Abstract
Glycoprotein D (gD) of herpes simplex virus contains three utilized sites (Asn-X-Ser/Thr) for addition of asparagine-linked carbohydrates (N-CHO). Previously, we used oligonucleotide-directed mutagenesis to alter serine or threonine residues to alanine at each N-CHO addition site. Studies with monoclonal antibodies showed that a mutant protein lacking all three sites (now designated AAA) was structurally altered because of the amino acid change at residue 96 as well as the absence of the N-CHO. In this study, we constructed additional single mutations at site 1 (residues 94 and 96) and found that in most cases, the amino acid change itself adversely affected the conformation of gD. However, changing asparagine 94 to glutamine (Q) at site 1 had the least effect on gD. We constructed a second triple mutant, QAA, which lacked all three N-CHO signals. The antigenic conformation of QAA was similar to that of gD produced in the presence of tunicamycin (TM-gD). However, binding of MAbs to the AAA protein or to single mutants altered at site 1 was reduced compared with TM-gD. Wild-type gD and QAA proteins were equally susceptible to digestion by trypsin or Staphylococcus aureus V8 protease. In contrast, the AAA protein was more sensitive to trypsin but less sensitive to V8, again suggesting conformational alterations of the AAA protein. Despite what appeared to be large changes in structure, each mutant complemented the infectivity of a virus lacking gD (F-gD beta). We conclude that the N-CHO and amino acids at N-CHO site 1 play an important role in forming and/or maintaining gD structure, but none of the N-CHO are required for gD to function in the complementation assay.