In a recent study posted to the bioRxiv* preprint server, researchers discovered that the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is susceptible to cleavage at the disulfide bonds. They also found that the vulnerability to reductive cleavage varies across variants, with the Omicron variant family being highly susceptible to reduction.

Background

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The SARS-CoV-2 Omicron variant family has become the globally dominant variant currently in circulation, although it mostly causes only mild symptoms and has lower mortality rates than previous variants. The Omicron subvariants carry various mutations that enable immune evasion and increase their transmissibility. Recent research has focused on studying the mutations in the spike protein region, especially the receptor binding motif (RBM) in the receptor binding domain (RBD), which alter its ability to bind to the angiotensin-converting enzyme2 (ACE2) receptor, possibly altering its transmissibility. Mutations in the spike protein region also reduce the efficacy of vaccine-induced and therapeutic monoclonal antibodies.

Studies have identified the ectodomain of the SARS-CoV-2 spike protein to contain 30 cysteine residues, which form paired disulfide bonds. These cysteine residues could potentially be conserved across variants since no mutations have been found in this region thus far. Previous studies with the human immunodeficiency virus have shown that virus-host interactions could change through alterations in the disulfide bonds. Determining the vulnerability of the SARS-CoV-2 spike protein of emergent variants to reductive cleavage at the disulfide bonds could present potential therapeutic avenues to treat SARS-CoV-2 infections.

About the study

In the present study, the researchers used a tri-part Nanoluciferase (tNLuc) assay to measure the change in the spike protein-ACE2 binding after the reductive cleavage of the spike protein disulfide bonds. Compared to traditionally used methods for binding affinity measurement, such as surface plasmon resonance, the tNLuc assay is cost-effective and does not require complicated equipment.

The tNLuc assay contains three components — the β10 tag, which is attached to the RBD protein or the spike ectodomain protein, the β9 tag, which is attached to the ACE2 protein, and the Δ11S which completes the functional luciferase when added to β9-β10 that are in proximity to each other during spike-ACE2 binding. The luminescence signal indicates functional spike-ACE2 binding, which would be lower or absent in the case of disulfide bond reduction.

The two reducing agents that were tested are dithiothreitol (DTT) and tris-(2-carboxyethyl)phosphine (TCEP). The ability of the reducing agents to cleave the disulfide bonds in the spike proteins of several SARS-CoV-2 variants was tested by incubating the β10-tagged spike proteins from the wild-type (WT), Alpha, Beta, Delta, and Gamma strains, as well as those from the Omicron subvariants BA.1, BA.2 and BA.4/BA.5, in varying concentrations of the reducing agents. This was followed by incubation with the β9-tagged ACE2 and Δ11S to measure luminescence.

The mutations in the RBM of the Omicron variants were examined to determine those that make the Omicron subvariants more vulnerable to disulfide reduction than earlier strains. Loss- and gain-of-function approaches were used to determine the roles of the identified mutations. Furthermore, the specific disulfide bonds in the Omicron RBD that undergo reductive cleavage were also identified using chemical-labeled mass spectrometry.

Results

The results revealed that the Omicron sub-variants BA.1, BA.2, and BA.4/BA.5 were more susceptible to reductive cleavage of disulfide bonds than other SARS-CoV-2 variants. Furthermore, mutations in the Omicron spike protein RBM facilitated the cleavage of the disulfide bonds on the cysteine residues between positions 480 and 488, and 379 and 425. This cleavage subsequently impaired the spike-protein-ACE2 binding and reduced the stability of the spike protein.

Sub-millimolar levels of both DTT and TCEP were shown to inhibit WT, Alpha, and Gamma strains of SARS-CoV-2, with the half-maximal inhibitory concentration (IC₅₀) values for the Alpha and Gamma strains being 0.5 and 0.56 log units lower than that for the WT strain, respectively. The Omicron subvariant BA.1 exhibited the lowest IC₅₀ values compared to the WT strain, with the IC₅₀ for TCEP and DTT being 0.8 and 0.68 log units lower than that of the WT strain, respectively. The BA.2 and the BA.4/BA.5 subvariants also exhibited a significant decrease in IC₅₀ values for TCEP and DTT.

Based on the evaluation of the Omicron mutations and their role in increasing the susceptibility of the Omicron spike proteins to reductive cleavage, the authors believe that the T478K and the E484A mutations in the Omicron spike protein RBD could be making the disulfide bonds on the C480 to C488 residues more vulnerable to cleavage.

Conclusions

Overall, the results suggested that mutations such as T478K, S477N, and E484A, which are present in most of the Omicron subvariants, might increase their susceptibility to reductive cleavage by redox agents such as TCEP and DTT. While mutations, in general, have increased the immune evasive abilities and transmissibility of Omicron subvariants, this mutation-enhanced vulnerability to disulfide cleavage presents potential target areas for treating SARS-CoV-2 Omicron infections.

*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.

Written by

Dr. Chinta Sidharthan

Chinta Sidharthan is a writer based in Bangalore, India. Her academic background is in evolutionary biology and genetics, and she has extensive experience in scientific research, teaching, science writing, and herpetology. Chinta holds a Ph.D. in evolutionary biology from the Indian Institute of Science and is passionate about science education, writing, animals, wildlife, and conservation. For her doctoral research, she explored the origins and diversification of blindsnakes in India, as a part of which she did extensive fieldwork in the jungles of southern India. She has received the Canadian Governor General’s bronze medal and Bangalore University gold medal for academic excellence and published her research in high-impact journals.