Epstein-Barr virus (EBV) is a human gamma-herpesvirus that primarily infects B cells 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 gamma-herpesviruses: 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 with 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 cells and epithelial cells. Furthermore, even though the gH/gL complex is conserved within the herpesvirus family, its exact role in entry and mechanism of fusion are not known yet. To understand more about the functionality of EBV gH/gL, the functional homology of gHs and gLs from human herpesvirus 8 (HHV8) and two nonhuman primate (rhesus and marmoset) gamma-herpesviruses in EBV-mediated virus-free cell fusion were investigated. Overall, gHs and gLs from the more homologous primate herpesviruses were better at complementing EBV gH and gL in fusion than HHV8 gH and gL. Interestingly, marmoset gH was able to complement fusion with epithelial cells, but not B cells. Further investigation of this led to the discovery that EBVgH is the binding partner of gp42 in the tripartite complex and the absence of fusion with B cells in the presence of marmoset gH is due to its inability to bind gp42.

Last modified

• 06/05/2018

Creator

• Jasmina Omerovic

DOI

• https://doi.org/10.21985/N2297W

Subject

• Integrated Graduate Program in the Life Sciences

Keyword

• Molecular
• Biology

Date created

• 2007-05-09

Resource type

• Dissertation

Rights statement

• In Copyright