 cells. The average diameter of the plaques are presented in the parentheses. (C and D) Replication competitions between Delta and Alpha-spike/Delta-backbone (C) and Alpha-spike/Delta-backbone and Alpha (D) on primary HAE cells. Equal PFU amounts of two viruses were mixed and inoculated onto HAE cells at an MOI of 5. After 2 h incubation, the cells were washed thrice with DPBS and maintained for 5 days. Secreted progeny viruses were collected daily by incubating the apical side of the HAE culture with 300 μL DPBS at 37°C for 30 min. Red dots represent individual cell cultures (n = 6) pooled from 2 independent biological repeats; 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 were calculated for group coefficient using linear regression model. ^∗∗∗p < 0.001.

Figure 3

Delta spike P681 reversion impairs viral fitness (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. (B) Plaques of Delta-P681 virus. The plaque images were taken on day 2.5 post infection of Vero E6 cells. The average diameter of the plaques are presented in the parentheses. For direct comparison of plaque sizes of different viruses, all plaques presented in different figures of this paper were performed from the same set of experiment. (C and D) Replication competitions between Delta and Delta-P681 (C) and Delta-P681 and Alpha (D) on HAE cultures. Equal PFU inputs of two viruses were mixed and inoculated onto HAE cultures at an MOI of 5. After 2 h incubation, the cells were washed thrice with DPBS and maintained for 5 days. Secreted progeny viruses were collected daily by incubating the apical side of the HAE culture with 300 μL DPBS at 37°C for 30 min. Red dots represent individual cell cultures (n = 6) pooled from 2 independent biological repeats; 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 were calculated for group coefficient using linear regression model. ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 4

The Delta spike P681R mutation improves spike protein processing (A) 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 (FL) spike, cleaved S1/S2, and S2′ proteins were annotated. (B) Intracellular spike cleavages. C-terminally HA-tagged USA/WA1-2020 wild-type, P681H, and P681R mutant spikes were expressed in 293T cells. Total cell lysates were analyzed by western blot using anti-HA antibody. For both (A) and (B), one representative image of three independent experiments is shown. The densitometry was quantified by ImageLab 6.0.1 (BioRad). The ratios of S1/S2 or S2 subunits over FL spike are indicated at the top of the western blots.