Use of gHgL for Attachment of Epstein-Barr Virus to Epithelial Cells Compromises Infection

Borza, Corina M.; Morgan, Andrew; Turk, Susan M.; Hutt-Fletcher, Lindsey

doi:10.1128/jvi.78.10.5007-5014.2004

Cited by 66 publications

(85 citation statements)

References 42 publications

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“…Nearby mutations in gH residues 65 and 69 disrupt E1D1 antibody binding. E1D1 inhibits membrane fusion with epithelial cells (but not B cells) (36) and blocks gH binding to integrin (37), consistent with the possibility that E1D1 and integrins may bind overlapping surfaces in this junctional region between D-I and D-II.…”

Section: Resultsmentioning

confidence: 66%

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.

146

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: 66%

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.

146

205

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“…Binding of gHtgL to AGS cells was done as described previously (5). Briefly, AGS cells were trypsinized, allowed to recover for 1 h at 37°C in growth medium, and incubated for 1 h with protein on ice.…”

Section: Methodsmentioning

confidence: 99%

“…What may be a "syntaxin-like bundle" has been identified in domain II (2), and the integrin binding site of EBV gHgL is also in a prominent loop in domain II (27). The monoclonal antibody E1D1 reduces binding to integrins (5), binds only to gH complexed with gL, and has a reduced ability to recognize gH mutated at residue 65, located in the domain I-domain II linker helix (34). The antibody thus presumably binds surfaces at the junctional region between domain I, which comprises the amino-terminal 65 residues of gH and the entirety of gL, and domain II.…”

mentioning

confidence: 99%

“…The antibody thus presumably binds surfaces at the junctional region between domain I, which comprises the amino-terminal 65 residues of gH and the entirety of gL, and domain II. The putative binding site for gp42 is also probably nearby, given the ability of gp42 to block integrin binding (5). Mutations that map to the domain I-domain II interface affect fusion (34), and it has been suggested that a domain I-domain II conformational change could be part of the mechanism for triggering membrane fusion (27).…”

mentioning

confidence: 99%

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Fusion of Epstein-Barr Virus with Epithelial Cells Can Be Triggered by αvβ5 in Addition to αvβ6 and αvβ8, and Integrin Binding Triggers a Conformational Change in Glycoproteins gHgL

Chesnokova

Hutt-Fletcher

2011

J Virol

Self Cite

114

128

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Fusion of herpesviruses with their target cells requires a minimum of three glycoproteins, namely, gB and a complex of gH and gL. Epstein-Barr virus (EBV) fusion with an epithelial cell requires no additional virus glycoproteins, and we have shown previously that it can be initiated by an interaction between integrin ␣v␤6 or ␣v␤8 and gHgL. We now report that integrin ␣v␤5 can also bind to gHgL and trigger fusion. Binding of gHgL to integrins is a two-step reaction. The first step, analyzed by surface plasmon resonance, was fast, with high association and low dissociation rate constants. The second step, detected by fluorescence spectroscopy of gHgL labeled at cysteine 153 at the domain I-domain II interface with the environmentally sensitive probes acrylodan and IANBD, involved a slower conformational change. Interaction of gHgL with neutralizing monoclonal antibodies or Fab fragments was also consistent with a two-step reaction involving fast high-affinity binding and a subsequent slower conformational change. None of the antibodies bound to the same epitope, and none completely inhibited integrin binding. However, binding of each decreased the rate of conformational change induced by integrin binding, suggesting that neutralization might involve a conformational change that precludes fusion. Overall, the data are consistent with the interaction of gHgL with an integrin inducing a functionally important rearrangement at the domain I-domain II interface. Epstein-Barr virus (EBV)is an orally transmitted human gammaherpesvirus that is carried by the majority of the adult population worldwide. Many primary infections are asymptomatic, but the virus is an etiologic agent of infectious mononucleosis and is also associated with and implicated in development of both lymphoid and epithelial malignancies (reviewed in reference 38). B lymphocytes and epithelial cells are its primary targets.Like all herpesviruses, EBV enters its target cells by fusion. Fusion with a B cell first requires endocytosis, is sensitive to chlorpromazine, and occurs at acidic pH. Fusion with an epithelial cell is not sensitive to chlorpromazine and occurs at neutral pH, possibly at the cell surface (28). In both cases, it requires the activities of trimers of the glycoprotein gB and heterodimers of the glycoproteins gH and gL (18). Homologs of these three glycoproteins are thought to be responsible for fusion of all herpesviruses and are referred to as the core fusion machinery (45). Glycoprotein B has been posited to be the protein that is proximal to the fusion event, in large part because of its structural similarity to the vesicular stomatitis virus fusion protein G (3, 16, 39), but full fusion activity is achieved only in the presence of gHgL.One of the major differences in entry of individual herpesviruses into various cell types is the way in which the core fusion machinery is initially activated. Several herpesviruses, including herpes simplex virus (HSV) and EBV, can require a fourth protein for activation of fusion. Herpes simplex viru...

