Assembly and Architecture of the EBV B Cell Entry Triggering Complex

Sathiyamoorthy, Karthik; Jiang, Junchen; Hu, Yao Xiong; Rowe, Cynthia L.; Möhl, Britta S.; Chen, Jia; Jiang, Wei; Mellins, Elizabeth; Longnecker, Richard; Zhou, Z. Hong; Jardetzky, Theodore S.

doi:10.1371/journal.ppat.1004309

Cited by 80 publications

(114 citation statements)

References 53 publications

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“…Infection of B cells requires an additional viral protein gp42 (75). gp42 is proteolytically cleaved releasing a secreted form that links gH-gL on the virion to HLA class II on B cells, thereby bringing the two membranes to close proximity (76). A single O-glycosite was identified on gp42 localized in one of the regions essential for high affinity binding to gH-gL and just C-terminally to the proteolytic cleavage site required for release of gp42 from the membrane (Fig.…”

Section: Mapping O-glycosites In Human Herpesvirusesmentioning

confidence: 99%

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

Bagdonaite

Nordén

Joshi

et al. 2016

Journal of Biological Chemistry

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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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Section: Mapping O-glycosites In Human Herpesvirusesmentioning

confidence: 99%

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

Bagdonaite

Nordén

Joshi

et al. 2016

Journal of Biological Chemistry

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“…The structural features of D-II and D-III are necessary for the binding of EBV gH/gL to either the epithelial cell receptor or gp42 (14,(24)(25)(26). Interestingly, residues involved in the binding of gp42, forming the EBV B cell entry complex (26), are in close proximity to the conserved DB and its contacting amino acids. To test if the mutated amino acids are involved in cell-specific function of gH, we used a virus-free cellbased fusion assay with luciferase as a reporter.…”

Section: The Db In D-iii Of Prv and Ebv Gh Is Surrounded By A Group Omentioning

confidence: 99%

Comparative Mutagenesis of Pseudorabies Virus and Epstein-Barr Virus gH Identifies a Structural Determinant within Domain III of gH Required for Surface Expression and Entry Function

Möhl

Schröter

Klupp

et al. 2016

J Virol

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Herpesviruses infect cells using the conserved core fusion machinery composed of glycoprotein B (gB) and gH/gL. The gH/gL complex plays an essential but still poorly characterized role in membrane fusion and cell tropism. Our previous studies demonstrated that the conserved disulfide bond (DB) C278/C335 in domain II (D-II) of Epstein-Barr virus (EBV) gH has an epithelial cell-specific function, whereas the interface of D-II/D-III is involved in formation of the B cell entry complex by binding to gp42. To extend these studies, we compared gH of the alphaherpesvirus pseudorabies virus (PrV) with gH of the gammaherpesvirus EBV to identify functionally equivalent regions critical for gH function during entry. We identified several conserved amino acids surrounding the conserved DB that connects three central helices of D-III of PrV and EBV gH. The present study verified that the conserved DB and several contacting amino acids in D-III modulate cell surface expression and thereby contribute to gH function. In line with this finding, we found that DB C404/C439 and T401 are important for cell-to-cell spread and efficient entry of PrV. This parallel comparison between PrV and EBV gH function brings new insights into how gH structure impacts fusion function during herpesvirus entry. IMPORTANCEThe alphaherpesvirus PrV is known for its neuroinvasion, whereas the gammaherpesvirus EBV is associated with cancer of epithelial and B cell origin. Despite low amino acid conservation, PrV gH and EBV gH show strikingly similar structures. Interestingly, both PrV gH and EBV gH contain a structural motif composed of a DB and supporting amino acids which is highly conserved within the Herpesviridae. Our study verified that PrV gH uses a minimal motif with the DB as the core, whereas the DB of EBV gH forms extensive connections through hydrogen bonds to surrounding amino acids, ensuring the cell surface expression of gH/gL. Our study verifies that the comparative analysis of distantly related herpesviruses, such as PrV and EBV, allows the identification of common gH functions. In addition, we provide an understanding of how functional domains can evolve over time, resulting in subtle differences in domain structure and function.A variety of enveloped viruses enter host cells by using a core fusion machinery to facilitate the initial receptor-binding step, followed by fusion of the host cell membrane with the virion envelope. Most enveloped viruses use one or two surface glycoproteins to bind to receptor and mediate fusion. In contrast, the herpesvirus core fusion machinery consists of the heterodimeric complex gH/gL and the fusogen gB, with an additional receptorbinding protein such as gD in the alphaherpesvirus pseudorabies virus (PrV) or gp42 in the gammaherpesvirus Epstein-Barr virus (EBV) (1). For infectious entry, PrV requires gB and gH/gL as well as gD, which binds to specific host cell receptors such as nectin-1 and -2 and HveD (2), whereas EBV requires gB, gH/gL, and gp42 for B cell entry, with gp42 binding the receptor...

