Replication of Orgyia pseudotsugata Baculovirus DNA: lef-2 and ie-1 Are Essential and ie-2, p34, and Op-iap Are Stimulatory Genes

Abstract

A transient DNA replication assay was used to identify genes located within m.u. 90.5-7.0 of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome that influenced replication of a reporter plasmid containing an OpMNPV origin of replication, when cotransfected into uninfected Lymantria dispar cells. The viral transactivator ie-1 and a 2.4-kb subclone were found to be essential for replication. The 2.4-kb region was sequenced and open reading frames were identified. Replication assays using subclones from this region identified a gene called late expression factor 2 (lef-2), as the essential replication gene. The OpMNPV lef-2 gene encodes a protein with a predicted molecular weight of 22.7 kDa (204 amino acids) and exhibits 54.7% amino acid sequence identity with its homolog in the genome of the Autographa californica MNPV. Transcriptional mapping using both Northern blot and S1 nuclease protection assays demonstrated that OpMNPV lef-2 was expressed at both early and late times postinfection as a transcript of about 1.6 kb. The early transcript initiated approximately 30 nt downstream of a TAATA box, whereas the late transcript initiated from within a late promoter consensus motif. In addition, we identified three genes stimulatory for DNA replication including two OpMNPV transcriptional activators (ie-2 and p34) and Op-iap, which is the functional analog of the AcMNPV p35 gene that inhibits apoptosis in AcMNPV-infected Spodoptera frugiperda cells.