title: Site-directed and linker insertion mutagenesis of herpes simplex virus type 1 glycoprotein H.
abstract: The gH-gL complex of herpes simplex virus type 1 (HSV-1) is essential for virion infectivity and virus-induced cell fusion, but functional domains of the gH molecule remain to be defined. We have addressed this question by mutagenesis. A set of linker insertion mutants in HSV-1 gH was generated and tested in transient assays for their ability to complement a gH-negative virus. Insertions at three sites in the C-terminal third of the external domain affected the ability of gH to function in cell-cell fusion and virus entry, while insertions at six sites in the N-terminal half of the external domain induced conformational changes in gH such that it was not recognized by monoclonal antibody LP11, although expression at the cell surface was unchanged. A recombinant virus in which a potential integrin-binding motif, RGD, in gH was changed to the triplet RGE entered cells as efficiently as the wild type, indicating that HSV-1 entry is not mediated by means of the gH-RGD motif binding to cell surface integrins. Furthermore, mutagenesis of the glycosylation site which is positionally conserved in all herpesvirus gH sequences in close proximity to the transmembrane domain generated a recombinant virus that grew in vitro with wild-type single-step kinetics.
authors.affiliation: Department of Pathology, University of Cambridge, United Kingdom.
authors.forename: M
authors.initials: M
authors.lastname: Galdiero
authors.forename: A
authors.initials: A
authors.lastname: Whiteley
authors.forename: B
authors.initials: B
authors.lastname: Bruun
authors.forename: S
authors.initials: S
authors.lastname: Bell
authors.forename: T
authors.initials: T
authors.lastname: Minson
authors.forename: H
authors.initials: H
authors.lastname: Browne
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D000914:Antibodies, Viral / Q000378:metabolism; D019556:COS Cells; D002460:Cell Line; D002522:Chlorocebus aethiops; D018259:Herpesvirus 1, Human / Q000235:genetics*; D006801:Humans; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000235:genetics; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D011134:Polysaccharides; D014759:Viral Envelope Proteins; C044200:glycoprotein H, herpes simplex virus type 1