N-Glycans Mediate the Ebola Virus-GP1 Shielding of Ligands to Immune Receptors and Immune Evasion

Abstract

The Ebola Virus (EBOV) glycoprotein (GP) sterically shields cell-membrane ligands to immune receptors such as human leukocyte antigen class-1 (HLA-I) and MHC class I polypeptide-related sequence A (MICA), thus mediating immunity evasion. It was suggested that the abundant N-glycosylation of the EBOV-GP is involved in this steric shielding. We aimed to characterize (i) the GP N-glycosylation sites contributing to the shielding, and (ii) the effect of mutating these sites on immune subversion by the EBOV-GP. The two highly glycosylated domains of GP are the mucin-like domain (MLD) and the glycan cap domain (GCD) with three and six N-glycosylation sites, respectively. We mutated the N-glycosylation sites either in MLD or in GCD or in both domains. We showed that the glycosylation sites in both the MLD and GCD domains contribute to the steric shielding. This was shown for the steric shielding of either HLA-I or MICA. We then employed the fluorescence resonance energy transfer (FRET) method to measure the effect of N-glycosylation site removal on the distance in the cell membrane between the EBOV-GP and HLA-I (HLA.A * 0201 allele). We recorded high FRET values for the interaction of CFP-fused HLA.A * 0201 and YFP-fused EBOV-GP, demonstrating the very close distance (<10 nm) between these two proteins on the cell membrane of GP-expressing cells. The co-localization of HLA-I and Ebola GP was unaffected by the disruption of steric shielding, as the removal of N-glycosylation sites on Ebola GP revealed similar FRET values with HLA-I. However, these mutations directed to N-glycosylation sites had restored immune cell function otherwise impaired due to steric shielding over immune cell ligands by WT Ebola GP. Overall, we showed that the GP-mediated steric shielding aimed to impair immune function is facilitated by the N-glycans protruding from its MLD and GCD domains, but these N-glycans are not controlling the close distance between GP and its shielded proteins.

Glycan shield of the ebolavirus envelope glycoprotein GP

“…The effective shielding would perhaps be better illustrated by the antibodies that don’t bind or neutralize ebolavirus infection because of the steric clashes with glycans. Several studies have illustrated this by showing greatly enhanced sensitivity of (pseudo) ebolavirus to serum neutralization after glycan removal by mutagenesis [31, 32].…”

“…The copyright holder for this preprint this version posted February 7, 2022. ; https://doi.org/10.1101/2022.02.07.479410 doi: bioRxiv preprint cell attachment through C-type lectins DC-SIGN/L-SIGN [27][28][29] and have been implicated in shielding GP from binding by neutralizing antibodies [28,[30][31][32][33][34][35][36]. Whereas the overall sequences and especially the N-linked glycosylation sites in the base, head and glycan cap are relatively well conserved among ebolavirus species, those in the MLD are highly variable.…”

“…There are up to 17 N-linked glycosylation sites in ebolavirus GP, 15 of which are in the GP1 subunit, primarily in the glycan cap and MLD (see Figure 1A). The N-linked glycans mediate host-cell attachment through C-type lectins DC-SIGN/L-SIGN [27][28][29] and have been implicated in shielding GP from binding by neutralizing antibodies [28,[30][31][32][33][34][35][36]. Whereas the overall sequences and especially the N-linked glycosylation sites in the base, head and glycan cap are relatively well conserved among ebolavirus species, those in the MLD are highly variable.…”

Glycans of SARS-CoV-2 Spike Protein in Virus Infection and Antibody Production

“…These "self"-glycans are generally thought to be a strategy to escape the host immune response (Wang, 2020). For example, human immunodeficiency virus (HIV-1) (Stewart-Jones et al, 2016), hepatitis C virus (Falkowska et al, 2007), and Ebola virus (Iraqi et al, 2020) exhibit extensive N-linked glycans that cover some of the critical virus-neutralizing epitopes to block antibody recognition. Similarly, coronavirus S glycans also mask the protein surface and consequently limit antibody access to protein-neutralizing epitopes (Grant et al, 2020;Wang, 2020;Watanabe et al, 2020).…”

Isoform-specific O-glycosylation dictates Ebola virus infectivity

“…1A). A few specific N-glycosites affecting EBOV conformational stability or immunogenicity have been identified and shown to provide steric shielding of host cell ligands for immune effector cells (Dowling et al, 2007; Iraqi et al, 2020). The MLD is also predicted to be heavily glycosylated, though no specific sites have been identified, and the studies on O-glycosylation have been limited to deletion of MLD (Dowling et al ., 2007; Feldmann et al, 1994; Lee and Saphire, 2009).…”