Analysis of an Autographa californica Nucleopolyhedrovirus lef-11 Knockout: LEF-11 Is Essential for Viral DNA Replication

ABSTRACT The Autographa californica nucleopolyhedrovirus (Ac M NPV) lef-11 gene was previously identified by transient late expression assays as a gene important for viral late gene expression. The lef-11 gene was not previously identified as necessary for DNA replication in transient origin-dependent plasmid DNA replication assays. To examine the role of lef-11 in the context of the infection cycle, we generated a deletion of the lef-11 gene by recombination in an Ac M NPV genome propagated as a BACmid in Escherichia coli . The resulting Ac M NPV lef-11- null BACmid (vAc lef11KO ) was unable to propagate in cell culture, although a “repair” Ac M NPV BACmid (vAc lef11KO-REP ), which was generated by transposition of the lef-11 gene into the polyhedrin locus of the vAc lef11KO BACmid, was able to replicate in a manner similar to wild-type or control Ac M NPV viruses. Thus, the lef-11 gene is essential for viral replication in Sf9 cells. The vAc lef11KO BACmid was examined to determine if the defect in viral replication resulted from a defect in DNA replication or from a defect in late transcription. The lef-11- null BACmid and control BACmids were transfected into Sf9 cells, and viral DNA replication was monitored. The viral DNA genome of the lef-11- null BACmid (vAc lef11KO ) was not amplified, whereas replication and amplification of the genomes of the repair BACmid (vAc lef11KO-REP ), wild-type Ac M NPV, and a nonpropagating gp64- null control BACmid (vAc GUSgp64KO ) were readily detected. Northern blot analysis of transcripts from selected early, late, and very late genes showed that late and very late transcription was absent in cells transfected with the lef-11- null BACmid. Thus, in contrast to prior studies using transient replication and late expression assays, studies of a lef-11- null BACmid indicate that LEF-11 is required for viral DNA replication during the infection cycle.