Soluble Epstein-Barr Virus Glycoproteins gH, gL, and gp42 Form a 1:1:1 Stable Complex That Acts Like Soluble gp42 in B-Cell Fusion but Not in Epithelial Cell Fusion

ABSTRACT Epstein-Barr virus (EBV) is a herpesvirus that infects cells by fusing its lipid envelope with the target cell membrane. The fusion process requires the actions of viral glycoproteins gH, gL, and gB for entry into epithelial cells and additionally requires gp42 for entry into B cells. To further study the roles of these membrane-associated glycoproteins, purified soluble forms of gp42, gH, and gL were expressed that lack the membrane-spanning regions. The soluble gH/gL protein complex binds to soluble gp42 with high affinity, forming a stable heterotrimer with 1:1:1 stoichiometry, and this complex is not formed by an N-terminally truncated variant of gp42. The effects of adding soluble gp42, gH/gL, and gH/gL/gp42 were examined with a virus-free cell-cell fusion assay. The results demonstrate that, in contrast to gp42, membrane fusion does not proceed with secreted gH/gL. The addition of soluble gH/gL does not inhibit or enhance B-cell or epithelial cell fusion when membrane-bound gH/gL, gB, and gp42 are present. However, the soluble gH/gL/gp42 complex does activate membrane fusion with B cells, similarly to soluble gp42, but it does not inhibit fusion with epithelial cells, as observed for gp42 alone. A gp42 peptide, derived from an N-terminal segment involved in gH/gL interactions, binds to soluble gH/gL and inhibits EBV-mediated epithelial cell fusion, mimicking gp42. These observations reveal distinct functional requirements for gH/gL and gp42 complexes in EBV-mediated membrane fusion.

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Mutations of Epstein-Barr Virus gH That Are Differentially Able To Support Fusion with B Cells or Epithelial Cells

Journal of Virology ◽

10.1128/jvi.79.17.10923-10930.2005 ◽

2005 ◽

Vol 79 (17) ◽

pp. 10923-10930 ◽

Cited By ~ 37

Author(s):

Liguo Wu ◽

Corina M. Borza ◽

Lindsey M. Hutt-Fletcher

Keyword(s):

B Cells ◽

Epithelial Cell ◽

Epithelial Cells ◽

B Cell ◽

Cell Fusion ◽

Epstein Barr Virus ◽

Common Mechanism ◽

Barr Virus ◽

The Core ◽

Epstein Barr

ABSTRACT The core fusion machinery of all herpesviruses consists of three conserved glycoproteins, gB and gHgL, suggesting a common mechanism for virus cell fusion, but fusion of Epstein-Barr virus (EBV) with B cells and epithelial cells is initiated differently. Fusion with B cells requires a fourth protein, gp42, which complexes with gHgL and interacts with HLA class II, the B-cell coreceptor. Fusion with an epithelial cell does not require gp42 but requires interaction of gHgL with a novel epithelial cell coreceptor. Epithelial cell fusion can be inhibited by gp42 binding to gHgL and by antibodies to gH that fail to block B-cell fusion. This suggests that regions of gHgL initiating fusion with each cell are separable from each other and from regions involved in fusion itself. To address this possibility we mapped the region of gH recognized by a monoclonal antibody to gH that blocks EBV fusion with epithelial cells but not B cells by making a series of chimeras with the gH homolog of rhesus lymphocryptovirus. Proteins with mutations engineered within this region included those that preferentially mediate fusion with B cells, those that preferentially mediate fusion with epithelial cells, and those that mediate fusion with neither cell type. These results support the hypothesis that the core fusion function of gH is the same for B cells and epithelial cells and that it differs only in the way in which it is triggered into a functionally active state.

