Epstein-Barr virus (EBV) infects B lymphocytes and epithelial cells. While the glycoproteins required forentry 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 gammaherpesviruses (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 by 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 lymphocytes and epithelial cells. Epstein-Barr virus (EBV) is a gammaherpesvirus that has tropism for both B lymphocytes and epithelial cells (18, 42).Similar to other enveloped viruses, EBV entry into the target cells occurs in two steps: the initial attachment of the virus to the cell surface and the subsequent fusion of the viral envelope and the cell membrane (18, 51). EBV infection can occur by cell-free virus or through cell-cell spread, but fusion is required for both entry pathways (18, 42). The initiation of infection by EBV is driven by interaction of viral glycoproteins with cellsurface receptors (42). EBV encodes as many as 11 glycoproteins, but only a subset is required for efficient EBV entry (15,42). In general, more is known about the mechanism of EBV entry into B lymphocytes than into epithelial cells. Infection of B lymphocytes is initiated by the attachment of glycoprotein gp350/220 to the CD21/CR2 receptor on the B cells (9,43,45). This interaction enhances infection efficiency of B cells, but it is not absolutely required for infection to occur (16). Subsequent to this binding, viral glycoprotein gp42 binds to the B-cell surface protein HLA class II, which triggers fusion mediated by a concerted action of three glycoproteins (gB, gH, and gL) (19,43). The glycoprotein requirement for EBV entry differs between epithelial and B cells; gB, gH, and gL are essential for infection of both epithelial and B cells, while gp42 is required only for B-cell entry (12,20,24,47). Even though gB, gH, and gL are conserved throughout the herpesvirus family, the structure and the exact mechanism of action of these three glycoproteins in the fusion process are still unclear.

The latent membrane protein 2A (LMP2A) of EBV plays a key role in regulating viral latency and EBV pathogenesis by functionally mimicking a constitutively active B cell Ag receptor. When expressed as a B cell-specific transgene in mice, LMP2A drives B cell development, resulting in the bypass of normal developmental checkpoints. In this study, we have demonstrated that expression of LMP2A in transgenic mice results in B cell development that exclusively favors B-1 cells. This switch to B-1 cell development occurs at the pre-B-cell stage of normal B cell development in the bone marrow, a B cell stage much earlier than appreciated for B-1 commitment. This finding indicates that all pre-B cells have the capacity to assume a B-1 cell phenotype if they encounter the appropriate signal during normal development. Furthermore, these studies offer insight into EBV latency and pathogenesis in the human host.

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