The role of a single N-linked glycosylation site for a functional epitope of herpes simplex virus type 1 envelope glycoprotein gC.
A monoclonal antibody, B1C1, binding to an epitope of antigenic site II of the herpes simplex virus type 1 (HSV-1) glycoprotein gC-1, is a potent inhibitor of two important biological functions of gC-1: its binding to cell surface heparan sulfate and its binding to the receptor for complement factor C3b. Here, we have analyzed a B1C1-resistant HSV-1 variant (HSV-12762/B1C1B4.2), obtained after passage of wild type HSV-1 (HSV-12762) in the presence of high concentrations of B1C1. The transport of newly synthesized mutant gC-1 to the cell surface was comparable to that of wild type glycoprotein, but no binding of surface-associated mutant gC-1 to B1C1 was detected. However, mutant and wild type gC-1 bound equally well to other site II Mabs. Attachment of wild type but not mutant virus was inhibited by B1C1. Sequencing of the mutant gC-1 gene revealed only one nucleotide change, resulting in replacement of Thr150 by an Ile, in turn destroying an N-glycosylation site at Asn148. Loss of one complex type N-linked glycan was confirmed by endoglycosidase digestion and subsequent SDS-polyacrylamide gel electrophoresis. Circular dichroism analysis of purified gC-1 from cells infected with mutant or wild type virus did not reveal any difference in secondary structure between mutant and wild type gC-1. It was not possible to obtain a B1C1-resistant phenotype by nucleotide-directed mutagenesis of gC-1 where Asn148 was changed to a glutamine. These data demonstrated that the threonine of the glycosylation site and not the N-linked glycan in itself was essential for B1C1 binding
Olofsson
S
S
Department of Clinical Virology, University of Göteborg, Guldhedsgatan 10 B, S-413 46 Göteborg, Sweden.
Bolmstedt
A
A
Biller
M
M
Mårdberg
K
K
Leckner
J
J
Malmström
B G
BG
Trybala
E
E
Bergström
T
T
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000378:metabolism / Q000494:pharmacology; D001216:Asparagine; D001483:Base Sequence; D001666:Binding Sites, Antibody; D002236:Carbohydrate Conformation; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry*; D006031:Glycosylation; D006497:Heparan Sulfate / Q000378:metabolism; D007163:Immunosorbent Techniques; D008969:Molecular Sequence Data; D017354:Point Mutation; D011134:Polysaccharides / Q000737:chemistry; D017433:Protein Structure, Secondary; D017463:Receptors, Complement 3b / Q000378:metabolism; D013329:Structure-Activity Relationship; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D000911:Antibodies, Monoclonal; D001216:Asparagine; D000939:Epitopes; D006497:Heparan Sulfate; D011134:Polysaccharides; D017463:Receptors, Complement 3b; D014759:Viral Envelope Proteins; C038535:glycoprotein gC, herpes simplex virus type 1