Glycoengineered keratinocyte library reveals essential functions of specific glycans for all stages of HSV-1 infection.

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.

Hepatitis E virus is an infrequent but potentially serious infection in allogeneic hematopoietic stem cell transplant recipients.

Hepatitis E virus (HEV) can cause chronic infection and liver cirrhosis in immunocompromised individuals. The frequency and clinical importance of HEV was studied retrospectively in a cohort of 236 Swedish allogeneic hematopoietic stem cell transplantation (HSCT) recipients. In blood samples collected at 6 months after HSCT, HEV RNA was identified in 8/236 (3.4%) patients, and 11/236 (4.7%) patients had detectable anti-HEV IgG and/or IgM, eight of whom were HEV RNA negative. Two of the patients with positive HEV RNA died with ongoing signs of hepatitis: one of acute liver and multiple organ failure, the other of unrelated causes. The remaining six patients with HEV RNA had cleared the infection at 7-24 (median 8.5) months after HSCT. HEV infection was associated with elevated alanine aminotransferase at 6 months after HSCT (OR 15, 1.3-174, p = 0.03). Active graft-versus-host disease of the liver at 6 months after HSCT was present in 3/8 (38%) patients with HEV RNA, but was not significantly associated with HEV infection. In conclusion, HEV infection is an important differential diagnosis in patients with elevated liver enzymes after HSCT. Although spontaneous clearance was common, the clinical course may be severe.

Recombinant Glycoprotein E of Varicella Zoster Virus Contains Glycan-Peptide Motifs That Modulate B Cell Epitopes into Discrete Immunological Signatures.

A recombinant subunit vaccine (Shingrix®) was recently licensed for use against herpes zoster. This vaccine is based on glycoprotein E (gE) of varicella zoster virus (VZV), the most abundantly expressed protein of VZV, harboring sites for N- and O-linked glycosylation. The subunit vaccine elicits stronger virus-specific CD4+ T cell response as well as antibody B cell response to gE, compared to the currently used live attenuated vaccine (Zostavax®). This situation is at variance with the current notion since a live vaccine, causing an active virus infection, should be far more efficient than a subunit vaccine based on only one single viral glycoprotein. We previously found gE to be heavily glycosylated, not least by numerous clustered O-linked glycans, when it was produced in human fibroblasts. However, in contrast to Zostavax®, which is produced in fibroblasts, the recombinant gE of Shingrix® is expressed in Chinese hamster ovary (CHO) cells. Hence, the glycan occupancy and glycan structures of gE may differ considerably between the two vaccine types. Here, we aimed at (i) defining the glycan structures and positions of recombinant gE and (ii) identifying possible features of the recombinant gE O-glycosylation pattern contributing to the vaccine efficacy of Shingrix®. Firstly, recombinant gE produced in CHO cells ("Shingrix situation") is more scarcely decorated by O-linked glycans than gE from human fibroblasts ("Zostavax situation"), with respect to glycan site occupancy. Secondly, screening of immunodominant B cell epitopes of gE, using a synthetic peptide library against serum samples from VZV-seropositive individuals, revealed that the O-linked glycan signature promoted binding of IgG antibodies via a decreased number of interfering O-linked glycans, but also via specific O-linked glycans enhancing antibody binding. These findings may, in part, explain the higher protective efficacy of Shingrix®, and can also be of relevance for development of subunit vaccines to other enveloped viruses.

Global Mapping of O-Glycosylation of Varicella Zoster Virus, Human Cytomegalovirus, and Epstein-Barr Virus.

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.

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.

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.

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 Thr-87 and Thr-91 and one GalNAc unit to either Thr-99 or Thr-101, forming a core glycopeptide for subsequent additions of in all 11 GalNAc residues to selected Ser and Thr residues of the Thr-76-Lys-107 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 followup 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. Because the GalNAc occupancy pattern is of relevance for receptor binding of gC-1, the data provide a model to delineate biosynthetic steps of O-linked glycosylation of the gC-1 mucin domain in HSV-1-infected target cells.

Involvement of viral glycoprotein gC-1 in expression of the selectin ligand sialyl-Lewis X induced after infection with herpes simplex virus type 1.

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.

Induction of sialyl-Lex expression by herpes simplex virus type 1 is dependent on viral immediate early RNA-activated transcription of host fucosyltransferase genes.

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.