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

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.