| 1. |
- Bagdonaite, I., et al.
(författare)
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A Strategy for O-Glycoproteomics of Enveloped Viruses-the O-Glycoproteome of Herpes Simplex Virus Type 1
- 2015
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Ingår i: Plos Pathogens. - : Public Library of Science (PLoS). - 1553-7366 .- 1553-7374. ; 11:4
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Tidskriftsartikel (refereegranskat)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.
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| 2. |
- Nordén, Rickard, 1977, et al.
(författare)
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Activation of host antiviral RNA-sensing factors necessary for herpes simplex virus type 1-activated transcription of host cell fucosyltransferase genes FUT3, FUT5, and FUT6 and subsequent expression of sLex in virus-infected cells.
- 2009
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 19:7, s. 776-88
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Tidskriftsartikel (refereegranskat)abstract
- Herpes simplex virus type 1 (HSV-1) induces expression of a selectin receptor, the carbohydrate epitope sialyl Lewis X (sLe(x)), at the surface of infected cells. The molecular background to this phenomenon is that a viral immediate early RNA interacts with as yet unidentified host factors, eventually resulting in transcription of three dormant host fucosyltransferase genes (FUT3, FUT5, and FUT6), whose gene products are rate-limiting for synthesis of sLe(x). The aim of the present study was to define the immediate targets for the viral RNA in this process. We found that the Protein Kinase R (PKR) inhibitors 2-aminopurine (2-AP) and C16 inhibited FUT3, FUT5, and FUT6 expression as well as HSV-1-induced expression of sLe(x), indicating a primary role of PKR as a viral RNA target. The PKR-dependent activation of the FUT genes seemed neither to involve PKR effects on translation nor to involve NF-kappaB- or JNK-dependent activation. IMD-0354, known as an inhibitor of the NF-kappaB-activating factor IKK-2, induced FUT transcription via a novel IKK-2-independent mechanism, irrespective of whether the cells were virus-infected or not. Altogether, the results suggested that PKR is the primary target for HSV-1 early RNA during induction of FUT3, FUT5, and FUT6, and that the subsequent steps in the transcriptional activation of these host genes involve a hitherto unknown IMD-0354, yet IKK-2-independent, pathway.
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| 3. |
- Nordén, Rickard, 1977, et al.
(författare)
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Involvement of viral glycoprotein gC-1 in expression of the selectin ligand sialyl-Lewis X induced after infection with herpes simplex virus type 1.
- 2013
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Ingår i: APMIS : acta pathologica, microbiologica, et immunologica Scandinavica. - : Wiley. - 1600-0463. ; 121:4, s. 280-289
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Tidskriftsartikel (refereegranskat)abstract
- Several herpesviruses induce expression of the selectin receptor sialyl-Lewis X (sLe(x) ) by activating transcription of one or more of silent host FUT genes, each one encoding a fucosyltransferase that catalyses the rate-limiting step of sLe(x) synthesis. The aim here was to identify the identity of the glycoconjugate associated with sLe(x) glycoepitope in herpes simplex virus type 1 (HSV-1) infected human diploid fibroblasts, using immunofluorescence confocal microscopy. Cells infected with all tested HSV-1 strains analysed demonstrated bright sLe(x) fluorescence, except for two mutant viruses that were unable to induce proper expression of viral glycoprotein gC-1: One gC-1 null mutant and another mutant expressing gC-1 devoid of its major O-glycan-containing region (aa 33-116). The sLe(x) reactivity of HSV-1 infected cells was abolished by mild alkali treatment. Altogether the results indicated that the detectable sLe(x) was associated with O-linked glycans, situated in the mucin region of gC-1. No evidence for sLe(x) (i) in other HSV-1 glycoproteins with mucin domains such as gI-1 or (ii) in host cell glycoproteins/glycolipids was found. Thus, the mucin domain of HSV-1 gC-1 may support expression of selectin ligands such as sLe(x) and other larger O-linked glycans in cell types lacking endogenous mucin domain-containing glycoproteins, optimized for O-glycan expression, provided that the adequate host glycosyltransferase genes are activated.
