title: Identification of a high-efficiency baculovirus DNA replication origin that functions in insect and mammalian cells.
abstract: The p143 gene from Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) 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 the p143 coding sequence, p143-3, underwent virus-dependent DNA replication in Spodoptera frugiperda IPLB-Sf-21 (Sf-21) cells. Deletion analysis of the p143-3 fragment showed that subfragment p143-3.2a contained the essential sequence of this putative ori. Sequence analysis of this region revealed a unique distribution of imperfect palindromes with high AT contents. No sequence homology or similarity between p143-3.2a and any other known ori was detected, suggesting that it is a novel baculovirus ori. Further study showed that the p143-3.2a ori can replicate more efficiently in infected Sf-21 cells than baculovirus homologous regions (hrs), the major baculovirus ori, or non-hr oris during virus replication. Previously, hr on its own was unable to replicate in mammalian cells, and for mammalian viral oris, viral proteins are generally required for their proper replication in host cells. However, the p143-3.2a ori was, surprisingly, found to function as an efficient ori in mammalian cells without the need for any viral proteins. We conclude that p143 contains a unique sequence that can function as an ori to enhance gene expression in not only insect cells but also mammalian cells.
  
  
  OBJECTIVE
  Baculovirus DNA replication relies on both hr and non-hr oris; however, so far very little is known about the latter oris. Here we have identified a new non-hr ori, the p143 ori, which resides in the coding region of p143. By developing a novel DNA replication-enhanced reporter system, we have identified and located the core region required for the p143 ori. This ori contains a large number of imperfect inverted repeats and is the most active ori in the viral genome during virus infection in insect cells. We also found that it is a unique ori that can replicate in mammalian cells without the assistance of baculovirus gene products. The identification of this ori should contribute to a better understanding of baculovirus DNA replication. Also, this ori is very useful in assisting with gene expression in mammalian cells.
authors.affiliation: Department of Entomology, National Taiwan University, Taipei, Taiwan.
authors.forename: Yueh-Lung
authors.initials: YL
authors.lastname: Wu
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan.
authors.forename: Carol-P
authors.initials: CP
authors.lastname: Wu
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan.
authors.forename: Yu-Hui
authors.initials: YH
authors.lastname: Huang
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan.
authors.forename: Sheng-Ping
authors.initials: SP
authors.lastname: Huang
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan.
authors.forename: Huei-Ru
authors.initials: HR
authors.lastname: Lo
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan.
authors.forename: Hao-Shuo
authors.initials: HS
authors.lastname: Chang
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan.
authors.forename: Pi-Hsiu
authors.initials: PH
authors.lastname: Lin
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan.
authors.forename: Ming-Cheng
authors.initials: MC
authors.lastname: Wu
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan.
authors.forename: Chia-Jung
authors.initials: CJ
authors.lastname: Chang
authors.affiliation: Institute of Molecular Biology, Academia Sinica, Nankang, Taipei, Taiwan Department of Life Sciences, National Chung Hsing University, Taichung, Taiwan Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan mbycchao@imb.sinica.edu.tw.
authors.forename: Yu-Chan
authors.initials: YC
authors.lastname: Chao
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D002460:Cell Line; D004252:DNA Mutational Analysis; D004261:DNA Replication; D007313:Insecta; D008322:Mammals; D018741:Replication Origin; D017384:Sequence Deletion