IDENTIFICATION OF FUNCTIONAL REGIONS OF HERPES-SIMPLEX VIRUS GLYCOPROTEIN GO BY USING LINKER-INSERTION MUTAGENESIS

Glycoprotein gD is a component of the herpes simplex virus (HSV) envel ope essential for virus entry into susceptible cells. Previous studies using deletion and point mutations identified a functional domain of HSV-1 gD (gD-1) from residues 231 to 244. However, many of the deletio n mutations had global effects on gD-1 structure, thus precluding asse ssment of the functional role of large portions of the protein. In thi s study, we constructed a large panel of linker-insertion mutants in t he genes for gD-1 and HSV-2 gD (gD-2). The object was to create mutati ons which would have only localized effects on protein structure but m ight have profound effects on gD function. The mutant proteins were ex pressed in transiently transfected L cells. Monoclonal antibodies (MAb s) were used as probes of gD structure. We also examined protein aggre gation and appearance of the mutant glycoproteins on the transfected c ell surface. A complementation assay measured the ability of the mutan t proteins to rescue the infectivity of the gD-null virus, FgD beta, i n trans. Most of the mutants were recognized by one or more MAbs to di scontinuous epitopes, were transported to the transfected cell surface , and rescued FgD beta virus infectivity. However, some mutants which retained structure were unable to complement FgD beta. These mutants w ere clustered in four regions of gD. Region III (amino acids 222 to 24 6) overlaps the region previously defined by gD-1 deletion mutants. Th e others, from 27 through 43 (region I), from 125 through 161 (region II), and from 277 to 310 (region IV), are newly described. Region IV, immediately upstream of the transmembrane anchor sequence, was previou sly postulated to be part of a putative stalk structure. However, resi dues 277 to 300 are directly involved in gD function. The linker-inser tion mutants were useful for mapping MAb AP7, a previously ungrouped n eutralizing MAb, and provided further information concerning other dis continuous epitopes. The mapping data suggest that regions I through I V are physically near each other in the folded structure of gD and may form a single functional domain.