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-l, is a potent inhibitor of two important biological functions of gC-l: 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-1(2762/B1C1B4.2)), Obtained after passage of wild type HSV-1 (HSV-1(2762)) in the presence of high concentrations of B1C1, The transport of newly synthesized mutant gC-l to the cell surface was comparable to that of wild type glycoprotein, but no binding of surface-associated mutant gC-l to B1C1 was detected. However, mutant and wild type gC-l 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-l gene revealed only one nucleotide change, resulting in replacement of Thr150 by an ne, 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-l from cells infected with mutant or wild type virus did not reveal any difference in secondary structure between mutant and wild type gC-l, It was not possible to obtain a B1C1-resistant phenotype by nucleotide-directed mutagenesis of gC-l 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.