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Compatibility of the gH homologues of Epstein–Barr virus and related lymphocryptoviruses

Journal of General Virology ◽

10.1099/vir.0.82949-0 ◽

2007 ◽

Vol 88 (8) ◽

pp. 2129-2136 ◽

Cited By ~ 7

Author(s):

Liguo Wu ◽

Lindsey M. Hutt-Fletcher

Keyword(s):

B Cells ◽

Epithelial Cells ◽

B Cell ◽

Cell Fusion ◽

Epstein Barr Virus ◽

Common Marmoset ◽

Barr Virus ◽

Human B Cells ◽

The Common ◽

Epstein Barr

Glycoprotein gH, together with its chaperone gL and a third glycoprotein gB, is essential for cell–cell fusion and virus–cell fusion mediated by herpesviruses. Epstein–Barr virus (EBV), the prototype human lymphocryptovirus, requires a fourth glycoprotein gp42 to support fusion with B cells in addition to epithelial cells. Two other lymphocryptoviruses, the rhesus lymphocryptovirus (Rh-LCV) and the common marmoset lymphocryptovirus (CalHV3), have been sequenced in their entirety and each has a gp42 homologue. Combinations of proteins from EBV, Rh-LCV and CalHV3 were able to mediate fusion of epithelial cells, but, even when complexed with EBV gp42, only Rh-LCV and not CalHV3 proteins were able to mediate fusion with human B cells. CalHV3 gL was also unable to function effectively as a chaperone for EBV or Rh-LCV gH. The Rh-LCV gH homologue supported more fusion than EBV gH with an epithelial cell and supported the highest levels of fusion with a B cell. Chimeric constructs made from Rh-LCV gH and EBV gH that have 85.4 % sequence identity should prove useful for mapping the regions of gH that are of importance to fusion as a whole and to B-cell fusion in particular.

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Binding-Site Interactions between Epstein-Barr Virus Fusion Proteins gp42 and gH/gL Reveal a Peptide That Inhibits both Epithelial and B-Cell Membrane Fusion

Journal of Virology ◽

10.1128/jvi.00575-07 ◽

2007 ◽

Vol 81 (17) ◽

pp. 9216-9229 ◽

Cited By ~ 38

Author(s):

Austin N. Kirschner ◽

Amanda S. Lowrey ◽

Richard Longnecker ◽

Theodore S. Jardetzky

Keyword(s):

Epithelial Cell ◽

Cell Membrane ◽

B Cell ◽

Membrane Fusion ◽

Cell Fusion ◽

Epstein Barr Virus ◽

Terminal Region ◽

Deletion Mutants ◽

Barr Virus ◽

Epstein Barr

ABSTRACT Herpesviruses require membrane-associated glycoproteins gB, gH, and gL for entry into host cells. Epstein-Barr virus (EBV) gp42 is a unique protein also required for viral entry into B cells. Key interactions between EBV gp42 and the EBV gH/gL complex were investigated to further elucidate their roles in membrane fusion. Deletion and point mutants within the N-terminal region of gp42 revealed residues important for gH/gL binding and membrane fusion. Many five-residue deletion mutants in the N-terminal region of gp42 that exhibit reduced membrane fusion activity retain binding with gH/gL but map out two functional stretches between residues 36 and 96. Synthetic peptides derived from the gp42 N-terminal region were studied in in vitro binding experiments with purified gH/gL and in cell-cell fusion assays. A peptide spanning gp42 residues 36 to 81 (peptide 36-81) binds gH/gL with nanomolar affinity, comparable to full-length gp42. Peptide 36-81 efficiently inhibits epithelial cell membrane fusion and competes with soluble gp42 to inhibit B-cell fusion. Additionally, this peptide at low nanomolar concentrations inhibits epithelial cell infection by intact virus. Shorter gp42 peptides spanning the two functional regions identified by deletion mutagenesis had little or no binding to soluble gH/gL and were also unable to inhibit epithelial cell fusion, nor could they complement gp42 deletion mutants in B-cell fusion. These studies identify key residues of gp42 that are essential for gH/gL binding and membrane fusion activation, providing a nanomolar inhibitor of EBV-mediated membrane fusion.

