Insertions in the SARS-CoV-2 Spike N-Terminal Domain May Aid COVID-19 Transmission

Coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2 is an ongoing pandemic that causes significant health/socioeconomic burden. Variants of concern (VOCs) have emerged which may affect transmissibility, disease severity and re-infection risk. Most studies focus on the receptor-binding domain (RBD) of the Spike protein. However, some studies suggest that the Spike N-terminal domain (NTD) may have a role in facilitating virus entry via sialic-acid receptor binding. Furthermore, most VOCs include novel NTD variants. Recent analyses demonstrated that NTD insertions in VOCs tend to lie close to loop regions likely to be involved in binding sialic acids. We extended the structural characterisation of these putative sugar binding pockets and explored whether variants could enhance the binding to sialic acids and therefore to the host membrane, thereby contributing to increased transmissibility. We found that recent NTD insertions in VOCs (i.e., Gamma, Delta and Omicron variants) and emerging variants of interest (VOIs) (i.e., Iota, Lambda, Theta variants) frequently lie close to known and putative sugar-binding pockets. For some variants, including the recent Omicron VOC, we find increases in predicted sialic acid binding energy, compared to the original SARS-CoV-2, which may contribute to increased transmission. We examined the similarity of NTD across a range of related Betacoronaviruses to determine whether the putative sugar-binding pockets are sufficiently similar to be exploited in drug design. Despite global sequence and structure similarity, most sialic-acid binding pockets of NTD vary across related coronaviruses. Typically, SARS-CoV-2 possesses additional loops in these pockets that increase contact with polysaccharides. Our work suggests ongoing evolutionary tuning of the sugar-binding pockets in the virus. Whilst three of the pockets are too structurally variable to be amenable to pan Betacoronavirus drug design, we detected a fourth pocket that is highly structurally conserved and could therefore be investigated in pursuit of a generic drug. Our structure-based analyses help rationalise the effects of VOCs and provide hypotheses for experiments. For example, the Omicron variant, which has increased binding to sialic acids in pocket 3, has a rather unique insertion near pocket 3. Our work suggests a strong need for experimental monitoring of VOC changes in NTD.