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Thep143 gene fromAutographa californica multinucleocapsid nucleopolyhedrovirus (Ac MNPV) has been found to increase the expression of luciferase, which is driven by the polyhedrin gene promoter, in a plasmid with virus coinfection. Further study indicated that this is due to the presence of a replication origin (ori ) in the coding region of this gene. Transient DNA replication assays showed that a specific fragment of thep143 coding sequence,p143 -3, underwent virus-dependent DNA replication inSpodoptera frugiperda IPLB-Sf-21 (Sf-21) cells. Deletion analysis of thep143 -3 fragment showed that subfragmentp143 -3.2a contained the essential sequence of this putativeori . Sequence analysis of this region revealed a unique distribution of imperfect palindromes with high AT contents. No sequence homology or similarity betweenp143 -3.2a and any other knownori was detected, suggesting that it is a novel baculovirusori . Further study showed that thep143 -3.2aori can replicate more efficiently in infected Sf-21 cells than baculovirus homologous regions (hr s), the major baculovirusori , or non-hr ori s during virus replication. Previously,hr on its own was unable to replicate in mammalian cells, and for mammalian viralori s, viral proteins are generally required for their proper replication in host cells. However, thep143 -3.2aori was, surprisingly, found to function as an efficientori in mammalian cells without the need for any viral proteins. We conclude thatp143 contains a unique sequence that can function as anori to enhance gene expression in not only insect cells but also mammalian cells.IMPORTANCE Baculovirus DNA replication relies on bothhr and non-hr ori s; however, so far very little is known about the latterori s. Here we have identified a new non-hr ori , thep143 ori , which resides in the coding region ofp143 . By developing a novel DNA replication-enhanced reporter system, we have identified and located the core region required for thep143 ori . Thisori contains a large number of imperfect inverted repeats and is the most activeori in the viral genome during virus infection in insect cells. We also found that it is a uniqueori that can replicate in mammalian cells without the assistance of baculovirus gene products. The identification of thisori should contribute to a better understanding of baculovirus DNA replication. Also, thisori is very useful in assisting with gene expression in mammalian cells.

Identification of a High-Efficiency Baculovirus DNA Replication Origin That Functions in Insect and Mammalian Cells