STRUCTURAL AND ANTIGENIC ANALYSIS OF A TRUNCATED FORM OF THE HERPES-SIMPLEX VIRUS GLYCOPROTEIN GH-GL COMPLEX

The herpes simplex virus (HSV) gH-gL complex is essential for virus in fectivity and is a major antigen for the host immune system, The assoc iation of gH with gL is required for correct folding, cell surface tra fficking, and membrane presentation of the complex. Previously, a mamm alian cell line was constructed which produces a secreted form of gHt- gL complex lacking the transmembrane and cytoplasmic tail regions of g H, gHt-gL retains a conformation similar to that of its full-length co unterpart in HSV-infected cells. Here, we examined the structural and antigenic properties of gHt-gL, We first determined its stoichiometry and carbohydrate composition. We found that the complex consists of on e molecule each of gH and gL, The N-linked carbohydrate (N-CHO) site o n gL and most of the N-CHO sites on gH are utilized, and both proteins also contain O-linked carbohydrate and sialic acid. These results sug gest that the complex is processed to the mature form via the Golgi ne twork prior to secretion. To determine the antigenically active sites of gH and gL, we mapped the epitopes of a panel of gH and gL monoclona l antibodies (MAbs), using a series of gH and gL C-terminal truncation variant proteins produced in transiently transfected mammalian cells. Sixteen gH MAbs (including H6 and 37S) reacted,vith the N-terminal po rtion of gH between amino acids 19 and 276, One of the gH MAbs, H12, r eacted with the middle portion of gH (residues 476 to 678), Nine gL MA bs (including 8H4 and VIII 62) reacted with continuous epitopes within the C-terminal portion of gL, and this region was further mapped with in amino acids 168 to 178 with overlapping synthetic peptides, Finally , plasmids expressing the gH and gL truncations were employed in cotra nsfection assays to define the minimal regions of both gH and gL requi red for complex formation and secretion. The first 323 amino acids of gH and the first 161 amino acids of gL can form a stable secreted hete ro-oligomer with gL and gH792, respectively, while gH323-gL168 is the smallest secreted hetero-oligomer. The first 648 amino acids of gH are required for reactivity with MAbs LP11 and 53S, indicating that a com plex of gH648-gL oligomerizes into the correct conformation. The data suggest that both antigenic activity and oligomeric structure require the amino-terminal portions of gH and gL.