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 prone to cleavage on the disulfide bonds. Additionally they found that the vulnerability to reductive cleavage varies across variants, with the Omicron variant family being highly prone to reduction.

Study: Omicron Spike Protein Is Vulnerable to Reduction. Image Credit: Fit Ztudio/Shutterstock

Background

The SARS-CoV-2 Omicron variant family has turn out to be the globally dominant variant currently in circulation, even though 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 within the spike protein region, especially the receptor binding motif (RBM) within the receptor binding domain (RBD), which alter its ability to bind to the angiotensin-converting enzyme2 (ACE2) receptor, possibly altering its transmissibility. Mutations within 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 present in this region to this point. Previous studies with the human immunodeficiency virus have shown that virus-host interactions could change through alterations within the disulfide bonds. Determining the vulnerability of the SARS-CoV-2 spike protein of emergent variants to reductive cleavage on the disulfide bonds could present potential therapeutic avenues to treat SARS-CoV-2 infections.

In regards to the study

In the current study, the researchers used a tri-part Nanoluciferase (tNLuc) assay to measure the change within the spike protein-ACE2 binding after the reductive cleavage of the spike protein disulfide bonds. In comparison with traditionally used methods for binding affinity measurement, reminiscent of surface plasmon resonance, the tNLuc assay is cost-effective and doesn’t require complicated equipment.

The tNLuc assay accommodates 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 which can be in proximity to one another during spike-ACE2 binding. The luminescence signal indicates functional spike-ACE2 binding, which could be lower or absent within the case of disulfide bond reduction.

The 2 reducing agents that were tested are dithiothreitol (DTT) and tris-(2-carboxyethyl)phosphine (TCEP). The flexibility of the reducing agents to cleave the disulfide bonds within 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, in addition to those from the Omicron subvariants BA.1, BA.2 and BA.4/BA.5, in various concentrations of the reducing agents. This was followed by incubation with the β9-tagged ACE2 and Δ11S to measure luminescence.

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

Results

The outcomes revealed that the Omicron sub-variants BA.1, BA.2, and BA.4/BA.5 were more prone to reductive cleavage of disulfide bonds than other SARS-CoV-2 variants. Moreover, mutations within 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 steadiness of the spike protein.

Sub-millimolar levels of each 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 bottom IC50 values in comparison with 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 major 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 consider that the T478K and the E484A mutations within the Omicron spike protein RBD might be making the disulfide bonds on the C480 to C488 residues more vulnerable to cleavage.

Conclusions

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

*Essential notice

bioRxiv publishes preliminary scientific reports that aren’t peer-reviewed and, subsequently, shouldn’t be thought to be conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:

• Igor Stagljar, Zhong Yao, Betty Geng, Edyta Marcon, Shuye Pu, Hua Tang, John Merluza, Alexander Bello, Jamie Snider, Ping Lu, and Heidi Wood. (2023). Omicron Spike Protein Is Vulnerable to Reduction. bioRxiv. doi: https://doi.org/10.1101/2023.01.06.522977 https://www.biorxiv.org/content/10.1101/2023.01.06.522977v1