The new CH.1.1 with the P681R mutation has already bee designated in the last 24 hours.
The P681R mutation is believed to enhance cell fusogenicity and cause rapid formation of syncytia.
The effect of the P681R mutation on viral fusion
The P681R mutation in the S protein is a unique feature of the B.1.617 lineage, including the B.1.617.2/Delta variant (Fig. 1d). As the P681R mutation is located in proximity to the furin cleavage site (FCS; residues RRAR positioned at 682–685) of the SARS-CoV-2 S protein16, we hypothesized that the P681R mutation is responsible for the promotion of cell–cell fusion, leading to the formation of larger syncytia.
To address this possibility, we generated a P681R-bearing artificial virus by reverse genetics (Extended Data Fig. 2b) and performed further virological experiments. Although the amounts of viral RNA in the culture supernatants of the D614G/P681R-infected Vero and VeroE6/TMPRSS2 cells were significantly lower compared with those of the D614G-infected cells at some timepoints, the growth of these two viruses was relatively comparable (Fig. 3a).
However, the floating syncytia (Fig. 3b) and plaques (Extended Data Fig. 2c) in the D614G/P681R-infected VeroE6/TMPRSS2 cells at 72 h.p.i. were significantly larger in size compared with the syncytia in the D614G-mutant-infected cells.
Moreover, immunofluorescence assays showed that D614G/P681R-infected VeroE6/TMPRSS2 cells exhibited larger multinuclear cells than D614G-infected cells (Extended Data Fig. 3b). These observations correspond well to the observations in the culture infected with the B.1.617.2/Delta variant (Fig. 2b and Extended Data Figs. 2a, 3a).
To clearly observe syncytium formation, we further generated GFP-expressing replication-competent D614G and D614G/P681R viruses. The levels of viral RNA in the supernatant and proportion of GFP-positive cells were similar in Vero, VeroE6/TMPRSS2 and Calu-3 cells (Extended Data Fig. 6).
However, at 24 h.p.i., significantly larger GFP-positive adherent syncytia were observed in VeroE6/TMPRSS2 cells infected with the GFP-expressing D614G/P681R virus (Fig. 3c). Moreover, the GFP-positive floating syncytia at 72 h.p.i. in VeroE6/TMPRSS2 cells infected with GFP-expressing D614G/P681R virus were significantly larger (2.4-fold) in size compared with those of VeroE6/TMPRSS2 cells infected with GFP-expressing D614G virus (Extended Data Fig. 7a).
Moreover, GFP-positive syncytia were observed in D614G/P681R-infected Calu-3 cells but not in D614G-infected Calu-3 cells at 72 h.p.i. (Extended Data Fig. 6c). These results suggest that the features of the B.1.617.2/Delta virus observed in in vitro cell culture experiments, particularly the formation of larger syncytia, are well reproduced by the insertion of the P681R mutation.
To further investigate the effect of the P681R mutation, the GFP-expressing viruses were inoculated into human primary nasal epithelial culture. Notably, the viral RNA levels of D614G/P681R virus on the apical side of culture at 2 and 3 d.p.i. were 12.3-fold and 7.0-fold higher, respectively, than those of parental D614G virus with statistical significance, and the rapid growth of D614G/P681R virus was supported by the observation of GFP expression (Extended Data Fig. 7b).
Although the viral RNA levels of D614G/P681R virus gradually decreased after 5 d.p.i., plaque-like spots were observed after 7 d.p.i., and the sizes of these plaque-like spots in the culture of D614G/P681R infection were significantly larger than the plaque-like spots in the culture of parental D614G virus infection (Extended Data Fig. 7b). These data suggest that the P681R mutation accelerates viral replication in human primary nasal epithelial culture and produces large plaque-like spots, which could be formed by cell-to-cell infection as the case of plaque formation.
To directly investigate the effect of the P681R mutation on the cleavage of the SARS-CoV-2 S protein, we prepared an HIV-1-based pseudovirus carrying the P681R mutation. Western blot analysis of the prepared pseudoviruses showed that the level of the cleaved S2 subunit was significantly increased in the presence of the P681R mutation (Extended Data Fig. 8a), suggesting that the P681R mutation facilitates furin-mediated cleavage of the SARS-CoV-2 S protein.
We next performed a single-round pseudovirus infection assay using target HOS-ACE2 cells with or without TMPRSS2 expression. The infectivity of both the D614G and D614G/P681R pseudoviruses was increased approximately tenfold by the expression of TMPRSS2 in the target cells (Extended Data Fig. 8b). However, the relative infectivity of the D614G and D614G/P681R pseudoviruses was not altered by TMPRSS2 expression (Extended Data Fig. 8b). These data suggest that the P681R mutation does not affect the infectivity of the viral particles.
We next addressed the effect of the P681R mutation on viral fusogenicity by a cell-based fusion assay. In the effector cells (that is, S-expressing cells), although the expression level of the D614G/P681R S protein was comparable to that of the D614G S protein, the level of the cleaved S2 subunit was significantly higher for the D614G/P681R mutant than for the D614G mutant (Fig. 3d).
