INCREASED TRANSCOMPLEMENTATION PROPERTIES OF PLASMIDS CARRYING HSV-1 ORIGIN OF REPLICATION AND PACKAGING SIGNALS

This report describes a very simple method that facilitates the analys is of the functional activity of essential genes of herpes simplex vir us type 1 (HSV-1) in cell culture. The method, which depends on the us e of complementing plasmids containing a virus origin of replication ( ori(s)) and a packaging signal (amplicon plasmids), was tested for its ability to detect complementation of the defective HSV-1 Cgal Delta 4 2 virus, a UL42 null mutant, by amplicon plasmids carrying either wild -type (pA-UL42) or mutated alleles (pA-Delta UL42) of the HSV-1 UL42 g ene. In nonpermissive Vero cells transfected with amplicon plasmid pA- UL42 and superinfected with the defective Cgal Delta 42 virus, both th e plasmid and the helper genomes were amplified and packaged, giving r ise to a self-complementing amplicon/helper virus population able to d isseminate and to form lytic plaques in cell culture. These plaques we re due to complementation and not to recombination between the defecti ve virus and the amplicon plasmid as confirmed by Southern blot analys is of individual plaques. No complementation was observed by superinfe ction of cells transfected with the noncomplementing pA-Delta UL42 amp licon plasmid. Instead, small foci were observed in cells transfected with plasmids which express wild-type UL42 but were unable to amplify or to become packaged, and the few true lytic plaques that were observ ed in these systems resulted from the spread of competent recombinant viruses. Results presented in this work indicate that (i) self-complem entation between a defective virus and an amplicon plasmid provides a strong and very sensitive method to assess functional activity of an H SV-1 essential gene in a one-step experiment and (ii) ori(s)-carrying plasmids could be instrumental in the production and selection of reco mbinant HSV-1 vectors. (C) 1996 Academic Press, Inc.