Structure-function analysis of soluble forms of herpes simplex virus glycoprotein D

Glycoprotein D (gD) of herpes simplex virus (HSV) is essential for virus entry, Truncated forms of go lacking the transmembrane and cytoplasmic tail regions have been shown to bind to cells and block plaque formation, Using complementation analysis and a panel of go mutants, we previously identified four regions of go (regions I to IV) which are important for virus entry, Here, we used baculovirus vectors to overexpress 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 126t), 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 immunosorbent assay and circular dichroism to analyze the structure of these proteins, and we used functional assays to study the role of go in binding, penetration, and cell-to-cell spread, gD-1 and gD-2 are similar in antigenic structure and thermal stability but vary in secondary structure, Mutant proteins with insertions in region I or II were most altered in structure and stability, while mutants with insertions in region 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 to cells, confirming that binding is not the only function of go. The region I mutant did not inhibit HSV plaque formation or cell-to-cell spread, suggesting that this region is necessary for the function of go in these processes, Surprisingly, the other three mutant proteins functioned in all of the in vitro assays, indicating that the ability of go to bind to cells and inhibit infection does not correlate with its ability to initiate infection as measured by the complementation assay, The region IV mutant, gD-1(Delta 290-299t), had an unexpected enhanced inhibitory effect on HSV infection, Taken together, the results argue against a single functional domain in go. It is likely that different go structural elements are involved in successive steps of infection.