Functional studies of herpes simplex virus glycoproteins involved in virus entry

The entry of Herpes simplex virus (HSV) into mammalian cells is a multi-step process beginning with an attachment step involving glycoproteins gC and gB. A second step requires the interaction of glycoprotein gD with a cell surface molecule. I used two different approaches to characterize the role of gC and gD in HSV entry. First, I studied three HSV mutants in the mouse eye infection model to investigate the role of gD in pathogenesis: F-gD(QAA), a mutant virus which lacks all three N-linked carbohydrates (N-CHO) in gD, FgD$\beta\{-\}$ a gD null virus, and FgD$\beta\{+\}$ a complemented pseudotype containing gD in its viral envelope. I determined that N-CHO are not essential for establishment, maintenance or reactivation from latency. Furthermore, my results suggest that the presence of gD in viral envelopes is essential for pathogenesis. Second, using an in vitro approach, I studied the interaction of purified truncated gC with mammalian cell surface components. Truncated forms of gC and gD lacking the transmembrane and carboxyl regions were expressed in the baculovirus system. I used these proteins in several functional assays: binding to immobilized heparin, binding to uninfected cells, blocking of HSV attachment to cells, and blocking of HSV infection. Truncated gC1 and gC2 each bind specifically to several cell lines. Binding involves heparan sulfate proteoglycans, since heparitinase treatment of cells reduced gC binding, but had no effect on gD binding. Furthermore, the binding efficiency of gC to two heparan sulfate deficient L-cell lines, gro2C and sog9, both of which are resistant to HSV infection was markedly reduced. Purified gD, however, bound equally well to both cell lines. Saturating amounts of gC were able to interfere with HSV-1 attachment to cells; however, gC failed to block plaque formation. This suggests that gC functions during HSV attachment but not during penetration. Lastly, a baculovirus-expressed form of gC lacking residues 33-123 and an WSV-1 mutant which contains this mutation were used to determine that the N-terminal region of gC is critical for efficient HSV attachment to cells