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“…Our recently published study determined the EBV B cell entry triggering complex composed of HLA class II, gp42, and gH/gL by negative-stain electron microscopy (EM) (40). The study characterized the interacting interface between the gp42 hydrophobic pocket and gH based on the EM model and was functionally confirmed by introducing potential glycosylation sites in D-II and D-III of the gH interface.…”

Section: Discussionmentioning

confidence: 99%

The Conserved Disulfide Bond within Domain II of Epstein-Barr Virus gH Has Divergent Roles in Membrane Fusion with Epithelial Cells and B Cells

Möhl

Sathiyamoorthy

Jardetzky

et al. 2014

J Virol

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Epstein-Barr virus (EBV) infects target cells via fusion with cellular membranes.For entry into epithelial cells, EBV requires the herpesvirus conserved core fusion machinery, composed of glycoprotein B (gB) and gH/gL. In contrast, for B cell fusion it requires gB and gH/gL with gp42 serving as a cell tropism switch. The available crystal structures for gH/gL allow the targeted analysis of structural determinants of gH to identify functional regions critical for membrane fusion. Domain II of EBV gH contains two disulfide bonds (DBs). The first is unique for EBV and closely related gammaherpesviruses. The second is conserved across the beta-and gammaherpesviruses and is positioned to stabilize a putative syntaxin-like bundle motif. To analyze the role of these DBs in membrane fusion, gH was mutated by amino acid substitution of the DB cysteines. Mutation of the EBV-specific DB resulted in diminished gH/gL cell surface expression that correlated with diminished B cell and epithelial cell fusion. In contrast, mutation of the conserved DB resulted in wild-type-like B cell fusion, whereas epithelial cell fusion was greatly reduced. The gH mutants bound well to gp42 but had diminished binding to epithelial cells. Tyrosine 336, located adjacent to cysteine 335 of the conserved DB, also was found to be important for DB stabilization and gH/gL function. We conclude that the conserved DB has a cell type-specific function, since it is important for the binding of gH to epithelial cells initiating epithelial cell fusion but not for fusion with B cells and gp42 binding. IMPORTANCE EBV predominantly infects epithelial and B cells in humans, which can result in EBV-associated cancers, such as Burkitt andHodgkin lymphoma, as well as nasopharyngeal carcinoma. EBV is also associated with a variety of lymphoproliferative disorders, typically of B cell origin, observed in immunosuppressed individuals, such as posttransplant or HIV/AIDS patients. The gH/gL complex plays an essential but still poorly characterized role as an important determinant for EBV cell tropism. In the current studies, we found that mutants in the DB C278/C335 and the neighboring tyrosine 336 have cell type-specific functional deficits with selective decreases in epithelial cell, but not B cell, binding and fusion. The present study brings new insights into the gH function as a determinant for epithelial cell tropism during herpesvirus-induced membrane fusion and highlights a specific gH motif required for epithelial cell fusion. E pstein-Barr virus (EBV) causes infectious mononucleosis in young adults or adolescents, resulting in lifelong persistence following primary infection, which can lead to the development of various malignancies in B lymphocytes or epithelial cells (1). EBV enters epithelial cells via plasma membrane fusion, requiring the highly conserved core fusion machinery composed of gB and the heterodimeric gH/gL complex (1-3). In contrast, EBV infects B cells via endocytosis and, in addition to gB and gH/gL, requires gp42, which acts as ...

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Assembly and Architecture of the EBV B Cell Entry Triggering Complex

Cited by 80 publications

References 53 publications

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

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

Comparative Mutagenesis of Pseudorabies Virus and Epstein-Barr Virus gH Identifies a Structural Determinant within Domain III of gH Required for Surface Expression and Entry Function

The Conserved Disulfide Bond within Domain II of Epstein-Barr Virus gH Has Divergent Roles in Membrane Fusion with Epithelial Cells and B Cells

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