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A team of scientists from the University of Texas, USA, recently revealed that the peak mutation P681R is responsible for increased infectivity of the delta variant of the coronavirus 2 severe acute respiratory syndrome (SARS-CoV-2 ). The P681R spike mutation increases 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 variants have been designated as variants of concern (VOC) due to their significantly increased transmissibility, infectivity and virulence. In the recent pandemic phase of coronavirus disease 2019 (COVID-19), the delta variant circulates primarily around the world. Shortly after its first identification in India in October 2020, the delta variant quickly spread to over 119 countries and gradually replaced the previously dominant alpha variant.
The study
In the present study, the scientists identified a potential peak mutation responsible for improving the fitness of the delta variants. Specifically, they used a reverse genetic approach to identify specific peak mutations responsible for the rapid overall replacement of the alpha variant by the delta variant.
Replication ability of SARS-CoV-2 variants
Scientists have 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 ratio of delta to alpha RNA at different times after infection, they observed that the delta variant has better replication capacity than the alpha variant.
In addition, the researchers studied the impact of advanced genetic mutations on replication capacity. To this end, they prepared a chimeric delta variant which contains the spike protein of the alpha variant and the delta variant backbone. By comparing the replication power of the chimeric variant with the original delta variant, they observed that the presence of an alpha peak considerably decreases the replication efficiency of the delta variant. Another interesting observation was that the replication efficiency of the chimeric variant is even lower than that of the alpha variant, which highlights the importance of the spikeless mutations of the delta variant in reducing viral replication capacity. overall.
Mutational analysis of SARS-CoV-2 variants
According to the results of whole genome sequencing, the delta variant contains several cutting edge mutations, including the T19R, G142D, E156G, F157-R158, L452R, T478K, D614G, P681R and D950N deletion. Among these mutations, P681R is present at the furin cleavage site.
Unlike other B-lineage beta-coronaviruses, SARS-CoV-2 gains functional benefits by acquiring the furin cleavage site. At this site, the S1 receptor binding subunit of the spike protein is cleaved and dissociated from the S2 fusion subunit, leading to increased entry of the virus into host cells.
Given the importance of the furin cleavage site in viral entry, scientists hypothesized that the P681R mutation could enhance the infectivity of the delta variant by increasing S1 / S2 cleavage at the site. furin. For validation, they generated a modified version of the delta variant carrying wild type P681 instead of mutated R681.
Importantly, by estimating the RNA ratio of the variants tested in infected cells, they observed that the delta version carrying wild type P681 has a significantly lower replication efficiency than the original delta variant with the P681R mutation. These observations highlight the importance of the P681R mutation in improving the replication capacity of the delta variant.
Functional impact of the P681R mutation
Scientists determined the functional impact of the P681R mutation by measuring the rate of tip cleavage in wild-type SARS-CoV-2 and the alpha, delta, and delta-P681 variants. The results revealed that the delta variant has the highest peak cleavage efficiency, followed by the wild-type alpha, delta-P681 and SARS-CoV-2. As mentioned by scientists, a relatively higher peak cleavage efficiency of the alpha variant compared to that of wild-type SARS-CoV-2 could be due to the presence of the P681H peak mutation.
Although the results of the study indicate that the delta variant gains in replicability by effectively possessing the spike protein at the furin cleavage site, there remains a possibility that the improved replication is due to an improvement in tip interaction – ACE2 (angiotensin 2 converting enzyme).
To rule out this possibility, the scientists performed a binding test using a recombinant viral tip and human ACE2. They observed that the alpha peak has a significantly higher affinity for ACE2 than the delta peak. These observations indicate that the replicability of the delta variants is not associated with an improvement in the tip – ACE2 interaction.
Importance of the study
The study identifies the peak P681R mutation as the main driver of mutation for improved replication efficiency of the delta variant. Additionally, the study indicates that newly emerging viral variants with mutations at the furin cleavage site could gain functional benefits by efficiently cleaving the full-length spike protein into S1 and S2 subunits. Thus, monitoring for cutting edge mutations affecting the efficiency of furin cleavage is paramount for effective monitoring of variants as the pandemic continues.
*Important Notice
bioRxiv publishes preliminary scientific reports which are not peer reviewed and, therefore, should not be considered conclusive, guide clinical practice / health-related behavior, or treated as established information.
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