THE 35-KILODALTON PROTEIN GENE (P35) OF AUTOGRAPHA-CALIFORNICA NUCLEAR POLYHEDROSIS-VIRUS AND THE NEOMYCIN RESISTANCE GENE PROVIDE DOMINANTSELECTION OF RECOMBINANT BACULOVIRUSES

Autographa californica nuclear polyhedrosis virus (AcMNPV) recombinant s were constructed to test the effectiveness of the AcMNPV 35-kilodalt on protein gene (35K gene) and the bacterial neomycin resistance gene (neo) as dominant selectable markers for baculoviruses. Insertion of t he AcMNPV apoptosis suppressor gene (p35) into the genome of p35-delet ion mutants inhibited premature host cell death and increased virus yi elds up to 1200-fold at low multiplicities in Spodoptera frugiperda (S F21) cell cultures. When placed under control of an early virus promot er, the bacterial neomycin resistance gene (neo) restored multiplicati on of AcMNPV in the same cells treated with concentrations of the anti biotic G418 that inhibited wild-type virus growth greater than 1000-fo ld. The selectivity of these dominant markers was compared by serial p assage of recombinant virus mixtures. After four passages, the proport ion of p35-containing virus increased as much as 2,000,000-fold relati ve to deletion mutants, whereas the proportion of neo-containing virus es increased 500-fold relative to wild-type virus under G418 selection . The strength and utility of p35 as a selectable marker was further d emonstrated by the construction of AcMNPV expression vectors using pol yhedrin-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 DN A at the intended site of recombination prior to transfection. These r esults indicate that p35 and neo facilitate the selection of baculovir us recombinants and that p35, in particular, is an effective marker fo r the generation of AcMNPV expression vectors.