A Strategy for O-Glycoproteomics of Enveloped Viruses—the O-Glycoproteome of Herpes Simplex Virus Type 1

Glycosylation of viral envelope proteins is important for infectivity and interaction with host immunity,however,our current knowledge of the functions of glycosylation is largely limited to n-glycosylation because it is difficult to predict and identify site-specific o-glycosylation. here,we present a novel proteome-wide discovery strategy for o-glycosylation sites on viral envelope proteins using herpes simplex virus type 1 (hsv-1) as a model. we identified 74 o-linked glycosylation sites on 8 out of the 12 hsv-1 envelope proteins. two of the identified glycosites found in glycoprotein b were previously implicated in virus attachment to immune cells. we show that hsv-1 infection distorts the secretory pathway and that infected cells accumulate glycoproteins with truncated o-glycans,nonetheless retaining the ability to elongate most of the surface glycans. with the use of precise gene editing,we further demonstrate that elongated o-glycans are essential for hsv-1 in human hacat keratinocytes,where hsv-1 produced markedly lower viral titers in hacat with abrogated o-glycans compared to the isogenic counterpart with normal o-glycans. the roles of o-linked glycosylation for viral entry,formation,secretion,and immune recognition are poorly understood,and the o-glycoproteomics strategy presented here now opens for unbiased discovery on all enveloped viruses. © 2015 bagdonaite et al.