GLYCOPROTEIN-B OF HERPES-SIMPLEX VIRUS TYPE-1 OLIGOMERIZES THROUGH THE INTERMOLECULAR INTERACTION OF A 28-AMINO-ACID DOMAIN

Herpes simplex virus type 1 glycoprotein B (gB) is an envelope compone nt that plays an essential role in virus infection, The biologically a ctive form of gB is an oligomer that contributes to the process of vir al envelope fusion with the cell surface membrane, resulting in viral penetration and initiation of the replication cycle. In previous studi es, two discontinuous sites for oligomer formation were identified: a nonessential upstream site located between residues 93 and 282 and an essential downstream site located between residues 596 and 711, In thi s study, in vitro-transcribed and -translated gB test molecules were u sed to characterize the more active essential membrane-proximal domain , A series of gB test polypeptides mutated in this downstream oligomer ization domain were assayed for their abilities to form oligomers with a mutant gB rapture polypeptide containing the analogous wild-type do main, Detection of oligomers was achieved by coimmunoprecipitation of two gB mutant molecules by using a monoclonal antibody specific for a hemagglutinin epitope tag introduced into the coding sequence of the c apture polypeptide. Analysis of the immune-precipitated products by so dium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated t hat the downstream oligomerization domain resided within residues 626 to 676. This region was further resolved into two segments, residues 6 26 to 653 and 653 to 675, each of which was independently sufficient t o form oligomers. However, residues 626 to 653 provided for a stronger interaction between gB monomers, Moreover, this stretch of 28 amino a cids was shown to form oligomers when introduced into the carboxy-term inal region of gB monomers lacking this domain at the normal site, thu s indicating that this domain was functionally independent of its natu ral location within the gB molecule, Further analysis of the sequence within residues 596 to 653 by using mutant test polypeptides altered i n individual amino acids revealed that cysteines 9 and 10, located at positions 596 and 633, respectively, were not required for oligomer fo rmation but contributed to dimer formation and/or stabilization. The r esults of this study suggest that oligomerization of gB monomers is in duced by interactions between contiguous residues localized within the ectodomain near the site of molecule insertion into the viral envelop e membrane.