Herpes simplex virus type 1 DNA amplified as bacterial artificial chromosome in Escherichia coli: Rescue of replication-competent virus progeny and packaging of amplicon vectors

Herpes simplex virus type 1 (HSV-1)-based amplicon vectors contain only sim ilar to 1% of the 152-kb HSV-1 genome, and consequently, replication and pa ckaging into virions depends on helper functions. These helper functions ha ve been provided conventionally by a helper virus, usually a replication-de fective mutant of HSV-1, or more recently, by a set of five cosmids that ov erlap and represent the genome of HSV-1 deleted for DNA cleavage/packaging signals (pac). In the absence of pac signals, potential HSV-1 genomes that are reconstituted from the cosmids via homologous recombination are not pac kageable, The resulting amplicon stocks are, therefore, virtually free of c ontaminating helper virus. To simplify this packing system, the HSV-1 genom e was cloned and maintained stably as a single-copy, F plasmid-based bacter ial artificial chromosome in E. coli. Such a plasmid containing the HSV-1 g enome deleted for the pac signals (fHSV Delta pac) did not generate replica tion-competent progeny virus on transfection into mammalian cells, but rath er, it was able to support the packaging of cotransfected amplicon DNA that contained a functional pac signal. The resulting amplicon vector stocks ha d titers of up to 10(7) transducing units per milliliter of culture medium and efficiently transduced neural cells in the rat brain, as well as hepato cytes in the rat. The capacity of generating infectious and replication-com petent HSV-1 progeny following transfection into mammalian cells was restor ed after insertion of a pac signal into fHSV Delta pac.