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“…A soluble form of gL fused to the constant region fragment of IgG bound better to human embryonic kidney (HEK)-293, AGS, and NU-GC-3 cell lines when compared with the B cell lines, Raji and BJAB (17). Recently, soluble gH͞gL was found to bind to both AGS and SVKCR2 cell lines, but not EBV-negative Akata cells, suggesting a specific receptor for gH͞gL (gHgLR) is present on epithelial cells (18). Despite the conservation of gH and gL, the only binding partner of this complex identified thus far is the binding of human herpesvirus (HHV)-6 to CD46 (19), but this does not appear to be functionally significant.…”

mentioning

confidence: 99%

Cell-surface expression of a mutated Epstein–Barr virus glycoprotein B allows fusion independent of other viral proteins

McShane

Longnecker

2004

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

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Epstein-Barr virus (EBV) infects human B lymphocytes and epithelial cells. We have compared the requirements for EBV glycoprotein-induced cell fusion between Chinese hamster ovary effecter cells and human B lymphoblasts or epithelial cells by using a virus-free cell fusion assay. EBV-encoded gB, gH, gL, and gp42 glycoproteins were required for efficient B cell fusion, whereas EBV gB, gH, and gL glycoproteins were required for Chinese hamster ovary effecter cell fusion with epithelial cell lines (AGS and SCC68) or the human embryonic kidney cell line 293-P. Fusion with human embryonic kidney 293-P cells was greater than fusion observed with B cells, indicative of an important role for cell contact. An antibody directed against the gH and gL complex inhibited epithelial cell fusion. Increased surface expression of gB alone as a result of truncations or point mutants in the carboxyl-terminal tail allowed gB-mediated fusion with epithelial cells, albeit at a lower level than with coexpression of gB, gH, and gL. Overall, gB appears to be the critical component for EBV glycoprotein-mediated cell fusion. viral entry ͉ herpesvirus T he entry process of human herpesviruses entails a two-step process: binding followed by fusion. Specific herpesvirusencoded glycoproteins and cell-surface receptors are important for both events (1-3). Entry may occur by free virus infecting a cell or by cell-cell spread (2). In either case, fusion is a prerequisite for infection. Despite herpesvirus genomes encoding many viral glycoproteins, the glycoproteins implicated in entry and fusion are limited and conserved (1). Most herpesviruses require the conserved glycoproteins gH, gL, and gB, as well as an additional, family-specific viral glycoprotein, such as gD for ␣-herpesviruses or gp42 for the ␥-herpesvirus Epstein-Barr virus (EBV), for efficient entry and fusion (1). These nonconserved glycoproteins result in cell specificity by recognizing distinct cellular receptors. For example, EBV glycoprotein 42 binds to HLA class II on B cells, an interaction essential for the infection of B cells (4, 5). Additionally, EBV gp350͞220 binds to the B cell-surface receptor CD21͞CR2, and this binding mediates the initial interaction of EBV with B cells and is thought to tether the virus to promote the subsequent binding of gp42 to HLA class II (6, 7). The gp42-class II interaction is then proposed to trigger membrane fusion, a reaction mediated by gH (EBV gp85), gL (EBV gp25), and gB (EBV gp110) (8). The exact role each of these glycoproteins plays in fusion is unclear.EBV can enter cells by means of two routes, depending on the host cell type. In primary human lymphocytes, EBV is endocytosed before fusion, whereas, in B cells in culture, the virion envelope directly fuses with the plasma membrane (9, 10). Similarly to B cells in culture, fusion of EBV with epithelial cells occurs by direct fusion of the virion envelope with the plasma membrane. Despite B lymphocytes being the primary site for latency, the tropism of EBV for epithelial cells is suppor...

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Use of gHgL for Attachment of Epstein-Barr Virus to Epithelial Cells Compromises Infection

Cited by 66 publications

References 42 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

Fusion of Epstein-Barr Virus with Epithelial Cells Can Be Triggered by αvβ5 in Addition to αvβ6 and αvβ8, and Integrin Binding Triggers a Conformational Change in Glycoproteins gHgL

Cell-surface expression of a mutated Epstein–Barr virus glycoprotein B allows fusion independent of other viral proteins

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