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| 4. |
- Nordén, Rickard, 1977, et al.
(författare)
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NF kappa B-mediated activation of the cellular FUT3, 5 and 6 gene cluster by herpes simplex virus type 1
- 2017
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 0959-6658 .- 1460-2423. ; 27:11, s. 999-1005
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Tidskriftsartikel (refereegranskat)abstract
- Herpes simplex virus type 1 has the ability to induce expression of a human gene cluster located on chromosome 19 upon infection. This gene cluster contains three fucosyltransferases (encoded by FUT3, FUT5 and FUT6) with the ability to add a fucose to an N-acetylglucosamine residue. Little is known regarding the transcriptional activation of these three genes in human cells. Intriguingly, herpes simplex virus type 1 activates all three genes simultaneously during infection, a situation not observed in uninfected tissue, pointing towards a virus specific mechanism for transcriptional activation. The aim of this study was to define the underlying mechanism for the herpes simplex virus type 1 activation of FUT3, FUT5 and FUT6 transcription. The transcriptional activation of the FUT-gene cluster on chromosome 19 in fibroblasts was specific, not involving adjacent genes. Moreover, inhibition of NF kappa B signaling through panepoxydone treatment significantly decreased the induction of FUT3, FUT5 and FUT6 transcriptional activation, as did siRNA targeting of p65, in herpes simplex virus type 1 infected fibroblasts. NF kappa B and p65 signaling appears to play an important role in the regulation of FUT3, FUT5 and FUT6 transcriptional activation by herpes simplex virus type 1 although additional, unidentified, viral factors might account for part of the mechanism as direct interferon mediated stimulation of NF kappa B was not sufficient to induce the fucosyltransferase encoding gene cluster in uninfected cells.
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| 5. |
- Nordén, Rickard, 1977, et al.
(författare)
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O-linked glycosylation of the mucin domain of the herpes simplex virus type 1 specific glycoprotein gC-1 is temporally regulated in a seed-and-spread manner.
- 2015
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Ingår i: The Journal of biological chemistry. - 1083-351X. ; 290:8, s. 5078-5091
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Tidskriftsartikel (refereegranskat)abstract
- The herpes simplex virus type 1 (HSV-1) glycoprotein gC-1, participating in viral receptor interactions and immunity interference, harbors a mucin-like domain with multiple clustered O-linked glycans. Using HSV-1 infected diploid human fibroblasts, an authentic target for HSV-1 infection, and a protein immunoaffinity procedure, we enriched fully glycosylated gC-1 and a series of its biosynthetic intermediates. This fraction was subjected to trypsin digestion and a LC-MS/MS glycoproteomics approach. In parallel, we characterized the expression patterns of the 20 isoforms of human GalNAc transferases responsible for initiation of O-linked glycosylation. The gC-1 O-glycosylation was regulated in an orderly manner initiated by synchronous addition of one GalNAc unit each to T87 and T91, and one GalNAc unit to either T99 or T101, forming a core glycopeptide for subsequent additions of in all 11 GalNAc residues to selected Ser and Thr residues of the T76-L107 stretch of the mucin domain. The expression patterns of GalNAc transferases in the infected cells suggested that initial additions of GalNAc were carried out by initiating GalNAc transferases, in particular GalNAc-T2, whereas subsequent GalNAc additions were carried out by follow up transferases, in particular GalNAc-T10. Essentially all of the susceptible Ser or Thr residues had to acquire their GalNAc units before any elongation to longer O-linked glycans of the gC-1-associated GalNAc units was permitted. Since the GalNAc occupancy pattern is of relevance for receptor binding of gC-1, the data provides a model to delineate biosynthetic steps of O-linked glycosylation of the gC-1 mucin domain in HSV-1 infected target cells.
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| 6. |
- Nordén, Rickard, 1977, et al.