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The KGD Motif of Epstein-Barr Virus gH/gL Is Bifunctional, Orchestrating Infection of B Cells and Epithelial Cells

mBio ◽

10.1128/mbio.00290-11 ◽

2012 ◽

Vol 3 (1) ◽

Cited By ~ 32

Author(s):

Jia Chen ◽

Cynthia L. Rowe ◽

Theodore S. Jardetzky ◽

Richard Longnecker

Keyword(s):

Amino Acids ◽

B Cells ◽

Epithelial Cell ◽

Epithelial Cells ◽

Membrane Fusion ◽

Epstein Barr Virus ◽

Cell Infection ◽

Induced Membrane ◽

Barr Virus ◽

Epstein Barr

ABSTRACTEpstein-Barr virus (EBV), a member of the herpesvirus family, is the causative agent of common human infections and specific malignancies. EBV entry into target cells, including B cells and epithelial cells, requires the interaction of multiple virus-encoded glycoproteins. Glycoproteins H and L (gH/gL) cooperate with glycoprotein B (gB) to mediate fusion of the viral envelope with target cell membranes. Both the gH/gL complex and gB are required for fusion, whereas glycoprotein 42 (gp42) acts as a tropism switch and is required for B cell infection and inhibits epithelial cell infection. Our previous studies identified a prominent KGD motif located on the surface of gH/gL. In the current study, we found that this motif serves as a bifunctional domain on the surface of gH/gL that directs EBV fusion of B cells and epithelial cells. Mutation of the KGD motif to AAA decreased fusion with both epithelial and B cells and reduced the binding of gH/gL to epithelial cells and to gp42. We also demonstrate that deletion of amino acids 62 to 66 of gp42 selectively reduces binding to wild-type gH/gL, but not the KGD mutant, suggesting that the KGD motif of gH/gL interacts with the N-terminal amino acids 62 to 66 of gp42.IMPORTANCEEpithelial and B cells are the major targets of Epstein-Barr virus (EBV) infection in the human host. EBV utilizes different glycoprotein complexes to enter these cell types. For B cell fusion, EBV uses complexes containing gp42, gH/gL, and gB, whereas just gH/gL and gB are required for epithelial cell fusion. In the current study, a bifunctional domain consisting of a prominent KGD motif on the surface of the gH/gL structure was identified; this domain affects interactions with gp42 or epithelial receptors, ultimately dictating with which cell type virus-induced fusion can occur. These studies will lead to a better understanding of the mechanism of EBV-induced membrane fusion and herpesvirus-induced membrane fusion in general.

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Hippo signaling effectors YAP and TAZ induce Epstein-Barr Virus (EBV) lytic reactivation through TEADs in epithelial cells

PLoS Pathogens ◽

10.1371/journal.ppat.1009783 ◽

2021 ◽

Vol 17 (8) ◽

pp. e1009783

Author(s):

Nicholas Van Sciver ◽

Makoto Ohashi ◽

Nicholas P. Pauly ◽

Jillian A. Bristol ◽

Scott E. Nelson ◽

...

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

B Cell ◽

Epstein Barr Virus ◽

Hippo Signaling ◽

Oral Keratinocytes ◽

Barr Virus ◽

Ebv Infection ◽

Lytic Reactivation ◽

Epstein Barr

The Epstein-Barr virus (EBV) human herpesvirus is associated with B-cell and epithelial-cell malignancies, and both the latent and lytic forms of viral infection contribute to the development of EBV-associated tumors. Here we show that the Hippo signaling effectors, YAP and TAZ, promote lytic EBV reactivation in epithelial cells. The transcriptional co-activators YAP/TAZ (which are inhibited by Hippo signaling) interact with DNA-binding proteins, particularly TEADs, to induce transcription. We demonstrate that depletion of either YAP or TAZ inhibits the ability of phorbol ester (TPA) treatment, cellular differentiation or the EBV BRLF1 immediate-early (IE) protein to induce lytic EBV reactivation in oral keratinocytes, and show that over-expression of constitutively active forms of YAP and TAZ reactivate lytic EBV infection in conjunction with TEAD family members. Mechanistically, we find that YAP and TAZ interact with, and activate, the EBV BZLF1 immediate-early promoter. Furthermore, we demonstrate that YAP, TAZ, and TEAD family members are expressed at much higher levels in epithelial cell lines in comparison to B-cell lines, and find that EBV infection of oral keratinocytes increases the level of activated (dephosphorylated) YAP and TAZ. Finally, we have discovered that lysophosphatidic acid (LPA), a known YAP/TAZ activator that plays an important role in inflammation, induces EBV lytic reactivation in epithelial cells through a YAP/TAZ dependent mechanism. Together these results establish that YAP/TAZ are powerful inducers of the lytic form of EBV infection and suggest that the ability of EBV to enter latency in B cells at least partially reflects the extremely low levels of YAP/TAZ and TEADs in this cell type.

