Pseudorabies virus glycoprotein C attachment-proficient revertants isolated through a simple, targeted mutagenesis...

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.