Disparate shielding efficacies of viral glycosylation
Viral envelope proteins are glycosylated to varying degrees, but depending on their overall mass, surface area, and volume, the overall density of glycan shielding may differ significantly. For example, both LASV GPC and coronavirus S proteins consist of 25% glycan by molecular weight. However, given the significantly larger protein surface area and volume of coronavirus S proteins, coverage of the glycan “shield” over the proteinaceous surface is considerably sparser in comparison to the smaller LASV GPC, which occludes a far greater proportion of the protein surface with fewer glycans. To demonstrate that the presence of glycosylation plays a major role in the immune response to these different glycoproteins, we studied the glycome of several biomedically important coronaviruses and compared their glycan compositions in a structural context.
We then investigated the glycan shield densities of seven viral class I fusion proteins using a global structural approach which was calculated by dividing the number of amino-acids that interact with glycans by the number of solvent-accessible amino-acid residues of each respective glycoprotein and plotted this against oligomannose abundance. A strong correlation was observed (Fig. 6) and viruses historically classified as “evasion strong”56 had significantly elevated glycan shield densities and oligomannose abundance, which underscores the importance of glycan shielding in immune evasion.
Fig. 6 Comparison of the glycan shields of viral class I fusion proteins.
Glycan shield densities were calculated using Proteins, Interfaces, Structures and Assemblies (PISA)83 analyses of fully glycosylated models of SARS S, MERS S, HKU1 S, LASV GPC, HIV-1 Env (BG505), Influenza H3N2 hemagglutinin (Victoria 2011), SIV Env (PDB ID 5X58, 5X59, 5I08, 5VK2, 4ZMJ, 4O5N, 6OHY, respectively)9,11,53,84–86. Oligomannose abundances of viral glycoproteins were ascertained by HILIC-UPLC analysis of PNGase F released N-linked glycans that were fluorescently labelled with procainamide24,45,53 (SI Fig. 5). The number of amino-acid residues interacting with N-linked glycans was divided by the number of solvent-accessible amino-acid residues of the glycoprotein as a measure for global glycan shield density. All viral glycoproteins analysed were expressed as trimers in HEK293F cells apart from LASV GPC, which was derived from virus-like particles from Madin-Darby canine kidney II cells.
Whether the restricted glycan shielding observed on coronaviruses is linked to the zoonosis of the pathogens is unknown. However, it is tempting to speculate, for example, that MERS has not evolved a dense shield since it would not offer as much of a protective advantage against camel nanobodies (also known as single-domain antibodies) which could more easily penetrate it. Investigation of the host immune response to viruses in their natural reservoirs may offer a route to understanding why coronavirus glycosylation does not reach the density of other viruses such as HIV-1. In addition, it may be that functional constraints, such as maintaining flexibility of the receptor-binding domains, limit the accretion of glycans on coronavirus spikes, which would render it incapable of performing its primary functions, including receptor-binding and membrane fusion. This phenomenon has been observed on other viral glycoproteins, including influenza HAs, where there is a limit to the accumulation of glycosylation sites that can be incorporated in vivo57,58, compared with in vitro59, with H3N2 and H1N1 HAs replacing existing PNGs rather than continually adding them upon the glycoprotein22,58. The importance of glycosylation in modulation of viral infectivity and immune responses have also been investigated during influenza vaccine research22,60 and should be considered in coronavirus vaccine research.
More topically, it is interesting to note the conservation of N-linked glycosylation sites on S proteins from the SARS-CoV-2 and SARS (SI Fig. 6). SARS-CoV-2 possesses a total of 22 N-linked glycan sites compared with 23 on SARS, with 18 of these sites being in common. As such, it is likely that these glycans on this novel coronavirus would shield similar immunogenic epitopes that are observed on SARS S. As expected, most of the differences between the two viruses are observed on the S1 subunit, due to its amenability to substitutions while still remaining functionally competent. Furthermore, likely targets for the majority of antibodies targeting the spike are located on S1, resulting in greater levels of immune pressure upon this subunit. This notion is further reflected in terms of glycosylation, with all of the glycan sites conserved on the S2 subunit between SARS and SARS-CoV-2, whereas the S1 subunit exhibits glycan site additions and deletions (SI Fig. 7). Bioinformatic analysis of current SARS-CoV-2 spike genes (n = 566 as of 17 March 2020) from nextstrain61 (https://nextstrain.org/ncov) revealed low sequence diversity and no changes in glycosylation sites (SI Fig. 8).
Although it is difficult to directly compare viruses in terms of immunogenic responses, on the one hand, SARS and MERS coronaviruses readily elicit neutralizing antibodies following infection or immunization62–65. Indeed, many potential MERS-CoV vaccine candidates are able to elicit high titres of serum IgG upon immunization but fail to produce sufficient mucosal immunity65. In contrast, the high mutation rate66 and the evolving glycan shield of HIV-120,39, which firmly exemplifies it as “evasion strong” virus, hinders the development of broadly neutralizing antibodies67.Viruses classified as “evasion strong”26,56 may then differ due to varied efficacies of protein surface shielding by glycans.
Overall, this study adds further evidence suggesting that extensive N-linked glycan modifications of SARS and MERS CoV S proteins do not constitute an effective shield, in comparison to glycan shields of certain other viruses, which is reflected by the overall structure, density and oligomannose abundance across the corresponding trimeric glycoproteins. We also demonstrate that amino-acid diversification indeed occurs at antibody accessible regions on the trimer, which confirms that glycans play a role in occluding specific regions if vulnerability on the glycoprotein. Furthermore, comparisons between glycan shields from a number of viruses highlight the importance of a glycan shield in immune evasion and reveal structural principles that govern glycosylation status.