IDENTIFICATION AND CHARACTERIZATION OF A 2ND PUTATIVE ORIGIN OF DNA-REPLICATION IN A BACULOVIRUS OF ORGYIA-PSEUDOTSUGATA

A 7.5-kb region (96.8-2.5 m.u.), called Op5, of the Orgyia pseudotsuga ta multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome that c ontains an origin of DNA replication was characterized. This region re plicates several times more efficiently and is unrelated to the previo usly identified putative origin of replication located on the viral Hi ndIII-N fragment. In contrast to HindIII-N, the origin on Op5 contains repeated sequences with limited sequence identity to the homologous r egions from AcMNPV. In isolation, these repeated sequences were not su fficient for origin activity in OpMNPV-infected Lymantria dispar cells , but required an additional 1.3 kb of sequences located to the left o f the repeats. Four regions of the OpMNPV genome that crosshybridize w ith the repeated region were also found to replicate in our infection- dependent DNA replication assay. A deletion clone of Op5 that replicat es efficiently in OpMNPV-infected L. dispar cells, was found to replic ate at less than 2% the replication level of AcMNPV hr2 in AcMNPV-infe cted Spodoptera frugiperda cells. (C) 1995 Academic Press, Inc.