Increased transcomplementation properties of plasmids carrying HSV-1 origin of replication and packaging signals.
This report describes a very simple method that facilitates the analysis of the functional activity of essential genes of herpes simplex virus type 1 (HSV-1) in cell culture. The method, which depends on the use 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 42 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 gene. In nonpermissive Vero cells transfected with amplicon plasmid pA-UL42 and superinfected with the defective Cgal delta 42 virus, both the plasmid and the helper genomes were amplified and packaged, giving rise to a self-complementing amplicon/helper virus population able to disseminate and to form lytic plaques in cell culture. These plaques were due to complementation and not to recombination between the defective virus and the amplicon plasmid as confirmed by Southern blot analysis of individual plaques. No complementation was observed by superinfection of cells transfected with the noncomplementing pA-delta UL42 amplicon 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 observed in these systems resulted from the spread of competent recombinant viruses. Results presented in this work indicate that (i) self-complementation between a defective virus and an amplicon plasmid provides a strong and very sensitive method to assess function activity of an HSV-1 essential gene in a one-step experiment and (ii) oris-carrying plasmids could be instrumental in the production and selection of recombinant HSV-1 vectors.
Berthomme
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H
Centre de Génétique Moléculaire et Cellulaire, UMR 106-CNRS, Université Claude Bernard-Lyon I, France.
Fournel
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Epstein
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D000818:Animals; D002522:Chlorocebus aethiops; D004279:DNA, Viral / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D006378:Helper Viruses / Q000235:genetics; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000502:physiology; D006801:Humans; D018741:Replication Origin / Q000235:genetics*; D014162:Transfection; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics; D019065:Virus Assembly / Q000235:genetics*
D004279:DNA, Viral; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus