scholarly journals Different Functional Domains in the Cytoplasmic Tail of Glycoprotein B Are Involved in Epstein–Barr Virus-Induced Membrane Fusion

Virology ◽  
2001 ◽  
Vol 290 (1) ◽  
pp. 106-114 ◽  
Author(s):  
Keith M. Haan ◽  
Suk Kyeong Lee ◽  
Richard Longnecker
Keyword(s):  
Membrane Fusion ◽  
Epstein Barr Virus ◽  
Cytoplasmic Tail ◽  
Glycoprotein B ◽  
Functional Domains ◽  
Induced Membrane ◽  
Barr Virus ◽  
Epstein Barr

scholarly journals Modulation of Epstein-Barr Virus Glycoprotein B (gB) Fusion Activity by the gB Cytoplasmic Tail Domain

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Nicholas J. Garcia ◽  
Jia Chen ◽  
Richard Longnecker
Keyword(s):  
Membrane Fusion ◽  
Epstein Barr Virus ◽  
Cytoplasmic Tail ◽  
Host Cells ◽  
Glycoprotein B ◽  
Tail Domain ◽  
Induced Membrane ◽  
Barr Virus ◽  
Cellular Expression ◽  
Epstein Barr

ABSTRACTEpstein-Barr virus (EBV), along with other members of the herpesvirus family, requires a set of viral glycoproteins to mediate host cell attachment and entry. Viral glycoprotein B (gB), a highly conserved glycoprotein within the herpesvirus family, is thought to be the viral fusogen based on structural comparison of EBV gB and herpes simplex virus (HSV) gB with the postfusion crystal structure of vesicular stomatitis virus fusion protein glycoprotein G (VSV-G). In addition, mutational studies indicate that gB plays an important role in fusion function. In the current study, we constructed a comprehensive library of mutants with truncations of the C-terminal cytoplasmic tail domain (CTD) of EBV gB. Our studies indicate that the gB CTD is important in the cellular localization, expression, and fusion function of EBV gB. However, in line with observations from other studies, we conclude that the degree of cell surface expression of gB is not directly proportional to observed fusion phenotypes. Rather, we conclude that other biochemical or biophysical properties of EBV gB must be altered to explain the different fusion phenotypes observed.IMPORTANCEEpstein-Barr virus (EBV), like all enveloped viruses, fuses the virion envelope to a cellular membrane to allow release of the capsid, resulting in virus infection. To further characterize the function of EBV glycoprotein B (gB) in fusion, a comprehensive library of mutants with truncations in the gB C-terminal cytoplasmic tail domain (CTD) were made. These studies indicate that the CTD of gB is important for the cellular expression and localization of gB, as well as for the function of gB in fusion. These studies will lead to a better understanding of the mechanism of EBV-induced membrane fusion and herpesvirus-induced membrane fusion in general, which will ultimately lead to focused therapies guided at preventing viral entry into host cells.

scholarly journals Analysis of Epstein-Barr Virus Glycoprotein B Functional Domains via Linker Insertion Mutagenesis

2008 ◽  
Vol 83 (2) ◽  
pp. 734-747 ◽  
Author(s):  
Jessica J. Reimer ◽  
Marija Backovic ◽  
Charuhas G. Deshpande ◽  
Theodore Jardetzky ◽  
Richard Longnecker
Keyword(s):  
B Cells ◽  
Epstein Barr Virus ◽  
Glycoprotein B ◽  
Functional Domains ◽  
Insertion Mutagenesis ◽  
Fusion Activity ◽  
Wild Type ◽  
Protein Fusion ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr Virus (EBV) glycoprotein B (gB) is essential for viral fusion events with epithelial and B cells. This glycoprotein has been studied extensively in other herpesvirus family members, but functional domains outside of the cytoplasmic tail have not been characterized in EBV gB. In this study, a total of 28 linker insertion mutations were generated throughout the length of gB. In general, the linker insertions did not disrupt intracellular expression and variably altered cell surface expression. Oligomerization was disrupted by insertions located between residues 561 and 620, indicating the location of a potential site of oligomer contacts between EBV gB monomers. In addition, a novel N-glycosylated form of wild-type gB was identified under nonreducing Western blot conditions that likely represents a mature form of the protein. Fusion activity was abolished in all but three variants containing mutations in the N-terminal region (gB30), within the ectodomain (gB421), and in the intracellular C-terminal domain (gB832) of the protein. Fusion activity with variants gB421 and gB832 was comparable to that of the wild type with epithelial and B cells, and only these two mutants, but not gB30, were able to complement gB-null virus and subsequently function in virus entry. The mutant gB30 exhibited a low level of fusion activity with B cells and was unable to complement gB-null virus. The mutations generated here indicate important structural domains, as well as regions important for function in fusion, within EBV gB.

