Random mutagenesis of the gene encoding a viral ligand for multiple cell entry receptors to obtain viral mutants altered for receptor usage

doi:10.1073/pnas.0407892101

Random mutagenesis of the gene encoding a viral ligand for multiple cell entry receptors to obtain viral mutants altered for receptor usage

Miri Yoon and
Patricia G. Spear*

Department of Microbiology–Immunology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611

Contributed by Patricia G. Spear, October 25, 2004

Abstract

Herpes simplex virus type 1 (HSV-1) can enter cells expressing any one of multiple entry receptors, including the herpesvirus entry mediator (HVEM), nectin-1, and sites in heparan sulfate generated by specific 3-O-sulfotransferases. The viral ligand for these receptors is glycoprotein D (gD). To define structural requirements for functional interactions of gD with its receptors and to obtain viral mutants altered for receptor usage, we generated a library of HSV-1 mutants with random mutations in the gD gene. Viral isolates selected on a monkey cell line (Vero) were screened for the loss of ability to infect cells expressing each of the HSV-1 receptors. The 10 HSV-1 mutants obtained had 12 mutations in gD, affecting 11 amino acids. All mutations reduced or abrogated viral entry through HVEM and 3-O-sulfated heparan sulfate, indicating that similar features of gD are critical for functional interactions with both these receptors. None of the mutations reduced viral entry through nectin-1, whereas a subset of the mutations conferred ability to use nectin-2 as an entry receptor. These and other results show that features of gD, including conformation of the N terminus, critical for functional interactions with HVEM/3-O-sulfated heparan sulfate, differ from those critical for interactions with nectin-1.

Footnotes

↵ * To whom correspondence should be addressed. E-mail: p-spear{at}northwestern.edu.
Author contributions: M.Y. and P.G.S. designed research; M.Y. performed research; M.Y. and P.G.S. analyzed data; and M.Y. and P.G.S. wrote the paper.
Abbreviations: FRT, FLP recombination target; gD, glycoprotein D; HSV-1, herpes simplex virus type 1; HVEM, herpesvirus entry mediator; 3-O-S HS, 3-O-sulfated heparan sulfate; pfu, plaque-forming units.