New Study Warns That SARS-CoV-2 Variants Are Strategically Evolving Towards Better Furin Cleavage Efficiency. Attention To The S:655Y Mutation Warranted

SARS-CoV-2 Variants: A new study led by researchers from Icahn School of Medicine at Mount Sinai-New York along with scientists from the University of California-San Francisco and Kansas State University warns that the SARS-CoV-2 variants are strategically evolving towards better Furin cleavage efficiency.

 
Typically for efficient cell entry and membrane fusion, SARS-CoV-2 spike (S) protein needs to be cleaved at two different sites, S1/S2 and S2’ by different cellular proteases such as furin and TMPRSS2.
 
Polymorphisms in the S protein can affect cleavage, viral transmission, and pathogenesis.
 
The study team investigated the role of arising S polymorphisms in vitro and in vivo to understand the emergence of SARS-CoV-2 variants. First, they showed that the S:655Y is selected after in vivo replication in the mink model. This mutation is present in the Gamma Variant Of Concern (VOC) but it also occurred sporadically in early SARS-CoV-2 human isolates.
 
In order to better understand the impact of this polymorphism, the study team analyzed the in vitro properties of a panel of SARS-CoV-2 isolates containing S:655Y in different lineage backgrounds.
 
Study findings demonstrated that this mutation enhances viral replication and spike protein cleavage. Viral competition experiments using hamsters infected with WA1 and WA1-655Y isolates showed that the variant with 655Y became dominant in both direct infected and direct contact animals.
 
The study team also investigated the cleavage efficiency and fusogenic properties of the spike protein of selected VOCs containing different mutations in their spike proteins.
 
Study findings showed that all VOCs have evolved to acquire an increased spike cleavage and fusogenic capacity despite having different sets of mutations in the S protein.
 
The study demonstrates that the S:655Y is an important adaptative mutation that increases viral cell entry, transmission, and host susceptibility. Moreover, SARS-COV-2 VOCs showed a convergent evolution that promotes the S protein processing.
 
The study findings were published on a preprint server and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2021.08.05.455290v1
 
The SARS-CoV-2 is a novel coronavirus that has caused infections and deaths in millions of people worldwide since its emergence in Wuhan, China, in late December 2019.
 
The novel coronavirus affects not only humans but also cats, dogs, ferrets, hamsters, and non-human primates. The virus was even found to affect minks, and cases of mink-to-human cross-species transmission have been reported.
 
Co-corresponding author, Dr Teresa Aydillo from the Department of Microbiology, Icahn School of Medicine at Mount Sinai told Thailand Medical News, "The spike (S) glycoprotein of SARS-CoV-2 is the main determinant of host tropism and susceptibility, and the main target of antibody response s.”
 
Hence the emergence of adaptive mutations in the spike protein has a strong effect on host tropism and viral transmission.
 
The S or spike protein comprises of two subunits: S1 subunit having the receptor-binding domain (RBD) that helps it to bind to the angiotensin-converting enzyme 2 (ACE2), which is present on the cell surface of the host, and the S2 subunit that helps in the fusion of the cellular membrane and the viral membrane.
 
Dr Aydillo added, "To fuse with the host cell, the S protein needs to be cleaved by cellular proteases at the S1/S2 and S2' sites.”
 
Importantly the S1/S2 site consists of a multibasic furin motif that can undergo processing by the furin proteases or by transmembrane serine proteases.
 
To date, several SARS-CoV-2 lineages termed as Variants of Concern (VOC) have emerged that have increased the virus's transmissibility. These variants have arisen mainly due to spike protein mutations.
 
The study team characterized the spike polymorphisms of SARS-CoV-2 both in vitro and in vivo in order to understand viral pathogenicity, transmissibility, and fitness.
 
It should be noted that the SARS-CoV-2 variants have arisen due to several mutations in the spike protein. The first mutant that became dominant in March 2020 was S:655Y, following which many more polymorphisms arose in late 2020.
 
Dr Aydillo further added, "The N501Y substitution convergently evolved in early emerging VOCs Alpha (B.1.1.7), Beta (B.1.351) and Gamma (P.1) variants and has been associated with an enhanced spike affinity for the cellular ACE2 receptor. These mutations were located in the receptor-binding motif (RBM) of the RBD and led to the decrease in neutralizing antibody responses that were elicited by the virus. Similarly, the later SARS-CoV-2 Kappa (B.1.617.1) and Delta (B.1.617.2) variants have also shown a significantly reduced sensitivity to convalescent and immune sera.”
 
The research was carried out using the African green monkey cell line, human cell line, hamsters, and minks. The viral samples were collected from human nasopharyngeal swabs collected in March 2020 and February 2021. Variants of SARS-CoV-2 were obtained from different labs.
 
Subsequently, the cell cultures, as well as hamsters and minks, were infected, and the viral titer was also calculated using the plaque assay. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to quantify the level of SARS-CoV-2 RNA present after infection.
 
To determine which mutations are mostly present at the furin cleavage site of SARS-CoV-2, mass spectrometry was utilized.
 
Multiple sequence alignment was also done with the mink and human variants to identify and compare the spike mutations in them. In the case of hamsters, the frequency of variants was determined using the Oxford Nanopore Sequencing method.
 
The study findings showed that the H655Y variant had a growth advantage in both human and monkey cell lines. Therefore, it can be confirmed that the S:655Y mutation alone led to enhanced growth and higher replication.
It was also observed in the hamster model as well that the efficiency of 655Y was much more than its ancestors.
 
Pertaining to the furin cleavage site, higher amounts of cleavage at this site were found in the case of alpha, delta, and kappa variants. Although the beta variant did not contain a change in the furin cleavage site, instead it contained a change in residue 701.
 
The study team concluded that the 655Y spike polymorphism was a significant cause of the SARS-CoV-2 transmission and infection.
 
Dr Aydillo commented, "The selection and increasing frequency of S:655Y in the human population and following SARS-CoV-2 infection of different animal models such as cats, mice, and minks suggests this mutation is associated with an improvement of viral fitness and adaptation to diverse hosts through an increased cleavage of the spike protein.”
 
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