Elongation of O-linked glycans affects HSV-1 secretion/infectivity.

<p>(A) HaCaT wild-type or HaCaT mutant keratinocytes, lacking elongation of mucin-type O-liked glycosylation (HaCaT sc D5 and HaCaT sc E5) were infected with MOI of 10 of HSV-1 Syn17+ produced in HaCaT wt. Medium was harvested at 12 and 20 hours post-infection and number of infectious particles were quantified using plaque titration on Vero culture monolayer and expressed as plaque forming units per mL of medium (PFU/mL). Bar graphs represent mean values of 3 biological replicates assayed by 2 technical replicates each + SD. A 2-way ANOVA with Tukey’s multiple comparison test was used to compare differences between means. NS—<i>p</i> > 0.05, *—<i>p</i> < 0.05, **—<i>p</i> < 0.01, ***—<i>p</i> < 0.001, ****—<i>p</i> < 0.0001. Results are representative of at least two independent experiments. (B) Numbers of viral DNA copies in the medium were quantified by qPCR and using a standard curve based on amplification of known copy numbers of HSV-1 DNA fragments cloned in Topo TA plasmids. Copy numbers of viral DNA are expressed as copies/mL. Bar graphs represent mean values of 3 biological replicates assayed by 3 technical replicates each + SD. A 2-way ANOVA with Tukey’s multiple comparison test was used to compare differences between means. NS—<i>p</i> > 0.05, *—<i>p</i> < 0.05, **—<i>p</i> < 0.01, ***—<i>p</i> < 0.001, ****—<i>p</i> < 0.0001. Results are representative of at least two independent experiments.</p

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

<div><p>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.</p></div

Identified O-linked glycosylation sites on HSV-1 envelope glycoproteins.

<p>The cartoon depicts approximate localization of the 74 identified O-linked glycosylation sites in the context of known structural elements of 8 HSV-1 envelope glycoproteins [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004784#ppat.1004784.ref038" target="_blank">38</a>–<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004784#ppat.1004784.ref042" target="_blank">42</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004784#ppat.1004784.ref048" target="_blank">48</a>]. The remaining 4 HSV-1 envelope glycoproteins without identified O-glycosylation are not depicted, although some of them are predicted to be N-glycosylated (gJ, gK, gM, gN). O-glycosylation sites marked with an asterisk can potentially have a slightly different location due to the ambiguity of the site identification within the peptide stretch. Sequence-predicted N-linked glycosylation sites are indicated.</p

O-glycan processing upon HSV-1-induced fragmentation.

<p>(A-C) Immunolabeling of HSV-1 Syn17+ infected (MOI of 10) HEL fibroblasts fixed and permeabilized at indicated time points (hpi—hours post infection). Mock-infected cells were used as control. Cells were double labeled with antibodies and lectins and analyzed by confocal microscopy in order to investigate the cellular localization of Tn structures upon HSV-1 infection. (A) Green—HPA (Tn structure (GalNAcα1-O-Ser/Thr)); red—giantin (cis-/medial-Golgi marker); blue—DAPI. Scale bars: 20 μm for lower magnification images and 5 μm for higher magnification images. (B) Green—HPA; purple—GRP94 (ER marker); blue—DAPI. Scale bars as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004784#ppat.1004784.g004" target="_blank">Fig 4A</a>. (C) Green—HPA; red—TGN46 (trans-Golgi network marker); blue—DAPI. Scale bars as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004784#ppat.1004784.g004" target="_blank">Fig 4A</a>. (D, E) Cell surface expression of common O-glycoforms. HEL fibroblasts were mock- or HSV-1 Syn17+ infected (MOI of 10) and harvested at indicated time points. (D) Immunofluorescent cell surface staining using a panel of carbohydrate specific antibodies (Tn, mAb 5F4; STn, mAb 3F1; T, mAb 3C9; ST, mAb 3C9 plus neuraminidase treatment). 4C4 mAb for Golgi-resident glycosyltransferase GalNAc-T2 was used as a control for cell membrane integrity. Permeabilized cells (Perm) were used as a positive control for GalNAc-T2 staining. Scale bar—20 μm. (E) Cell surface carbohydrate profile of HEL fibroblasts analyzed by flow cytometry (Tn, HPA-FITC; T, mAb 3C9; ST, mAb 3C9 plus neuraminidase treatment). HSV-1 infected samples were gated (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004784#ppat.1004784.s002" target="_blank">S1 Fig</a>) on HSV-1-positive cells (except for HPA-FITC labeled samples). (F-G) HEL fibroblasts were mock- or HSV-1 Syn17+ infected (MOI of 10) and then fixed and permeabilized at indicated time points (hpi—hours post infection). Cells were double labeled with antibodies and analyzed by confocal microscopy in order to investigate the Golgi microorganization upon HSV-1 infection. (F) Green—GM130 (cis-Golgi marker); red—giantin (cis-/medial-Golgi marker); blue—DAPI. Scale bars as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004784#ppat.1004784.g004" target="_blank">Fig 4A</a>. (G) Green—β4GalT1 (trans-Golgi marker); red—giantin (cis-/medial-Golgi marker); blue—DAPI. Scale bars as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004784#ppat.1004784.g004" target="_blank">Fig 4A</a>. Enlarged micrographs marked with an asterisk do not correspond to the merged images to the left.</p

Overview of site identification using different enrichment strategies.

<p>Overview of site identification using different enrichment strategies.</p

Number of sites identified on individual envelope glycoproteins.

<p>Number of sites identified on individual envelope glycoproteins.</p