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A strategy for O-glycoproteomics of enveloped viruses--the O-glycoproteome of herpes simplex virus type 1.

Bagdonaite I, Nordén R, Joshi HJ, Dabelsteen S, Nyström K, Vakhrushev SY, Olofsson S, Wandall HH - PLoS Pathog. (2015)

Bottom Line: We identified 74 O-linked glycosylation sites on 8 out of the 12 HSV-1 envelope proteins.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.

View Article: PubMed Central - PubMed

Affiliation: Copenhagen Center for Glycomics, Institute of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Identified O-linked glycosylation sites on HSV-1 envelope glycoproteins.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 [38–42, 48]. 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.
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ppat.1004784.g002: Identified O-linked glycosylation sites on HSV-1 envelope glycoproteins.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 [38–42, 48]. 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.

Mentions: Fig 2 presents a graphic depiction of the HSV-1 O-glycoproteome. A total of 34 out of 74 identified O-glycosylation sites were localized on the four HSV-1 membrane proteins, gB, gD, gH and gL, which are all essential for viral infectivity in vitro [33–36] (Table 2). Twenty-one glycosylation sites were identified in gB, which is essential for fusion with host cell membrane [33]. The identified glycosylation sites include two positions, T53 and T480 (S1 Table), which have previously been proposed to be important for the interaction with the paired immunoglobulin-like type 2 receptor α based on the finding that Ala substitutions resulted in loss of interaction [37]. In addition, O-linked glycans were found throughout the ectodomain and localized to both ordered and unstructured regions of the molecule [38] (Fig 2). Interestingly, several gB O-glycosylation sites were highly conserved between 8 members of the human herpesviruses (Figs 3 and S2).


A strategy for O-glycoproteomics of enveloped viruses--the O-glycoproteome of herpes simplex virus type 1.

Bagdonaite I, Nordén R, Joshi HJ, Dabelsteen S, Nyström K, Vakhrushev SY, Olofsson S, Wandall HH - PLoS Pathog. (2015)

Identified O-linked glycosylation sites on HSV-1 envelope glycoproteins.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 [38–42, 48]. 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4382219&req=5

ppat.1004784.g002: Identified O-linked glycosylation sites on HSV-1 envelope glycoproteins.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 [38–42, 48]. 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.
Mentions: Fig 2 presents a graphic depiction of the HSV-1 O-glycoproteome. A total of 34 out of 74 identified O-glycosylation sites were localized on the four HSV-1 membrane proteins, gB, gD, gH and gL, which are all essential for viral infectivity in vitro [33–36] (Table 2). Twenty-one glycosylation sites were identified in gB, which is essential for fusion with host cell membrane [33]. The identified glycosylation sites include two positions, T53 and T480 (S1 Table), which have previously been proposed to be important for the interaction with the paired immunoglobulin-like type 2 receptor α based on the finding that Ala substitutions resulted in loss of interaction [37]. In addition, O-linked glycans were found throughout the ectodomain and localized to both ordered and unstructured regions of the molecule [38] (Fig 2). Interestingly, several gB O-glycosylation sites were highly conserved between 8 members of the human herpesviruses (Figs 3 and S2).

Bottom Line: We identified 74 O-linked glycosylation sites on 8 out of the 12 HSV-1 envelope proteins.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.

View Article: PubMed Central - PubMed

Affiliation: Copenhagen Center for Glycomics, Institute of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen, Denmark.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus