title: Pseudorabies virus glycoprotein C attachment-proficient revertants isolated through a simple, targeted mutagenesis scheme.
abstract: Pseudorabies virus (PRV) glycoprotein C (gC) initiates virus attachment to cells by binding to heparan sulfate (HS) proteoglycans. The gC:HS interaction is not essential since gC null mutants still infect; however, they are more easily removed from cells during the initial stages of infection. The expendability of gC has facilitated a genetic mapping of the HS-binding domain, which is composed of three independent heparin-binding domains (HBDs) of six to eight amino acids each. Previous results suggested that at least one of the HBDs (HBD 1) functioned in a context-dependent manner. To define the context better, a reversion analysis was performed in which a defective gC containing a nonfunctional but intact HBD 1 regained HS-binding ability. To increase the reversion frequency, an efficient method for targeted, yet random mutagenesis of the gC gene was developed. The method involves random mutagenesis of a plasmid-borne copy of gC, and highly efficient recombination of the plasmid-borne genes into the virus genome at the site of a double-strand break in the viral gC locus. Revertants were recovered readily, and their gC alleles suggested that HS-binding could be restored by several different amino acid substitutions. This approach should be applicable to targeted mutagenesis of other herpesvirus genes.
authors.affiliation: Department of Molecular Sciences, University of Tennessee Health Science Center, Memphis, TN 38163 USA. Cary.Rue@ars.usda.gov
authors.forename: Cary A
authors.initials: CA
authors.lastname: Rue
authors.forename: Patrick
authors.initials: P
authors.lastname: Ryan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism; D011558:Herpesvirus 1, Suid / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity*; D007668:Kidney / Q000166:cytology / Q000821:virology; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D013552:Swine; D014759:Viral Envelope Proteins / Q000235:genetics* / Q000378:metabolism; D014773:Virology / Q000379:methods
chemical_list: D019812:Heparan Sulfate Proteoglycans; D014759:Viral Envelope Proteins; C042966:pseudorabies virus glycoproteins; D008769:Methylnitronitrosoguanidine