Consistent with the results of the pseudovirus assay (Extended Data Fig. 8a), these results suggest that the P681R mutation facilitates S cleavage. Flow cytometry analysis showed that the surface expression level of D614G/P681R S was significantly lower than that of D614G S (Extended Data Fig. 8c). Nevertheless, the cell-based fusion assay using the target cells without TMPRSS2 demonstrated that D614G/P681R S is 2.1-fold more fusogenic than D614G S—a statistically significant difference (P = 0.0002, Welch’s t-test) (Fig. 3e).
Moreover, a mathematical modelling analysis of the fusion assay data showed that the initial fusion velocity of D614G/P681R S (0.83 ± 0.03 per hour) was significantly faster (2.8-fold) than that of D614G S (0.30 ± 0.03 per hour; P = 4.0 × 10−6, Welch’s t-test) (Extended Data Fig. 8d, e). These data suggest that the P681R mutation enhances and accelerates SARS-CoV-2 S-mediated fusion.
Furthermore, when we used targeted cells expressing TMPRSS2, both the fusion efficacy (about 1.2-fold) and initial fusion velocity (about 2.0-fold) were increased in both the D614G and D614G/P681R S proteins (Extended Data Fig. 8d, e). These results suggest that TMPRSS2 facilitates the fusion mediated by SARS-CoV-2 S and human ACE2 and that this TMPRSS2-dependent acceleration and promotion of viral fusion is not specific for the P681R mutant.
Neutralization of the P681R mutant
Resistance to neutralizing antibodies in the sera of COVID-19 convalescent individuals and vaccinated individuals is a hallmark of VOCs17,18, and it has recently been shown that the B.1.617.2/Delta variant is relatively resistant to vaccine-induced neutralization19,20. To determine whether the P681R mutation contributes to this virological phenotype, we performed a neutralization assay.
The D614G/P681R pseudovirus was partially resistant (1.2–1.5-fold) to three monoclonal antibodies targeting the receptor-binding domain of the SARS-CoV-2 S protein (Extended Data Fig. 9a). Furthermore, neutralization experiments using 19 serum samples collected after two rounds of BNT162b2 vaccination showed that the D614G/P681R pseudovirus was significantly more resistant than the D614G pseudovirus to vaccine-induced neutralizing antibodies (P < 0.0001, Wilcoxon matched-pairs signed-rank test) (Extended Data Fig. 9b, c). These results suggest that the P681R-bearing pseudovirus is relatively resistant to neutralizing antibodies.
Pathogenicity of the P681R mutant
To assess the effect of the P681R mutation on viral replication and the pathogenicity of SARS-CoV-2, we intranasally infected Syrian hamsters with the D614G and D614G/P681R viruses. The D614G-infected hamsters exhibited no weight loss, although a slight decrease in body weight by 7 d.p.i. was observed for one of the hamsters (5.0%) (Fig. 4a).
By contrast, all of the hamsters infected with the D614G/P681R virus experienced gradual body weight loss, and the hamsters showed a weight loss of 4.7–6.9% at 7 d.p.i., significantly greater compared with the weight loss of hamsters that were infected with the D614G virus (P = 0.011) (Fig. 4a).
The weight loss of D614G/P681R-infected hamsters was significantly greater compared with that of D614G-infected hamsters on average across all timepoints (P = 0.00015, multiple regression) (Fig. 4a). We also assessed pulmonary function in infected hamsters by using a whole-body plethysmography system to measure enhanced pause (PenH), which is a surrogate marker of bronchoconstriction or airway obstruction.
Infected hamsters of both groups showed increases in the lung PenH value, but the PenH values of D614G/P681R-infected hamsters were significantly higher than those of the D614G-infected hamsters on average across all timepoints (P = 0.038, multiple regression) (Fig. 4b). At 7 d.p.i., the D614G/P681R-infected hamsters had significantly higher PenH values than the D614G-infected hamsters (P = 0.043). At 3 d.p.i., both viruses replicated efficiently in the lungs and nasal turbinates of the infected hamsters, and no significant difference in viral replication was observed between the two groups (Fig. 4c). At 7 d.p.i., no differences in viral titres in the nasal turbinates were found between the two groups; however, the lung titres in the D614G/P681R-infected group were significantly higher than those in the D614G-infected group (P = 0.0013) (Fig. 4c).
Histopathological examination revealed cell infiltration in and around the bronchi/bronchioles at 3 d.p.i. in both groups, but solid bronchioloalveolar epithelial hyperplasia including type II pneumocytes was prominent at 7 d.p.i. in the D614G/P681R-infected hamsters (Fig. 4d).
Microcomputed tomography (microCT) analysis revealed lung abnormalities in all of the infected hamsters on 7 d.p.i. that were consistent with commonly reported imaging features of COVID-19 pneumonia21 (Extended Data Fig. 10a). Lung abnormalities included multifocal nodular ground glass opacity with a peripheral, bilateral, multilobar, peribronchial distribution with regions of lung consolidation.
The CT severity scores of the D614G-infected and D614G/P681R-infected hamsters ranged from 8 to 14, with an overall average CT severity score of 10.5 (median 9.5) (Extended Data Fig. 10b). The D614G/P681R-infected hamsters had a higher CT severity score (mean 11 (range 9–14, median 10.5)), compared with the D614G-infected hamsters (mean 10 (range 8–13, median 9.5)). Two of the D614G/P681R-infected hamsters developed a small-volume pneumomediastinum, probably secondary to severe pulmonary damage, micropulmonary rupture and gas tracking into the mediastinum.