REPLICATION OF ORGYIA-PSEUDOTSUGATA BACULOVIRUS DNA - LEF-2 AND IE-1 ARE ESSENTIAL AND IE-2, P34, AND OP-IAP ARE STIMULATORY GENES

A transient DNA replication assay was used to identify genes located w ithin m.u. 90.5-7.0 of the Orgyia pseudotsugata multinucleocapsid nucl ear polyhedrosis virus (OpMNPV) genome that influenced replication of a reporter plasmid containing an OpMNPV origin of replication, when co transfected into uninfected Lymantria dispar cells. The viral transact ivator ie-1 and a 2.4-kb subclone were found to be essential for repli cation. The 2.4-kb region was sequenced and open reading frames were i dentified. Replication assays using subclones from this region identif ied a gene called late expression factor 2 (lef-2), as the essential r eplication gene. The OpMNPV lef-2 gene encodes a protein with a predic ted 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 Aut ographa 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 transcri pt 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 th ree genes stimulatory for DNA replication including two OpMNPV transcr iptional activators (ie-2 and p34) and Op-iap, which is the functional analog of the AcMNPV p35 gene that inhibits apoptosis in AcMNPV-infec ted Spodoptera frugiperda cells. (C) 1995 Academic Press, Inc.