In silico analysis of mutations near S1/S2 cleavage site in SARS‐CoV‐2 spike protein reveals increased propensity of glycosylation in Omicron strain

Abstract

Cleavage of the severe respiratory syndrome coronavirus-2 (SARS-CoV-2) spike protein has been demonstrated to contribute to viral-cell fusion and syncytia formation. Studies have shown that variants of concern (VOC) and variants of interest (VOI) show differing membrane fusion capacity. Mutations near cleavage motifs, such as the S1/S2 and S2' sites, may alter interactions with host proteases and, thus, the potential for fusion. The biochemical basis for the differences in interactions with host proteases for the VOC/VOI spike proteins has not yet been explored. Using sequence and structure-based bioinformatics, mutations near the VOC/VOI spike protein cleavage sites were inspected for their structural effects. All mutations found at the S1/S2 sites were predicted to increase affinity to the furin protease but not TMPRSS2. Mutations at the spike residue P681 in several strains, such P681R in the Delta strain, resulted in the disruption of a proline-directed kinase phosphorylation motif at the S1/S2 site, which may lessen the impact of phosphorylation for these variants. However, the unique N679K mutation in the Omicron strain was found to increase the propensity for O-linked glycosylation at the S1/S2 cleavage site, which may prevent recognition by proteases. Such glycosylation in the Omicron strain may hinder entry at the cell surface and, thus, decrease syncytia formation and induce cell entry through the endocytic pathway as has been shown in previous studies. Further experimental work is needed to confirm the effect of mutations and posttranslational modifications on SARS-CoV-2 spike protein cleavage sites.

Difference in TMPRSS2 usage by Delta and Omicron variants of SARS-CoV-2: Implication for a sudden increase among children

“…Mostly identical reference strains (>99.5% homology) were found among 100 strains computed in a Betacoronavirus BLAST search, suggesting the imported strains spread in the study area without any mutation. The profile of key amino acids in the spike protein affecting S2' cleavage was identical to the reported ones [25][26][27]. Therefore, the properties of the Delta and Omicron variants reported elsewhere is presumably applicable to the strains in the present study.…”

Comparison of SARS-CoV-2 variants of concern in primary human nasal cultures demonstrates Delta as most cytopathic and Omicron as fastest replicating

“…Thus, our results indicate that factors in addition to the cleavage site play a role in these processes. Sequence and structure-based bioinformatics studies have proposed that mutations in Omicron encoding substitutions at the S1/S2 site may help evade recognition by proteases and prevent syncytia formation (48), which may explain our observation of less than expected levels of spike cleavage and propensity towards endosomal entry. However, more recent studies using nasal organoids have reported increased syncytia formation with the Omicron BA.5 subvariant compared to the first Omicron BA.1 variant used in our study (49, 50).…”

Interaction of SARS-CoV-2 with host cells and antibodies: experiment and simulation

“…The P681R mutation may boost membrane fusion of Delta by facilitating cleavage of the precursor protein S to the active S1/S2 conformation. 429 An important role of the P681R mutation in increased pathogenicity and fusogenicity in hamsters was also demonstrated by Saito et al 430 Using bioinformatics tools, Beaudoin et al 431 demonstrated that variants of concern and variants of interest display different membrane fusion capacity. Mutations at or near cleavage sites can alter interaction with host proteases and thus fusion potential.…”

“…All mutations found at the S1/S2 site were predicted to increase affinity for furin protease but not for TMPRSS2. 431 This interesting prediction needs further verification in in vitro and in vivo experiments.…”