(författare)
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Virus-induced appearance of the selectin ligand sLeX in herpes simplex virus type 1-infected T cells: Involvement of host and viral factors.
- 2013
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 23:3, s. 310-321
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Tidskriftsartikel (refereegranskat)abstract
- Circulating leukocytes that express selectin ligands such as the carbohydrate epitope sialyl Lewis X (sLeX) may interact with endothelial selectins, resulting in transmigration of the leukocyte across the endothelial wall to adjacent tissue. Due to the potential of selectin-ligand interactions as targets in viral pathogenesis, we aimed at determining whether herpes simplex virus type 1 (HSV1) is able to induce appearance of sLeX at the surface of infected leukocytes. We found that HSV1 infection of a T cell line resulted in transcriptional activation of human fucosyltransferase genes FUT3, FUT6 and FUT7, the two latter genes encoding fucosyltransferases rate limiting for sLeX synthesis. Flow cytometry and confocal microscopy demonstrated that HSV1 infection resulted in a two-fold rise in the proportion of sLeX-positive cells. Increased levels of FUT3, FUT6 and FUT7 RNA were detected already at 3 h post infection, and treatment with cycloheximide, a translation inhibitor, blocked HSV1-induced increase in expression of FUT3, FUT6 and FUT7 RNA, suggesting involvement of viral or cellular proteins. Studies with infectious viral mutants indicated that the viral immediate early (α) protein ICP0 is essential for initiation of FUT7 though not for FUT3 or FUT6 transcription. In CD3+ cells, derived from peripheral blood mononuclear cells, HSV1 infection induced expression of FUT3, FUT5 and FUT6, whereas FUT7 was not altered. The mean sLeX fluorescence intensity of CD3+ cells was significantly higher in HSV1-infected CD3+ cells. This suggests that infected leukocytes during HSV1 viremia may express selectin ligands with possible but as yet unproven roles in viral pathogenesis.
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| 7. |
- Nyström, Kristina, 1977, et al.
(författare)
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Induction of sialyl-Lex expression by herpes simplex virus type 1 is dependent on viral immediate early RNA-activated transcription of host fucosyltransferase genes. : Induction of sialyl-Lewis x expression by HSV-1
- 2009
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Ingår i: Glycobiology. - : Oxford University Press (OUP). - 1460-2423 .- 0959-6658. ; 19:8, s. 847-59
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Tidskriftsartikel (refereegranskat)abstract
- We have previously shown that varicella-zoster virus (VZV) and cytomegalovirus (CMV) infection of diploid human fibroblasts (HEL) results in neo-expression of Lewis antigens sialyl Lewis x (sLe(x)) and Lewis y (Le(y)), respectively, after transcriptional activation of different combinations of dormant human fucosyltransferase genes (FUT1, FUT3, FUT5, and FUT6), whose gene products are responsible for the synthesis of Le antigens. Here, we show that herpes simplex virus type 1 (HSV-1) also induces sLe(x) expression dependent on induction of FUT3, FUT5, and FUT6 transcription in infected cells. HSV-1 induction of FUT5 was subsequently used as a model system for analyzing the mechanism of viral activation of dormant fucosyltransferase genes. We show that this is a rapid process, which gives rise to elevated FUT5 RNA levels already at 90 min postinfection. Augmented FUT5 transcription was found to be dependent on transcription of viral genes, but not dependent on the immediate early proteins ICP0 and ICP4, as demonstrated by experiments with HSV-1 mutants defective in expression of these genes. Augmented FUT5 transcription takes place in cycloheximide-treated HSV-1-infected cells, suggesting a more direct role for IE viral RNA during activation of cellular FUT5.
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| 8. |
- Samuelsson, Ebba, 1991, et al.
