Identification and functional analysis of a non-hr origin of DNA replication in the genome of Spodoptera exigua multicapsid nucleopolyhedrovirus.

Abstract

The genome of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) was screened for the presence of putative origins of DNA replication (oris). Using a transient DNA replication assay, several fragments were identified that underwent SeMNPV-dependent DNA replication in Spodoptera frugiperda cells (Sf-AE-21). Preliminary sequence data revealed the presence of multiple copies of homologous repeats (hrs). Restriction fragment Xbal-F2 showed a distinct sequence reminiscent of Autographa californica and Orgyla pseudotsugata MNPV (AcMNPV and OpMNPV) non-hr oris. Deletion analysis of this fragment indicated that the essential sequences of this putative non-hr ori mapped within a region of 800 bp. Sequence analysis of this region showed a unique distribution of six different (im)-perfect palindromes, several polyadenylation motifs and the occurrence of multiple direct repeats. No sequence homology or similarities to other reported baculovirus oris were detected. The spatial and modular distribution of these motifs are similar to those of the non-hr oris of AcMNPV and OpMNPV. Comparison of baculovirus non-hr and consensus eukaryotic oris revealed no consensus ori but indicated that each of the non-hrs studied so far is unique. From the structural similarity, however, it was concluded that the SeMNPV Xbal-F2 ori represents a baculovirus non-hr type ori. In addition, evidence is provided that SeMNPV renders more specificity to baculovirus DNA replication than AcMNPV.