scholarly journals A soluble form of Epstein-Barr virus gH/gL inhibits EBV-induced membrane fusion and does not function in fusion

Virology ◽  
2013 ◽  
Vol 436 (1) ◽  
pp. 118-126 ◽  
Author(s):  
Cynthia L. Rowe ◽  
Sarah A. Connolly ◽  
Jia Chen ◽  
Theodore S. Jardetzky ◽  
Richard Longnecker
Keyword(s):  
Membrane Fusion ◽  
Epstein Barr Virus ◽  
Soluble Form ◽  
Induced Membrane ◽  
Barr Virus ◽  
Epstein Barr

scholarly journals Mutational Analyses of Epstein-Barr Virus Glycoprotein 42 Reveal Functional Domains Not Involved in Receptor Binding but Required for Membrane Fusion

2004 ◽  
Vol 78 (11) ◽  
pp. 5946-5956 ◽  
Author(s):  
Amanda L. Silva ◽  
Jasmina Omerović ◽  
Theodore S. Jardetzky ◽  
Richard Longnecker
Keyword(s):  
Membrane Fusion ◽  
Epstein Barr Virus ◽  
Mutational Analysis ◽  
Class Ii ◽  
Hla Class Ii ◽  
Hydrophobic Pocket ◽  
Induced Membrane ◽  
Lectin Domain ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with malignancies of both epithelial and lymphoid origin. Efficient infection of the latent host reservoir B lymphocytes involves the binding of glycoproteins gp350/220 for initial attachment, followed by the concerted action of gH, gL, gB, and gp42 for membrane fusion. The type II membrane protein gp42 is required for infection of B cells and assembles into a complex with gH and gL. The cellular host receptor for gp42, class II human leukocyte antigen (HLA), has been structurally verified by crystallization analyses of gp42 bound to HLA-DR1. Interestingly, the crystal structure revealed a hydrophobic pocket consisting of many aromatic and aliphatic residues from the predicted C-type lectin domain of gp42 that in other members of the C-type lectin family binds major histocompatibility complex class I or other diverse ligands. Although the hydrophobic pocket does not bind HLA class II, mutational analyses presented here indicate that this domain is essential for EBV-induced membrane fusion. In addition, mutational analysis of the region of gp42 contacting HLA class II in the gp42-HLA-DR1 cocrystal confirms that this region interacts with HLA class II and that this interaction is also important for EBV-induced membrane fusion.

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

mBio ◽  
2012 ◽  
Vol 3 (1) ◽  
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.

scholarly journals Cleavage and Secretion of Epstein-Barr Virus Glycoprotein 42 Promote Membrane Fusion with B Lymphocytes

2009 ◽  
Vol 83 (13) ◽  
pp. 6664-6672 ◽  
Author(s):  
Jessica Sorem ◽  
Theodore S. Jardetzky ◽  
Richard Longnecker
Keyword(s):  
Membrane Fusion ◽  
B Lymphocytes ◽  
Viral Entry ◽  
Epstein Barr Virus ◽  
Class Ii ◽  
Hla Class Ii ◽  
Full Length ◽  
Barr Virus ◽  
Infected Cells ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) membrane glycoprotein 42 (gp42) is required for viral entry into B lymphocytes through binding to human leukocyte antigen (HLA) class II on the B-cell surface. EBV gp42 plays multiple roles during infection, including acting as a coreceptor for viral entry into B cells, binding to EBV glycoprotein H (gH) and gL during the process of membrane fusion, and blocking T-cell recognition of HLA class II-peptide complexes through steric hindrance. EBV gp42 occurs in two forms in infected cells, a full-length membrane-bound form and a soluble form generated by proteolytic cleavage that is secreted from infected cells due to loss of the N-terminal transmembrane domain. Both the full-length and the secreted gp42 forms bind to gH/gL and HLA class II, and the functional significance of gp42 cleavage is currently unclear. We found that in a virus-free cell-cell fusion assay, enhanced secretion of gp42 promoted fusion with B lymphocytes, and mutation of the site of gp42 cleavage inhibited membrane fusion activity. The site of gp42 cleavage was found to be physically distinct from the residues of gp42 necessary for binding to gH/gL. These results suggest that cleavage and secretion of gp42 are necessary for the process of membrane fusion with B lymphocytes, providing the first indicated functional difference between full-length and cleaved, secreted gp42.

scholarly journals 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

2007 ◽  
Vol 81 (17) ◽  
pp. 9216-9229 ◽  
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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