Characterization of the Replication of Plasmids Containing hr Sequences in Baculovirus-Infected Spodoptera frugiperda Cells

Abstract

The replication of a series of plasmids, each containing one or more of 6 homologous regions ( hrs) from the Autographa californica multinucleocapsid nuclear polyhedrosis virus (AcMNPV) genome, was investigated in AcMNPV-infected Spodoptera frugiperda cells. Although a construct containing hr2 showed the most efficient replication, the sequences flanking the hr region appeared to influence the levels of replication. Plasmids with non- hr-containing AcMNPV inserts showed greatly reduced replication relative to hr-containing plasmids. A variety of features of hr -containing plasmid replication were characterized. These included the time course of plasmid replication and the influence of plasmid concentration and multiplicity of infection of the helper AcMNPV on replication efficiency. It was found that replicated plasmid DNA was detectable by 36 hr p.i. and plateaued by 60 hr p.i. Under our experimental conditions, plasmid levels of 0.5-2 μg/1.25 × 10 6 cells and multiplicities of infection of 0.1 to 10 produced optimal levels of plasmid replication. The transfection procedure was shown to delay viral DNA replication by about 12 hr. Experiments directed toward determining the form of the replicated hr -containing plasmid DNA were conducted using partial restriction enzyme digestion. The results indicated that the AcMNPV-replicated plasmids were composed of high molecular weight concatemers.