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Identification and functional analysis of a putative non-hr origin of DNA replication from the Spodoptera littoralis type B multinucleocapsid nucleopolyhedrovirus

Department of Biology, University of Victoria, PO Box 3020, Victoria, British Columbia, Canada V8W 3N5¹

Author for correspondence: David B. Levin.Fax +1 250 472 4075. e-mail dlevin{at}uvic.ca

A putative non-hr origin of DNA replication was identified in the Spodoptera littoralis multinucleocapsid nucleopolyhedrovirus (SpliNPV) genome by transient replication assays. The putative SpliNPV ori was mapped to the PstI-J fragment between 75·1–77·9 map units in the SpliNPV genome. While the DNA sequence of the putative SpliNPV ori aligned with regions within the non-hr oris of Autographa californica, Orgyia pseudotsugata and Spodoptera exigua multinucleocapsid nucleopolyhedroviruses, it has limited DNA sequence identity with these elements. The sequence of the putative SpliNPV non-hr ori fragment contains a unique distribution of imperfect palindromes, multiple direct repeats and putative transcription factor-binding sites. Transient expression assays indicated that the putative SpliNPV ori fragment repressed SpliNPV lef-3 promoter-mediated luciferase reporter gene expression. However, the putative SpliNPV ori fragment itself was capable of directing luciferase expression in the absence of a recognizable baculovirus promoter element in an orientation-independent fashion, suggesting that DNA sequence motifs within its sequence can activate transcription. Gel mobility shift analyses confirmed that proteins within nuclear extracts from both uninfected and virus-infected cells bound with specificity to the putative SpliNPV ori fragment.

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