Allele replacement: an application that permits rapid manipulation of herpes simplex virus type 1 genomes

Abstract

Herpes simplex virus (HSV) is a new platform for gene therapy. We cloned the human herpesvirus HSV-1 strain F genome into a bacterial artificial chromosome (BAC) and adapted chromosomal gene replacement technology to manipulate the viral genome. This technology exploits the power of bacterial genetics and permits generation of recombinant viruses in as few as 7 days. We utilized this technology to delete the viral packaging/cleavage (pac) sites from HSV-BAC. HSV-BAC DNA is stable in bacteria and the pac-deleted HSV-BAC (p45–25) is able to package amplicon plasmid DNA as efficiently as a comparable pac-deleted HSV cosmid set when transfected into mammalian cells. Moreover, the utility of bacterial gene replacement is not limited to HSV, since most herpesviruses can be cloned as BACs. Thus, this technology will greatly facilitate genetic manipulation of all herpesviruses for their use as research tools or as vectors in gene therapy.