Oligomer formation of the gB glycoprotein of herpes simplex virus type 1.
Oligomer formation of the gB glycoprotein of herpes simplex virus type 1 was studied by sedimentation analysis of radioactively labeled infected cell and virion lysates. Fractions from sucrose gradients were precipitated with a pool of gB-specific monoclonal antibodies and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Pulse-labeled gB from infected cell was synthesized as monomers and converted to oligomers posttranslationally. The oligomers from infected cells and from virions sedimented as dimers, and there was no evidence of higher-molecular-weight forms. To identify amino acid sequences of gB that contribute to oligomer formation, pairs of mutant plasmids were transfected into Vero cells and superinfected with a gB-null mutant virus to stimulate plasmid-specified gene expression. Radioactively labeled lysates were precipitated with antibodies and examined by SDS-PAGE. Polypeptides from cotransfections were precipitated with an antibody that recognized amino acid sequences present in only one of the two polypeptides. A coprecipitated polypeptide lacking the antibody target epitope was presumed to contain the sequences necessary for oligomer formation. Using this technique, two noncontiguous sites for oligomer formation were detected. An upstream site was localized between residues 93 and 282, and a downstream site was localized between residues 596 and 711. Oligomer formation resulted from molecular interactions between two upstream sites, between two downstream sites, and between an upstream and a downstream site. A schematic diagram of a gB oligomer is presented that is consistent with these data.
Highlander
S L
SL
Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor 48109.
Goins
W F
WF
Person
S
S
Holland
T C
TC
Levine
M
M
Glorioso
J C
JC
D000595:Amino Acid Sequence; D000818:Animals; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D015967:Gene Expression Regulation, Viral; D010957:Plasmids; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D018139:Simplexvirus / Q000032:analysis / Q000235:genetics*; D014162:Transfection; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics; D014771:Virion / Q000032:analysis
D014759:Viral Envelope Proteins; C040495:glycoprotein B, Simplexvirus