STRUCTURE-FUNCTION ANALYSIS OF SOLUBLE FORMS OF HERPES-SIMPLEX VIRUS GLYCOPROTEIN-D

Glycoprotein D (gD) of herpes simplex virus (HSV) is essential for vir us entry, Truncated forms of go lacking the transmembrane and cytoplas mic tail regions have been shown to bind to cells and block plaque for mation, Using complementation analysis and a panel of go mutants, we p reviously identified four regions of go (regions I to IV) which are im portant for virus entry, Here, we used baculovirus vectors to overexpr ess truncated forms of wild-type go from HSV type 1 (HSV-1) [gD-1(306t )] and HSV-2 [gD-2(306t)] and four mutants, gD-1(del 34t), gD-1(del 12 6t), gD-1(del 243t), and gD-1(Delta 290-299t), each having a mutation in one of the four functional regions, We used an enzyme-linked immuno sorbent assay and circular dichroism to analyze the structure of these proteins, and we used functional assays to study the role of go in bi nding, penetration, and cell-to-cell spread, gD-1 and gD-2 are similar in antigenic structure and thermal stability but vary in secondary st ructure, Mutant proteins with insertions in region I or II were most a ltered in structure and stability, while mutants with insertions in re gion III or IV were less altered, gD-1(306t) and gD-2(306t) inhibited both plaque formation and cell-to-cell transmission of HSV-1. In spite of obvious structural differences, all of the mutant proteins bound t o cells, confirming that binding is not the only function of go. The r egion I mutant did not inhibit HSV plaque formation or cell-to-cell sp read, suggesting that this region is necessary for the function of go in these processes, Surprisingly, the other three mutant proteins func tioned in all of the in vitro assays, indicating that the ability of g o to bind to cells and inhibit infection does not correlate with its a bility to initiate infection as measured by the complementation assay, The region IV mutant, gD-1(Delta 290-299t), had an unexpected enhance d inhibitory effect on HSV infection, Taken together, the results argu e against a single functional domain in go. It is likely that differen t go structural elements are involved in successive steps of infection .