The 35-kilodalton protein gene (p35) of Autographa californica nuclear polyhedrosis virus and the neomycin resistance gene provide dominant selection of recombinant baculoviruses.
Autographa californica nuclear polyhedrosis virus (AcMNPV) recombinants were constructed to test the effectiveness of the AcMNPV 35-kilodalton protein gene (35K gene) and the bacterial neomycin resistance gene (neo) as dominant selectable markers for baculoviruses. Insertion of the AcMNPV apoptosis suppressor gene (p35) into the genome of p35-deletion mutants inhibited premature host cell death and increased virus yields up to 1200-fold at low multiplicities in Spodoptera frugiperda (SF21) cell cultures. When placed under control of an early virus promoter, the bacterial neomycin resistance gene (neo) restored multiplication of AcMNPV in the same cells treated with concentrations of the antibiotic G418 that inhibited wild-type virus growth greater than 1000-fold. The selectivity of these dominant markers was compared by serial passage of recombinant virus mixtures. After four passages, the proportion of p35-containing virus increased as much as 2,000,000-fold relative to deletion mutants, whereas the proportion of neo-containing viruses increased 500-fold relative to wild-type virus under G418 selection. The strength and utility of p35 as a selectable marker was further demonstrated by the construction of AcMNPV expression vectors using polyhedrin-based transfer plasmids that contain p35. Recombinant viruses with foreign gene insertions at the polyhedrin locus accounted for 15 to 30% of the transfection progeny. The proportion of desired viruses was increased to greater than 90% by linearizing the parental virus DNA at the intended site of recombination prior to transfection. These results indicate that p35 and neo facilitate the selection of baculovirus recombinants and that p35, in particular, is an effective marker for the generation of AcMNPV expression vectors.
Institute for Molecular Virology, Graduate School and College of Agricultural and Life Sciences, University of Wisconsin, Madison 53706-1596.
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Lerch
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Friesen
D000818:Animals; D016367:Baculoviridae / Q000235:genetics* / Q000254:growth & development; D002460:Cell Line; D004274:DNA, Recombinant / Q000032:analysis; D004279:DNA, Viral / Q000032:analysis; D004352:Drug Resistance, Microbial / Q000235:genetics; D005814:Genes, Viral; D005819:Genetic Markers; D009036:Moths; D009355:Neomycin / Q000494:pharmacology*; D014764:Viral Proteins / Q000235:genetics*
D004274:DNA, Recombinant; D004279:DNA, Viral; D005819:Genetic Markers; D014764:Viral Proteins; D009355:Neomycin