Epstein-Barr virus (EBV) is the cause of infectious mononucleosis and is associated with the etiology of several human cancers of lymphoid and epithelial cell origin, including Burkitt's lymphoma and nasopharyngeal carcinoma. It is an extremely prevalent virus in human populations worldwide. All herpesviruses, including EBV, infect cells by fusing the viral lipid envelope with the target cell membrane using multiple viral proteins, which is distinct from most other enveloped viruses that typically use a single protein. The EBV fusion process requires the actions of the viral glycoproteins gH, gL, and gB for entry into epithelial cells, and additionally requires gp42 protein 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 heterotrimer with 1:1:1 stoichiometry. A virus-free cell-cell fusion assay demonstrated that soluble gp42 and gH/gL/gp42 complexes functioned to trigger B-cell fusion, but gp42 alone inhibited epithelial cell fusion. In contrast, soluble gH/gL did not inhibit or enhance B cell or epithelial cell fusion. By studying deletion and point mutants within the N-terminal region of gp42, two functional regions important for gH/gL binding and membrane fusion were identified. A peptide encompassing gp42 residues 36 to 81 was shown to bind gH/gL with nanomolar affinity, comparable to full-length gp42 protein, and efficiently inhibited epithelial cell membrane fusion and competed with soluble gp42 to inhibit B cell fusion. Additionally, this peptide at low nanomolar concentration inhibited epithelial cell infection by intact virus, demonstrating its excellent potential for future drug development. Finally, the structure of soluble gp42 in its unbound native form was determined by X-ray crystallography, complementing the previously solved receptor-bound form (gp42:HLA-DR1). A more ordered N-terminus was observed that makes specific hydrogen bond interactions with two neighboring gp42 molecules at residues involved in binding MHC class II receptor. The movement of a loop bordering a gp42 hydrophobic pocket thereby widening the region suggests a possible structural mechanism for gp42 triggering the fusion process.

Last modified

• 08/27/2018

Creator

• Austin Kirschner

DOI

• https://doi.org/10.21985/N2W132

Subject

• Interdepartmental Biological Sciences Program

Keyword

• membrane fusion
• peptide inhibitior
• antiviral
• viral entry
• Epstein-Barr virus
• glycoprotein structure

Date created

• 2007-08-15

Resource type

• Dissertation

Rights statement

• In Copyright