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

Abstract

A transient replication assay for the identification of baculovirus genes that are essential for replication of an origin-containing reporter plasmid was established for the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV). Using a replication origin located on the OpMNPV HindIII-N fragment, we identified a subset of cosmids 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. However, this limited set of DNA's was unable to support replication of a second origin-containing plasmid derived from a different region of the OpMNPV genome. Replication analysis of deletion clones of the HindIII-N fragment led to the identification of a gene, late expression factor 1 (lef-1), that is essential for the transactivation of DNA replication in this system. Transcriptional analysis of lef-1 mapped both early and late transcripts of about 1.75 kb. A motif characteristic of nucleoside triphosphate-binding sites present in the carboxy-terminal region of AcMNPV Lef-1 is not conserved in OpMNPV Lef-1.