P681R spike mutation enhances replication efficiency of SARS-CoV-2 delta variant

A team of scientists from the University of Texas, USA, has recently revealed that the P681R spike mutation is responsible for increased infectivity of the delta variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The P681R spike mutation increases the replication of the delta variant by increasing the dissociation of S1 and S2 subunits at the furin cleavage site. The study is currently available on the bioRxiv* preprint server.

Among the recently emerged variants of SARS-CoV-2, alpha, beta, gamma, and delta have been designated as the Variants of Concern (VOCs) because of their significantly increased transmissibility, infectivity, and virulence. In the recent coronavirus disease 2019 (COVID-19) pandemic phase, the delta variant is predominantly circulating globally. Soon after its first identification in India in October 2020, the delta variant has rapidly spread to more than 119 countries and gradually replaced the previously dominant alpha variant.  

The study

In the current study, the scientists have identified a potential spike mutation responsible for the improved fitness of delta variants. Specifically, they have employed a reverse genetic approach to identify specific spike mutations responsible for the rapid global replacement of the alpha by the delta variant.

Replication fitness of SARS-CoV-2 variants

The scientists prepared recombinant alpha and delta variants from infectious cDNA clones. They used a mixture of these recombinant viral variants to competitively infect human lung adenocarcinoma cells and primary human airway epithelial cells. By estimating the RNA ratio of delta versus alpha at different time points after infection, they observed that the delta variant has higher replication fitness than the alpha variant.

Furthermore, the researchers investigated the impact of spike gene mutations on replication fitness. For this purpose, they prepared a chimeric delta variant that contains the alpha variant's spike protein and the delta variant's backbone. By comparing the replication potency of the chimeric variant with the original delta variant, they observed that the presence of alpha-spike significantly decreases the replication efficiency of the delta variant. Another interesting observation was that the replication efficiency of the chimeric variant is even lower than the alpha variant, which highlights the significance of non-spike mutations of the delta variant in reducing overall viral replication fitness.

Mutational analysis of SARS-CoV-2 variants

According to whole genome sequencing findings, the delta variant contains multiple spike mutations, including T19R, G142D, E156G, F157-R158 deletion, L452R, T478K, D614G, P681R, and D950N. Of these mutations, P681R is present at the furin cleavage site.

In contrast to other beta-coronaviruses of the B lineage, SARS-CoV-2 gains functional advantages by acquiring the furin cleavage site. At this site, the S1 receptor-binding subunit of the spike protein is cleaved and dissociate from the S2 fusion subunit, leading to the enhanced entry of the virus into host cells.

Given the significance of the furin cleavage site in viral entry, the scientists hypothesized that the P681R mutation might enhance the infectivity of the delta variant by increasing S1/S2 cleavage at the furin site. For validation, they generated a modified version of the delta variant carrying wildtype P681 instead of mutated R681.

Importantly, by estimating the RNA ratio of tested variants in infected cells, they observed that the delta version carrying wildtype P681 has significantly lower replication efficiency than the original delta variant with P681R mutation. These observations highlight the significance of P681R mutation in improving the replication fitness of the delta variant.  

Functional impact of P681R mutation

The scientists determined the functional impact of P681R mutation by measuring the rate of spike cleavage in wildtype SARS-CoV-2 and variants alpha, delta, and delta-P681. The findings revealed that the delta variant has the highest spike cleavage efficiency, followed by alpha, delta-P681, and wildtype SARS-CoV-2. As mentioned by the scientists, a relatively higher spike cleavage efficiency of alpha variant compared to that of wildtype SARS-CoV-2 could be because of the presence of P681H spike mutation.

Although the study findings indicate that the delta variant gains replication fitness by efficiently possessing the spike protein at the furin cleavage site, there remains a possibility that the enhanced replication might be due to improved spike – ACE2 (angiotensin-converting enzyme 2) interaction.

To exclude this possibility, the scientists performed a binding assay using recombinant viral spike and human ACE2. They observed that the alpha spike has a significantly higher affinity for ACE2 than the delta spike. These observations indicate that the replication fitness of delta variants is not associated with improved spike – ACE2 interaction.

Study significance

The study identifies P681R spike mutation as the main driver mutation for enhanced replication efficiency of delta variant. Moreover, the study indicates that newly emerging viral variants with mutations at the furin cleavage site might gain functional benefits by efficiently cleaving full-length spike protein to S1 and S2 subunits. Thus, monitoring spike mutations affecting furin cleavage efficiency is paramount for effective variant surveillance as the pandemic continues.

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

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