Functional mapping of regions of the Autographa californica nuclear polyhedrosis viral genome required for DNA replication.

Previous results showed that plasmids containing one of the eight putative origins (ori's) of Autographa californica nuclear polyhedrosis virus (AcMNPV) are replicated after transfection into Spodoptera frugiperda cells if essential trans-acting factors are supplied by AcMNPV infection (Kool et al., Virology, 192, 94-101, 1993a; Kool et al., J. Gen. Virol., in press, 1993b; Leisy and Rohrmann, Virology, 196, 722-730, 1993). In this report a transient complementation assay is described in which four cotransfected cosmid clones, instead of AcMNPV infection, provided essential trans-acting factors for plasmid DNA replication. In this assay plasmid replication was found to be independent of the presence, in cis, of a viral ori. No replication of plasmids occurred when one of the cosmids was omitted from the transfection mixture. This result indicated that this assay is a valid approach for identification of AcMNPV replication genes. We further used the assay to define essential regions in the four required cosmids. Six regions of the AcMNPV genome, EcoRI-I (map unit 0.3-5.8), EcoRI-O (map unit 6.9-8.7), SstI-F (map unit 38.9-45.0), EcoRI-D (map unit 59.9-68.3), a BamHI-SstII fragment of BamHI-B (map unit 84.3-89.7), and EcoRI-B (map unit 90.0-100), with at least seven genes, were found to be essential for plasmid DNA replication. These regions contain the putative DNA polymerase gene (SstI-F), the helicase-like gene (EcoRI-D), and the region where most of the trans-activating immediate--early genes of AcMNPV are located (EcoRI-B). For SstI-F it was shown that this region contains besides the DNA polymerase gene at least one other replication gene. These results show that it will now be possible to define the set of AcMNPV genes necessary and sufficient for DNA replication.