Characterization of regions of herpes simplex virus type 1 glycoprotein E involved in binding the Fc domain of monomeric IgG and in forming a complex with glycoprotein I.
Glycoprotein E (gE) and glycoprotein I (gI) of herpes simplex virus type 1 form a molecular complex that binds the Fc domain of monomeric IgG. Two approaches were used to define regions of gE-1 involved in monomeric IgG binding and complex formation with gI-1. First, we constructed 22 in-frame gE-1 linker-insertion mutants and, in cotransfection experiments with gI-1, assayed each mutant for IgG monomer binding and the ability to complex with gI-1. Nine mutants with insertions between gE-1 amino acids 235 and 380 failed to bind IgG monomers, whereas mutants outside this region retained binding activity. Each mutant reacted with several gE-1 mAbs, was detected at the cell surface, and was fully processed. Only two gE-1 mutants with insertions at residues 235 and 264 lost the ability to co-immunoprecipitate with gI-1, which defines a region of gE-1 that complexes with gI-1. As an additional approach, we assayed 8 gE-1/gD-1 fusion proteins containing large overlapping gE-1 peptides inserted within the ectodomain of gD-1 for binding of IgG monomers and complex formation with gI-1. Three fusion proteins containing gE-1 peptides that overlap at residues 183-402 bound monomeric IgG. This region of gE-1 includes the Fc binding region defined by linker insertion mutagenesis. Five fusion proteins containing gE-1 peptides that overlap at residues 183-288 were co-immunoprecipitated with gI-1, confirming results of gE-1 linker insertion mutagenesis. These studies define two regions on gE-1 involved in Fc binding activity, one that interacts with gI-1, and another that binds IgG.
Basu
S
S
Department of Medicine, University of Pennsylvania, Philadelphia 19104.
Dubin
G
G
Basu
M
M
Nguyen
V
V
Friedman
H M
HM
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D007141:Immunoglobulin Fc Fragments / Q000378:metabolism*; D007074:Immunoglobulin G / Q000378:metabolism*; D007739:L Cells; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018139:Simplexvirus / Q000235:genetics / Q000276:immunology*; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; C044768:glycoprotein E, herpes simplex virus type 1; C064959:glycoprotein I, herpes simplex virus type 1