Characterization of the replication of a baculovirus mutant lacking the DNA polymerase gene

AbstractIn a previous study, the DNA polymerase gene (dnapol) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was identified as one of six genes required for plasmid replication in a transient replication assay (M. Kool, C. Ahrens, R.W. Goldbach, G.F. Rohrmann, J.M. Vlak, Identification of genes involved in DNA replication of the Autographa californica, Proc. Natl. Acad. Sci. U.S.A. 91, (1994) 11212–11216); however, another study based on a similar approach reported that the virally encoded polymerase was only stimulatory (A. Lu, L.K. Miller, The roles of 18 baculovirus late expression factor genes in transcription and DNA replication, J. Virol. 69, (1995) 975–982). To reconcile the conflicting data and determine if the AcMNPV DNA polymerase is required for viral DNA replication during the course of an infection, a dnapol-null virus was generated using bacmid technology. To detect viral DNA replication, a highly sensitive assay was designed based on real-time PCR and SYBR green chemistry. Our results indicate that a bacmid in which the dnapol ORF was deleted is unable to replicate its DNA when transfected into Spodoptera frugiperda (Sf-9) cells, although when the dnapol ORF was introduced into the polyhedrin (polh) locus, this repaired virus could propagate at levels similar to the control virus. These results confirm that the AcMNPV-encoded DNA polymerase is required for viral DNA replication and the host DNA polymerases cannot substitute for the viral enzyme in this process