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

Since the emergence of SARS-CoV-2, a number of dominant variants have sequentially emerged and spread around the world, including Alpha, Delta, and Omicron. It is important to identify viral mutations that contribute to such variant replacements. 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 outcompetes the Alpha variant in human lung epithelial cells and primary human airway tissues. Delta SARS-CoV-2 bearing the Alpha-spike glycoprotein replicates 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 reduces the replication of 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 Alpha spike is reduced compared to the Delta spike. Collectively, our results suggest P681R as a key mutation in enhancing Delta variant replication via increased S1/S2 cleavage. Thus, spike mutations that potentially affect furin cleavage efficiency must be closely monitored for future variant surveillance.