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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

Journal of Virology ◽

10.1128/jvi.00381-10 ◽

2010 ◽

Vol 84 (19) ◽

pp. 10375-10385 ◽

Cited By ~ 18

Author(s):

Fengling Liu ◽

Gaby Marquardt ◽

Austin N. Kirschner ◽

Richard Longnecker ◽

Theodore S. Jardetzky

Keyword(s):

Epithelial Cell ◽

Viral Infection ◽

B Cell ◽

Cell Fusion ◽

Viral Entry ◽

Fluorescence Polarization ◽

Epstein Barr Virus ◽

Host Cells ◽

Barr Virus ◽

Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) requires at a minimum membrane-associated glycoproteins gB, gH, and gL for entry into host cells. B-cell entry additionally requires gp42, which binds to gH/gL and triggers viral entry into B cells. The presence of soluble gp42 inhibits membrane fusion with epithelial cells by forming a stable heterotrimer of gH/gL/gp42. The interaction of gp42 with gH/gL has been previously mapped to residues 36 to 81 at the N-terminal region of gp42. In this study, we further mapped this region to identify essential features for binding to gH/gL by use of synthetic peptides. Data from fluorescence polarization, cell-cell fusion, and viral infection assays demonstrated that 33 residues corresponding to 44 to 61 and 67 to 81 of gp42 were indispensable for maintaining low-nanomolar-concentration gH/gL binding affinity and inhibiting B-cell fusion and epithelial cell fusion as well as viral infection. Overall, specific, large hydrophobic side chain residues of gp42 appeared to provide critical interactions, determining the binding strength. Mutations of these residues also diminished the inhibition of B-cell and epithelial cell fusions as well as EBV infection. A linker region (residues 62 to 66) between two gH/gL binding regions served as an important spacer, but individual amino acids were not critical for gH/gL binding. Probing the binding site of gH/gL and gp42 with gp42 peptides is critical for a better understanding of the interaction of gH/gL with gp42 as well as for the design of novel entry inhibitors of EBV and related human herpesviruses.

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Epstein-Barr Virus Fusion with Epithelial Cells Triggered by gB Is Restricted by a gL Glycosylation Site

Journal of Virology ◽

10.1128/jvi.01255-17 ◽

2017 ◽

Vol 91 (23) ◽

Cited By ~ 3

Author(s):

Britta S. Möhl ◽

Jia Chen ◽

Seo Jin Park ◽

Theodore S. Jardetzky ◽

Richard Longnecker

Keyword(s):

Epithelial Cell ◽

Epithelial Cells ◽

Membrane Fusion ◽

Cell Fusion ◽

Cell Receptor ◽

Glycosylation Site ◽

Fusion Activity ◽

Barr Virus ◽

Epstein Barr ◽

Kinetics Of

ABSTRACT Epstein-Barr virus (EBV) entry into epithelial cells is mediated by the conserved core fusion machinery, composed of the fusogen gB and the receptor-binding complex gH/gL. The heterodimeric gH/gL complex binds to the EBV epithelial cell receptor or gp42, which binds to the B-cell receptor, triggering gB-mediated fusion of the virion envelope with cellular membranes. Our previous study found that the gL glycosylation mutant N69L/S71V had an epithelial cell-specific hyperfusogenic phenotype. To study the influence of this gL mutant on the initiation and kinetics of gB-driven epithelial cell fusion, we established a virus-free split-green fluorescent protein cell-cell fusion assay that enables real-time measurements of membrane fusion using live cells. The gL_N69L/S71V mutant had a large increase in epithelial cell fusion activity of up to 300% greater than that of wild-type gL starting at early time points. The hyperfusogenicity of the gL mutant was not a result of alterations in complex formation with gH or alterations in cellular localization. Moreover, the hyperfusogenic phenotype of the gL mutant correlated with the formation of enlarged syncytia. In summary, our present findings highlight an important role of gL in the kinetics of gB-mediated epithelial cell fusion, adding to previous findings indicating a direct interaction between gL and gB in EBV membrane fusion. IMPORTANCE EBV predominantly infects epithelial cells and B lymphocytes, which are the cells of origin for the EBV-associated malignancies Hodgkin and Burkitt lymphoma as well as nasopharyngeal carcinoma. Contrary to the other key players of the core fusion machinery, gL has the most elusive role during EBV-induced membrane fusion. We found that the glycosylation site N69/S71 of gL is involved in restricting epithelial cell fusion activity, strongly correlating with syncytium size. Interestingly, our data showed that the gL glycosylation mutant increases the fusion activity of the hyperfusogenic gB mutants, indicating that this gL mutant and the gB mutants target different steps during fusion. Our studies on how gL and gB work together to modulate epithelial cell fusion kinetics are essential to understand the highly tuned tropism of EBV for epithelial cells and B lymphocytes and may result in novel strategies for therapies preventing viral entry into target host cells. Finally, making our results of particular interest is the absence of gL syncytial mutants in other herpesviruses.

