IDENTIFICATION OF ESSENTIAL TRANS-ACTING REGIONS REQUIRED FOR DNA-REPLICATION OF THE ORGYIA-PSEUDOTSUGATA MULTINUCLEOCAPSID NUCLEAR POLYHEDROSIS-VIRUS - LEF-1 IS AN ESSENTIAL REPLICATION GENE

A transient replication assay for the identification of baculovirus ge nes that are essential for replication of an origin-containing reporte r plasmid was established for the Orgyia pseudotsugata muitinucleocaps id nuclear polyhedrosis virus (OpMNPV). Using a replication origin loc ated on the OpMNPV HindIII-N fragment, we identified a subset of cosmi ds and plasmids from an OpMNPV cosmid library that was able to supply all the essential trans acting factors and support replication of the origin-containing plasmid in uninfected Lymantria dispar cells. Howeve r, this limited set of DNA's was unable to support replication of a se cond origin-containing plasmid derived from a different region of the OpMN PV genome. Replication analysis of deletion clones of the HindIII -N fragment led to the identification of a gene, late expression facto r 1 (lef-1), that is essential for the transactivation of DNA replicat ion in this system. Transcriptional analysis of lef-1 mapped both earl y and late transcripts of about 1.75 kb. A motif characteristic of nuc leoside triphosphate-binding sites present in the carboxyterminal regi on of AcMNPV Lef-1 is not conserved in OpMNPV Lef-1. (C) 1995 Academic Press, Inc.