title: Use of a negative selectable marker for rapid selection of recombinant vaccinia virus.
abstract: Vaccinia virus has been a powerful tool in molecular biology and vaccine development. The relative ease of inserting and expressing foreign genes combined with its broad host range has made it an attractive antigen delivery system against many heterologous diseases. Many different approaches have been developed to isolate recombinant vaccinia virus generated from homologous recombination; however, most are time-consuming, often requiring a series of passages or specific cell lines. Herein we introduce a rapid method for isolating recombinants using the antibiotic coumermycin and the interferon-associated PKR pathway to select for vaccinia virus recombinants. This method uses a negative selection marker in the form of a fusion protein, GyrB-PKR, consisting of the coumermycin dimerization domain of Escherichia coli gyrase subunit B fused to the catalytic domain of human PKR. Coumermycin-dependent dimerization of this protein results in activation of PKR and the phosphorylation of translation initiation factor, eIF2α. Phosphorylation of this factor leads to an inhibition of protein synthesis, and an inhibition of virus replication. In the presence of coumermycin, recombinants are isolated due to the loss of this coumermycin-sensitive gene by homologous recombination. We demonstrate that this method of selection is highly efficient and requires limited rounds of enrichment to isolate recombinant virus.
authors.affiliation: Center for Infectious Diseases and Vaccinology, Biodesign Institute, Arizona State University, Tempe, Arizona, USA.
authors.forename: Stacy D
authors.initials: SD
authors.lastname: White
authors.forename: Kip
authors.initials: K
authors.lastname: Conwell
authors.forename: Jeffrey O
authors.initials: JO
authors.lastname: Langland
authors.forename: Bertram L
authors.initials: BL
authors.lastname: Jacobs
mesh_terms: D049933:Aminocoumarins / Q000737:chemistry / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015852:Eukaryotic Initiation Factor-2 / Q000737:chemistry*; D005822:Genetic Vectors; D058507:Host Specificity / Q000187:drug effects; D011500:Protein Synthesis Inhibitors / Q000737:chemistry / Q000494:pharmacology; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000187:drug effects / Q000302:isolation & purification; D011995:Recombination, Genetic / Q000187:drug effects; D014616:Vaccinia virus / Q000187:drug effects / Q000254:growth & development / Q000302:isolation & purification*; D014779:Virus Replication / Q000187:drug effects / Q000235:genetics
chemical_list: D049933:Aminocoumarins; D015852:Eukaryotic Initiation Factor-2; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; C004628:coumermycin