Mapping the N-Terminal Residues of Epstein-Barr Virus gp42 That Bind gH/gL by Using Fluorescence Polarization and Cell-Based Fusion Assays

Liu, Fengling; Marquardt, Gaby; Kirschner, Austin N.; Longnecker, Richard; Jardetzky, Theodore S.

doi:10.1128/jvi.00381-10

Cited by 24 publications

(46 citation statements)

References 30 publications

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“…This putative site for gp42 binding is near to the integrin binding loop, and competitive binding between gp42 and integrin to gH/gL would be consistent with the observation that gp42 and gp42-derived peptides block fusion with epithelial cells (9,13,14,16).…”

Section: Resultsmentioning

confidence: 52%

“…Q54 is surface-exposed and surrounded by other prominently displayed D-I residues, which together could form a gB binding site. The binding region of gp42 for gH/gL has been mapped to a 33-aa peptide (14). We have not yet identified the corresponding binding site on gH/gL, but the prominent groove be- (D) EBV gL Q54 and K94 (yellow sticks and boxes) control the specificity of gB activation in membrane fusion.…”

Section: Resultsmentioning

confidence: 99%

“…gp42 has multiple functional sites for interaction with gH/gL, HLA class II, and potentially, another unknown binding ligand that could be engaged through a large surface-exposed hydrophobic pocket (6)(7)(8)(9)(10)(11)(12). The gp42 protein binds to the gH/gL complex with nanomolar affinity through its N-terminal region, and this interaction can be recapitulated with a synthetic peptide of ∼35 aa residues (10,13,14). EBV glycoprotein-mediated membrane fusion with epithelial cells does not require gp42 but only gB and gH/gL, and fusion can be completely blocked by saturating amounts of either gp42 or short gp42-derived peptides (13)(14)(15)(16), consistent with the hypothesis that gp42 levels in the virion regulate the cellular tropism of the virus in vivo (16).…”

mentioning

confidence: 99%

“…The gp42 protein binds to the gH/gL complex with nanomolar affinity through its N-terminal region, and this interaction can be recapitulated with a synthetic peptide of ∼35 aa residues (10,13,14). EBV glycoprotein-mediated membrane fusion with epithelial cells does not require gp42 but only gB and gH/gL, and fusion can be completely blocked by saturating amounts of either gp42 or short gp42-derived peptides (13)(14)(15)(16), consistent with the hypothesis that gp42 levels in the virion regulate the cellular tropism of the virus in vivo (16). Recent observations indicate that EBV gH/gL engages integrins αvβ6 and/or αvβ8 on epithelial cells to trigger membrane fusion and entry (17).…”

mentioning

confidence: 99%

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Crystal structure of the Epstein-Barr virus (EBV) glycoprotein H/glycoprotein L (gH/gL) complex

Matsuura

Kirschner

Longnecker

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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151

205

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The Epstein-Barr virus (EBV) is a γ-herpesvirus that infects B cells and epithelial cells and that has been linked to malignancies in both cell types in vivo. EBV, like other herpesviruses, has three glycoproteins, glycoprotein B (gB), gH, and gL, that form the core membrane fusion machinery mediating viral penetration into the cell. The gH and gL proteins associate to form a heterodimeric complex, which is necessary for efficient membrane fusion and also implicated in direct binding to epithelial cell receptors required for viral entry. To gain insight into the mechanistic role of gH/gL, we determined the crystal structure of the EBV gH/gL complex. The structure is comprised of four domains organized along the longest axis of the molecule. Comparisons with homologous HSV-2 gH/gL and partial pseudorabies virus gH structures support the domain boundaries determined for the EBV gH/gL structure and illustrate significant differences in interdomain packing angles. The gL subunit and N-terminal residues of gH form a globular domain at one end of the structure, implicated in interactions with gB and activation of membrane fusion. The C-terminal domain of gH, proximal to the viral membrane, is also implicated in membrane fusion. The gH/gL structure locates an integrin binding motif, implicated in epithelial cell entry, on a prominent loop in the central region of the structure. Multiple regions of gH/gL, including its two extreme ends, are functionally important, consistent with the multiple roles of gH/gL in EBV entry.

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Section: Resultsmentioning

confidence: 52%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Crystal structure of the Epstein-Barr virus (EBV) glycoprotein H/glycoprotein L (gH/gL) complex

Matsuura

Kirschner

Longnecker

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

151

205

View full text Add to dashboard Cite

show abstract

“…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. 1C) (77,78). Similar to betaherpesviruses, gM-gN protein complex is particularly important for EBV viral particle formation (79).…”

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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A fluorescence polarization assay using an engineered human respiratory syncytial virus F protein as a direct screening platform

Park

Matsuura

Lamb

et al. 2011

Analytical Biochemistry

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Human respiratory syncytial virus (hRSV) typically affects newborns and young children. Even though it can cause severe and, in some cases, lifelong respiratory infections, there are currently no FDA-approved therapeutics that control this virus. The hRSV F protein facilitates viral fusion, a critical extracellular event that can be targeted for therapeutic intervention by disrupting the assembly of a post-fusion 6-Helix bundle (6HB) within the hRSV F protein. Here we report the development of a fluorescence polarization assay using an engineered hRSV F protein 5-Helix Bundle (5HB). We generated the 5HB and validated its ability to form a 6HB in a fluorescence polarization assay. To test the potential of 5HB as a screening tool, we then investigated a series of truncated peptides derived from the “missing” sixth helix. Using this FP-based 5HB system, we have successfully demonstrated that short peptides can prevent 6HB formation and serve as potential hRSV fusion inhibitors. We anticipate this new 5HB system will provide an effective tool to identify and study potential antivirals to control hRSV infection.

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Mapping the N-Terminal Residues of Epstein-Barr Virus gp42 That Bind gH/gL by Using Fluorescence Polarization and Cell-Based Fusion Assays

Abstract: Epstein-Barr virus (EBV

Cited by 24 publications

References 30 publications

Crystal structure of the Epstein-Barr virus (EBV) glycoprotein H/glycoprotein L (gH/gL) complex

Crystal structure of the Epstein-Barr virus (EBV) glycoprotein H/glycoprotein L (gH/gL) complex

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

A fluorescence polarization assay using an engineered human respiratory syncytial virus F protein as a direct screening platform

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