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The Amino Terminus of Epstein-Barr Virus Glycoprotein gH Is Important for Fusion with Epithelial and B Cells

Journal of Virology ◽

10.1128/jvi.79.19.12408-12415.2005 ◽

2005 ◽

Vol 79 (19) ◽

pp. 12408-12415 ◽

Cited By ~ 52

Author(s):

Jasmina Omerović ◽

Lori Lev ◽

Richard Longnecker

Keyword(s):

B Cells ◽

Epithelial Cells ◽

Membrane Fusion ◽

B Lymphocytes ◽

Epstein Barr Virus ◽

Cell Types ◽

Fusion Activity ◽

Barr Virus ◽

N Terminus ◽

Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) infects B lymphocytes and epithelial cells. While the glycoproteins required for entry into these two cell types differ, the gH/gL glycoprotein complex is essential for entry into both epithelial and B cells. Analysis of gH protein sequences from three gammaherpesviruses (EBV, marmoset, and rhesus) revealed a potential coiled-coil domain in the N terminus. Four leucines located in this region in EBV gH were replaced by alanines by site-directed mutagenesis and analyzed for cell-cell membrane fusion with B cells and epithelial cells. Reduction in fusion activity was observed for mutants containing L65A and/or L69A mutations, while substitutions in L55 and L74 enhanced the fusion activity of the mutant gH/gL complexes with both cell types. All of the mutants displayed levels of cell surface expression similar to those of wild-type gH and interacted with gL and gp42. The observation that a conservative mutation of leucine to alanine in the N terminus of EBV gH results in fusion-defective mutant gH/gL complexes is striking and points to an important role for this region in EBV-mediated membrane fusion with B lymphocytes and epithelial cells.

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Membrane Anchoring of Epstein-Barr Virus gp42 Inhibits Fusion with B Cells Even with Increased Flexibility Allowed by Engineered Spacers

mBio ◽

10.1128/mbio.02285-14 ◽

2015 ◽

Vol 6 (1) ◽

Cited By ~ 4

Author(s):

Cynthia L. Rowe ◽

Jia Chen ◽

Theodore S. Jardetzky ◽

Richard Longnecker

Keyword(s):

