Genetic and Protein Engineering

We describe a way to introduce site-specific mutations to the genome of Autographa californica nuclear polyhedrosis virus. Particularly, the A. californica nuclear polyhedrosis virus gene for polyhedrin, the primary protein that forms viral occlusions in infected cells, was mutagenized by presenting deletions to the cloned DNA fragment which contains the gene. The mutagenized polyhedrin gene was utilized within the intact viral DNA by mixing fragment and viral DNAs, cotransfecting Spodoptera frugiperda cells, and screening for viral recombinants which in fact had been through allelic exchange. Recombinant infections with mutant polyhedrin genes were acquired by selecting the progeny virus that did not produce viral occlusions in infected cells (occlusion-negative mutants). Analyses of occlusion-negative mutants proven the polyhedrin gene wasn't needed

for creating infectious virus which deletion of certain sequences within the gene did not modify the control, or decrease the quantity of expression, of polyhedrin. An early on viral protein of 22,000 molecular weight was apparently not needed for virus replication in vitro, since the synthesis from the protein wasn't detected in cells have been infected with a mutant virus.