Isoform-specific O-glycosylation dictates Ebola virus infectivity

Abstract

Ebola virus glycoprotein is one of the most heavily O-glycosylated viral envelope glycoproteins. The glycoprotein possesses a large mucin-like domain responsible for the cytopathic effect on infected cells, yet its structure or potential role in early entry events is poorly defined. To understand the importance of elongated O-glycans and the individual O-glycosylation sites for viral infectivity, we performed a comprehensive characterization of site-specific glycosylation governed by the three key GalNAc-transferases, GalNAc-T1, -T2, and -T3, initiating O-glycan biosynthesis. Using TMT isobaric labelling we performed quantitative differential O-glycoproteomics on proteins produced in wild type HEK293 cells and cell lines ablated for each of the three GalNAc-Ts, as well as compared it to patterns on wild type virus-like particles. In total we found 38 and 41 O-glycosites on virus like particle-derived and recombinant GP, respectively, with well correlated sites and site-specific structures. Examination of the isoform-specific glycosylation demonstrate selective initiation of a subset of O-glycosites by each enzyme, with GalNAc-T1 having the largest contribution. We next demonstrate that O-linked glycan truncation and perturbed initiation retarded the production of viral particles and decreased infectivity of progeny virus. This work represents a comprehensive site-specific analysis of EBOV GP and sheds light on differential regulation of EBOV GP glycosylation initiated by host GalNAc-Ts. Together with the effect on viral propagation it opens prospective avenues for tailored intervention approaches and means for modulating immunogen O-glycan density.

Glycoproteomic landscape and structural dynamics of TIM family immune checkpoints enabled by mucinase SmE

“…4A (right). As others before have suggested, the glycan size and heterogeneity were much lower in areas of dense glycosylation; sparse O-glycosites afforded larger and more diverse glycan structures 64,65 .…”

Glycoproteomic landscape and structural dynamics of TIM family immune checkpoints enabled by mucinase SmE

“…Additionally, as others before have suggested, the glycan size and heterogeneity was much lower in areas of dense glycosylation; sparse O-glycosites afforded larger and more diverse glycan structures. 70,71 By obtaining the most abundant O-glycan at each glycosite, we built two all-atom computational models of the fully glycosylated transmembrane glycoproteins TIM-3 and TIM-4 (Figure 4A, left) to better understand the contribution of glycan density on the overall flexibility and length of TIMs. Each of these systems To identify the degree to which TIM-3 and TIM-4 mucin domains compress during simulation, we calculated a normalized end-to-end distance of each protein's mucin domain as a function of time (Figure 4B).…”