Isoform-specific O-glycosylation dictates Ebola virus infectivity

Abstract

Ebola virus glycoprotein is one of the most heavily O-glycosylated viral envelope glycoproteins. Using glycoengineered cell lines we demonstrate that O-linked glycan truncation and perturbed initiation retarded the production of viral particles and decreased infectivity of progeny virus. Next, using TMT isobaric labelling, we performed quantitative differential O-glycoproteomics on proteins produced in wild type HEK293 cells and cell lines ablated for three key GalNAc-transferases, GalNAc-T1, -T2, and -T3, as well as compared it to patterns on wild type virus-like particles. We demonstrate selective initiation of a subset of O-glycosites by each enzyme, with GalNAc-T1 having the largest contribution. This work represents a comprehensive site-specific analysis of EBOV GP, with 47 O-glycosites identified, 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.