Synthesis and processing of glycoprotein D of herpes simplex virus types 1 and 2 in an in vitro system.

We carried out studies of in vitro translation and processing of glycoprotein D (gD) of herpes simplex virus types 1 and 2 by using mRNA from cells infected for 6 h and a reticulocyte lysate translation system. Polypeptides of 49,000 daltons were immunoprecipitated with anti-gD-1 sera. Each in vitro-synthesized molecule had the same methionine tryptic peptide profile as the respective in vivo precursors, pgD-1 and pgD-2. In addition, the polypeptides synthesized in vitro were larger than the corresponding molecules synthesized in the presence of tunicamycin. This suggested that each of the gD polypeptides synthesized in vitro contained a transient N-terminal signal sequence. When the translation mixture was supplemented with pancreatic microsomes, each of the gD polypeptides was converted cotranslationally to a larger-molecular-weight form. Processing involved addition of three N-asparagine-linked oligosaccharides and removal of the signal peptide. When trypsin was added after in vitro processing, a polypeptide which was 3,000 daltons smaller than the in vitro-modified form of gD was immunoprecipitated. Experiments with endo-beta-N-acetylglucosaminidase H showed that this polypeptide still contained the three N-asparagine-linked oligosaccharides. Two monoclonal antibodies, 57S (group V) and 17O (group VII), were used to further orient gD in microsomes. The group V determinant was located in the trypsin-sensitive 3,000-dalton fragment, and the group VII determinant was located in the portion of gD which was protected from trypsin. We concluded that gD is oriented with the three glycosylation sites inside the vesicles and that 3,000 daltons containing the group V determinant are located outside. Immunofluorescence studies indicated that the group V determinant of gD is inside the plasma membrane of herpes simplex virus-infected cells and that the group VII determinant is outside. This cellular orientation is consistent with predictions based on the in vitro experiments.