B Cells ◽

B Cell ◽

Cell Fusion ◽

Steric Hindrance ◽

Epstein Barr Virus ◽

Class Ii ◽

Hla Class Ii ◽

Barr Virus ◽

Membrane Bound ◽

Epstein Barr

ABSTRACTWe recently described the architecture of the Epstein-Barr virus (EBV) fusion-triggering complex consisting of the EBV B cell receptor human leukocyte antigen (HLA) class II and the EBV-encoded proteins gp42 and gH/gL. The architecture of this structure positioned the main body of gp42, comprising the C-type lectin domain (CTLD), away from the membrane and distant from where the membrane-bound form of gp42 might be tethered. gp42 is a type II membrane glycoprotein, with functional gp42 formed by cleavage near the gp42 amino-terminal transmembrane domain. This cleavage results in an approximately 50-amino-acid unstructured region that is responsible for binding gH/gL with nanomolar affinity. Our previous studies had shown that membrane-bound gp42 is not functional in B cell fusion. To investigate whether we could restore gp42 function by extending it from the membrane, we introduced one, two, and four structured immunoglobulin-like domains from muscle protein titin into a membrane-bound form of gp42 and tested function in binding to gHgL and HLA class II and function in fusion. We hypothesized that cleavage of gp42 generates a soluble functional form that relieves steric hindrance imposed on gHgL by membrane-bound gp42. All of the linker mutants had a dominant-negative effect on gp42 function, indicating that gp42 fusion function could not be restored simply by the addition of one to four titin domains.IMPORTANCEEpstein-Barr virus (EBV) is associated with numerous diseases from benign mononucleosis to Burkitt’s and Hodgkin’s lymphoma, nasopharyngeal and gastric carcinoma, and lymphoproliferative disorders in patients with immune dysfunction resulting from immune suppression. Among the glycoproteins important for fusion, gp42, along with gH/gL, determines EBV tropism between epithelial and B cells. The function of gp42 is dependent on N-terminal cleavage, since membrane-bound gp42 cannot mediate fusion. We further investigated whether insertion of a linker into membrane-bound gp42 would relieve steric hindrance imposed on membrane-bound gp42 and restore fusion function. However, adding one, two, or four structured immunoglobulin-like domains to membrane gp42 did not restore fusion activity, indicating that the architecture and membrane orientation of the B cell fusion-triggering complex of EBV may be easily perturbed and that gp42 cleavage is essential for B cell fusion.

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ΔNp63α promotes Epstein-Barr virus latency in undifferentiated epithelial cells

PLoS Pathogens ◽

10.1371/journal.ppat.1010045 ◽

2021 ◽

Vol 17 (11) ◽

pp. e1010045

Author(s):

Nicholas Van Sciver ◽

Makoto Ohashi ◽

Dhananjay M. Nawandar ◽

Nicholas P. Pauly ◽

Denis Lee ◽

...

Keyword(s):

Transcription Factors ◽

Nasopharyngeal Carcinoma ◽

Epithelial Cell ◽

Epithelial Cells ◽

B Cell ◽

Epstein Barr Virus ◽

Viral Latency ◽

Human Herpesvirus ◽

Barr Virus ◽

Epstein Barr

Epstein-Barr virus (EBV) is a human herpesvirus that causes infectious mononucleosis and contributes to both B-cell and epithelial-cell malignancies. EBV-infected epithelial cell tumors, including nasopharyngeal carcinoma (NPC), are largely composed of latently infected cells, but the mechanism(s) maintaining viral latency are poorly understood. Expression of the EBV BZLF1 (Z) and BRLF1 (R) encoded immediate-early (IE) proteins induces lytic infection, and these IE proteins activate each other’s promoters. ΔNp63α (a p53 family member) is required for proliferation and survival of basal epithelial cells and is over-expressed in NPC tumors. Here we show that ΔNp63α promotes EBV latency by inhibiting activation of the BZLF1 IE promoter (Zp). Furthermore, we find that another p63 gene splice variant, TAp63α, which is expressed in some Burkitt and diffuse large B cell lymphomas, also represses EBV lytic reactivation. We demonstrate that ΔNp63α inhibits the Zp promoter indirectly by preventing the ability of other transcription factors, including the viral IE R protein and the cellular KLF4 protein, to activate Zp. Mechanistically, we show that ΔNp63α promotes viral latency in undifferentiated epithelial cells both by enhancing expression of a known Zp repressor protein, c-myc, and by decreasing cellular p38 kinase activity. Furthermore, we find that the ability of cis-platinum chemotherapy to degrade ΔNp63α contributes to the lytic-inducing effect of this agent in EBV-infected epithelial cells. Together these findings demonstrate that the loss of ΔNp63α expression, in conjunction with enhanced expression of differentiation-dependent transcription factors such as BLIMP1 and KLF4, induces lytic EBV reactivation during normal epithelial cell differentiation. Conversely, expression of ΔNp63α in undifferentiated nasopharyngeal carcinoma cells and TAp63α in Burkitt lymphoma promotes EBV latency in these malignancies.

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