Studies on the Herpes Simplex Virus Type 1 gB Glycoprotein

Abstract

The glycoprotein gB of HSV-1 is involved in viral entry and membrane fusion functions. It is glycosylated, forms homodimers, and is transported to both the inner nuclear membrane and plasma membrane in infected cells. The gB glycoprotein contains a potential membrane anchoring hydrophobic sequence of 69 amino acids, located near the carboxy terminus of the glycoprotein. This sequence is predicted to span the membrane bilayer three times, and can thus be divided into three distinct segments, each of which could span the bilayer. To define both the membrane anchoring sequence and the role of this 69 residue hydrophobic domain, a series of deletion mutants were constructed. These mutants have one, two or all three of the predicted membrane spanning segments deleted, in every combination possible, thereby creating a total of 7 deletion mutants. These mutant constructs were expressed in COS-1 cells using transient expression systems. All the mutant constructs were expressed and glycosylated in a manner similar to the wild type gB glycoprotein. Mutant glycoproteins lacking the third (or most carboxy terminal) predicted spanning segment of this 69 residue hydrophobic domain were found to be secreted from the cells, indicating that this segment may specify the membrane anchoring domain of the gB glycoprotein. Further, the mutant glycoproteins containing this third segment were localized in the nuclear envelope, while mutants lacking this segment were not. All the deletion mutants, except for one, were however defective in intracellular transport and processing of the N-linked oligosaccharides. The only mutant that showed any intracellular transport and processing had only the third segment deleted, but even this mutant was transported and processed much slower than the wild type glycoprotein. The mutant glycoproteins also failed to complement a gB-null virus. These results suggest that the carboxy terminus hydrophobic domain contains essential structural determinants of the gB glycoprotein.