(författare)
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Sialic Acid and Fucose Residues on the SARS-CoV-2 Receptor-Binding Domain Modulate IgG Antibody Reactivity
- 2022
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Ingår i: Acs Infectious Diseases. - : American Chemical Society (ACS). - 2373-8227. ; 8:9, s. 1883-1893
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Tidskriftsartikel (refereegranskat)abstract
- The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a conserved domain and a target for neutralizing antibodies. We defined the carbohydrate content of the recombinant RBD produced in different mammalian cells. We found a higher degree of complex-type N-linked glycans, with less sialylation and more fucosylation, when the RBD was produced in human embryonic kidney cells compared to the same protein produced in Chinese hamster ovary cells. The carbohydrates on the RBD proteins were enzymatically modulated, and the effect on antibody reactivity was evaluated with serum samples from SARS-CoV-2 positive patients. Removal of all carbohydrates diminished antibody reactivity, while removal of only sialic acids or terminal fucoses improved the reactivity. The RBD produced in Lec3.2.8.1-cells, which generate carbohydrate structures devoid of sialic acids and with reduced fucose content, exhibited enhanced antibody reactivity, verifying the importance of these specific monosaccharides. The results can be of importance for the design of future vaccine candidates, indicating that it is possible to enhance the immunogenicity of recombinant viral proteins.
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| 9. |
- Bagdonaite, I., et al.
(författare)
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Global Mapping of O-Glycosylation of Varicella Zoster Virus, Human Cytomegalovirus, and Epstein-Barr Virus
- 2016
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 291:23, s. 12014-12028
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Tidskriftsartikel (refereegranskat)abstract
- Herpesviruses are among the most complex and widespread viruses, infection and propagation of which depend on envelope proteins. These proteins serve as mediators of cell entry as well as modulators of the immune response and are attractive vaccine targets. Although envelope proteins are known to carry glycans, little is known about the distribution, nature, and functions of these modifications. This is particularly true for O-glycans; thus we have recently developed a "bottom up" mass spectrometry-based technique for mapping O-glycosylation sites on herpes simplex virus type 1. We found wide distribution of O-glycans on herpes simplex virus type 1 glycoproteins and demonstrated that elongated O-glycans were essential for the propagation of the virus. Here, we applied our proteome-wide discovery platform for mapping O-glycosites on representative and clinically significant members of the herpesvirus family: varicella zoster virus, human cytomegalovirus, and Epstein-Barr virus. We identified a large number of O-glycosites distributed on most envelope proteins in all viruses and further demonstrated conserved patterns of O-glycans on distinct homologous proteins. Because glycosylation is highly dependent on the host cell, we tested varicella zoster virus-infected cell lysates and clinically isolated virus and found evidence of consistent O-glycosites. These results present a comprehensive view of herpesvirus O-glycosylation and point to the widespread occurrence of O-glycans in regions of envelope proteins important for virus entry, formation, and recognition by the host immune system. This knowledge enables dissection of specific functional roles of individual glycosites and, moreover, provides a framework for design of glycoprotein vaccines with representative glycosylation.
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| 10. |
- Bagdonaite, Ieva, et al.
(författare)
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Glycoengineered keratinocyte library reveals essential functions of specific glycans for all stages of HSV-1 infection
- 2023
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Ingår i: Nature Communications. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Viral and host glycans represent an understudied aspect of host-pathogen interactions, despite potential implications for treatment of viral infections. This is due to lack of easily accessible tools for analyzing glycan function in a meaningful context. Here we generate a glycoengineered keratinocyte library delineating human glycosylation pathways to uncover roles of specific glycans at different stages of herpes simplex virus type 1 (HSV-1) infectious cycle. We show the importance of cellular glycosaminoglycans and glycosphingolipids for HSV-1 attachment, N-glycans for entry and spread, and O-glycans for propagation. While altered virion surface structures have minimal effects on the early interactions with wild type cells, mutation of specific O-glycosylation sites affects glycoprotein surface expression and function. In conclusion, the data demonstrates the importance of specific glycans in a clinically relevant human model of HSV-1 infection and highlights the utility of genetic engineering to elucidate the roles of specific viral and cellular carbohydrate structures.
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