Analysis of the function of cleavage and conformation of Epstein-Barr virus glycoproteins gB and gp42 in membrane fusion

Epstein-Barr virus (EBV) is a human gammaherpesvirus associated with infectious mononucleosis as well as malignancies of epithelial and B cell origin. EBV entry into epithelial and B cells requires glycoproteins gB, gH, and gL. EBV gB possesses a conserved furin cleavage motif and cleaved gB is present in EBV virions. The EBV gB furin cleavage motif was deleted by site-directed mutagenesis. Elimination of the gB cleavage motif reduced EBV-induced fusion with both epithelial and B cells in a cell fusion assay, with a greater effect on epithelial cell fusion observed. Proximal deletions of identical size on either side of the gB cleavage motif had no effect on fusion with either cell type. The observed decrease in cell fusion with the gB cleavage mutant is similar to that observed with multiple alphaherpesvirus gB cleavage mutants and supports a conserved function for cleaved gB.;For EBV entry into B cells, an additional glycoprotein, gp42, is required. EBV gp42 occurs in two forms, a full-length membrane-bound form and a soluble form generated by proteolytic cleavage, which is secreted from infected cells due to loss of the transmembrane domain. The functional significance of gp42 cleavage is currently unclear. To better understand the role of gp42 in membrane fusion, gp42 cleavage site mutants were assayed for ability to mediate fusion with B cells. In a cell fusion assay, enhanced secretion of gp42 promoted fusion, while gp42 cleavage site mutation inhibited fusion, suggesting that cleavage and secretion of gp42 is necessary for B-cell membrane fusion. These observations provide the first indicated functional difference between full-length and soluble gp42.;The recently solved crystal structure of unbound EBV gp42 allowed comparison to the structure of receptor-bound gp42. Conformational changes between the two structures, specifically around the gp42 hydrophobic pocket, suggested a structural mechanism for gp42 triggering of membrane fusion. Structure-based EBV gp42 mutants were constructed and assayed for receptor binding and membrane fusion activity. Some of the mutations had significant effects on these processes, but further study is necessary to determine the specific relationship between the observed gp42 conformational changes and membrane fusion activation.