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

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.

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

Journal of Virology ◽

10.1128/jvi.00195-09 ◽

2009 ◽

Vol 83 (13) ◽

pp. 6664-6672 ◽

Cited By ~ 25

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.

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Mutational Analysis of the HLA Class II Interaction with Epstein-Barr Virus Glycoprotein 42

Journal of Virology ◽

10.1128/jvi.77.13.7655-7662.2003 ◽

2003 ◽

Vol 77 (13) ◽

pp. 7655-7662 ◽

Cited By ~ 19

Author(s):

Marisa P. McShane ◽

Maureen M. Mullen ◽

Keith M. Haan ◽

Theodore S. Jardetzky ◽

Richard Longnecker

Keyword(s):

Epstein Barr Virus ◽

Mutational Analysis ◽

Hla Class I ◽

Class Ii ◽

Hla Class Ii ◽

Class I ◽

Viral Glycoprotein ◽

Barr Virus ◽

Lectin Family ◽

Epstein Barr

ABSTRACT Entry of Epstein-Barr virus (EBV) into B lymphocytes requires the binding of viral glycoprotein 42 (gp42), a C-type lectin family member, to HLA class II. Recently, the structure of the gp42:HLA-DR1 complex was determined. In order to confirm the interaction as determined in the structural study and to identify other potential interactive residues, a mutational analysis of HLA class II was performed. A secreted form of gp42 (sgp42) reacted with a conformation-specific monoclonal antibody and blocked EBV infection. The binding of sgp42 and EBV entry to two sets of HLA class II mutants were tested. The first set of mutants were based on the known interaction of the C-type lectin Ly49A with HLA class I, and the second set of mutants were based on the identified interface in the gp42:HLA-DR1 complex. As expected, none of the mutants that would be predicted to interfere with the interaction of Ly49A with class I affected the interaction of gp42 with HLA class II, whereas mutants in amino acids identified in the gp42:HLA-DR1 structure inhibited sg42 binding to class II. In general, sgp42 binding correlated with efficient entry of EBV, as demonstrated by the necessity of glutamic acid 46 or arginine 72 in class II molecules. Furthermore, other HLA class II residues buried within the interface of gp42 and HLA class II when mutated had either no effect or a decrease in both binding and entry and implicate a region of class II important in stabilizing the interaction with gp42. These studies provide insight into the entry and fusion processes of the critical interaction between gp42 and HLA class II.

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

mBio ◽

10.1128/mbio.00571-12 ◽

2013 ◽

Vol 4 (1) ◽

Cited By ~ 24

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.

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