A Strategy for O-Glycoproteomics of
Enveloped Viruses—the O-Glycoproteome of Herpes Simplex Virus Type 1
Ieva Bagdonaite1, Rickard Nordén2, Hiren J. Joshi1, Sally Dabelsteen3, Kristina Nyström2,
Sergey Y. Vakhrushev1, Sigvard Olofsson2, Hans H. Wandall1* 1 Copenhagen Center for Glycomics, Institute of Cellular and Molecular Medicine, University of
Copenhagen, Copenhagen, Denmark, 2 Department of Clinical Virology, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden, 3 Institute of Odontology, University of Copenhagen, Copenhagen,
Denmark * email@example.com
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.
Information on site-specific O-glycosylation of viral envelope glycoproteins is generally very limited despite important functions. We present a powerful mass-spectrometry based strategy to globally identify O-glycosylation sites on viral envelope proteins of a given virus in the context of a productive infection. We successfully utilized the strategy to map
O-linked glycosylation sites on the complex HSV-1 virus demonstrating that O-glycosylation is widely distributed on most envelope proteins. Moreover, we used genetically engineered keratinocytes lacking O-glycan elongation capacity to demonstrate that O-linked
PLOS Pathogens | DOI:10.1371/journal.ppat.1004784 April 1, 2015 1 / 22
Citation: Bagdonaite I, Nordén R, Joshi HJ,
Dabelsteen S, Nyström K, Vakhrushev SY, et al. (2015) A Strategy for O-Glycoproteomics of
Enveloped Viruses—the O-Glycoproteome of Herpes
Simplex Virus Type 1. PLoS Pathog 11(4): e1004784. doi:10.1371/journal.ppat.1004784
Editor: Félix A. Rey, Institut Pasteur, FRANCE
Received: November 3, 2014
Accepted: March 4, 2015
Published: April 1, 2015
Copyright: © 2015 Bagdonaite et al. This is an open access article distributed under the terms of the
Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability Statement: All relevant data are within the paper and its Supporting Information files.
Funding: This work was in part supported by The
Danish Research Councils (1331-00133B), http://ufm. dk/en/research-and-innovation/funding-programmesfor-research-and-innovation/find-danish-fundingprogrammes (IB, SD, HHW); a programme of excellence 2016 (Copenhagen as the next leader in precise genetic engineering CDO2016: 2016CDO04210) from the University of Copenhagen http://www.cdo.ku.dk/ http://research.ku.dk/strengths/ excellence-programmes/ (IB, HHW); The Danish
National Research Foundation (DNRF107) http://dg. glycans are indeed important for HSV-1 biology as HSV-1 particles produced in these cells had significantly lower titers compared to wild-type keratinocytes. These tools enable wider discovery and detailed analysis of the role of site-specific O-glycosylation in virology.
Enveloped viruses contain one or more membrane proteins important for adhesion and entry to host cells . The majority of envelope membrane proteins are predicted or confirmed to be covered with glycans with important functions in protein folding, transport, formation of infectious particles, entry into host cells, and shielding from the host’s immune system [2–7]. Numerous studies have addressed the structures and functions of N-linked glycans on membrane glycoproteins from different viruses [8–13], and N-glycosylation has attracted particular attention for the human immunodeficiency virus (HIV), where a cluster of N-glycans constitute the epitope for the 2G12 and other antibodies with broadly neutralizing function [14, 15]. In striking contrast, information on O-linked glycans and, in particular, where O-glycans are found is generally missing, which leaves a void in knowledge of the biological functions of O-glycosylation. This is in spite of substantial evidence suggesting that O-glycosylation is important for viral infectivity and virus-induced immunomodulation for several viruses [4, 7, 16–18].
Viral proteins destined for the virion surface travel through the host’s secretory pathway where they hijack the host cell’s glycosylation machinery and get decorated with glycans .
Protein glycosylation is controlled by hundreds of glycosyltransferases that reside in the secretory pathway and that, in a non-template fashion, orchestrate the diversity of glycan structures found on proteins . There is substantial evidence that many viral membrane proteins are
N-glycosylated, although there is surprisingly limited experimental evidence for actual glycosylation sites for many viruses with few exceptions [21, 22]. However, to a large extent the consensus sequence motif NXS/T (X—all amino acids except P) enables reliable prediction of Nglycosites . There is less evidence for the presence of O-glycosylation (GalNAc-type) on virus membrane glycoproteins, and this largely relies on the presence of mucin-like sequence motifs with high density of PST residues. Such are found in e.g. HSV-1 gC  and Ebola virus glycoprotein , but recent studies suggest that O-glycosylation is more prevalent in nonmucin-like regions and often exist as isolated sites or in small clusters . Site-specific O-glycosylation in such isolated or clustered positions may exert co-regulatory functions of basic processes such as pro-protein processing and ectodomain shedding , which may affect viral fusion protein activation and function [28, 29]. In contrast to N-linked glycosylation that can be predicted with reasonable certainty our knowledge of O-glycosylation is hampered by lack of simple consensus motifs for prediction of O-glycosites. O-glycosylation is unique in being controlled by 20 polypeptide GalNAc-transferases (GalNAc-Ts) that transfer GalNAc to select Ser, Thr and, possibly, Tyr residues . The initial GalNAc residues are further elongated, branched, and capped by a large number of different glycosyltransferases in subsequent processing steps. The large number of GalNAc-T isoenzymes with distinct peptide substrate specificities and cell expression patterns provides a high degree of differential regulation of Oglycosylation capacity directed by the repertoire of GalNAc-Ts in a given cell. This unprecedented complexity of protein glycosylation adds to the need for direct experimental analysis of