title: 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.
abstract: Herpes simplex virus type 1 (HSV-1)-based amplicon vectors contain only approximately 1% of the 152-kb HSV-1 genome, and consequently, replication and packaging into virions depends on helper functions. These helper functions have been provided conventionally by a helper virus, usually a replication-defective mutant of HSV-1, or more recently, by a set of five cosmids that overlap 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 packageable. The resulting amplicon stocks are, therefore, virtually free of contaminating helper virus. To simplify this packing system, the HSV-1 genome was cloned and maintained stably as a single-copy, F plasmid-based bacterial artificial chromosome in E. coli. Such a plasmid containing the HSV-1 genome deleted for the pac signals (fHSV delta pac) did not generate replication-competent progeny virus on transfection into mammalian cells, but rather, it was able to support the packaging of cotransfected amplicon DNA that contained a functional pac signal. The resulting amplicon vector stocks had 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 hepatocytes in the rat. The capacity of generating infectious and replication-competent HSV-1 progeny following transfection into mammalian cells was restored after insertion of a pac signal into fHSV delta pac.
authors.affiliation: Department of Neurosurgery, Massachusetts General Hospital, Harvard Medical School, Charlestown 02129, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Saeki
authors.forename: T
authors.initials: T
authors.lastname: Ichikawa
authors.forename: A
authors.initials: A
authors.lastname: Saeki
authors.forename: E A
authors.initials: EA
authors.lastname: Chiocca
authors.forename: K
authors.initials: K
authors.lastname: Tobler
authors.forename: M
authors.initials: M
authors.lastname: Ackermann
authors.forename: X O
authors.initials: XO
authors.lastname: Breakefield
authors.forename: C
authors.initials: C
authors.lastname: Fraefel
mesh_terms: D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D002876:Chromosomes, Bacterial / Q000235:genetics*; D003001:Cloning, Molecular; D003360:Cosmids; D003588:Cytopathogenic Effect, Viral; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000502:physiology; D006624:Hippocampus / Q000821:virology; D006801:Humans; D008099:Liver / Q000821:virology; D010957:Plasmids / Q000235:genetics; D051381:Rats; D014162:Transfection; D014709:Vero Cells; D014771:Virion / Q000235:genetics; D019065:Virus Assembly
chemical_list: D004279:DNA, Viral