title: Pivotal role of the non-hr origin of DNA replication in the genesis of defective interfering baculoviruses.
abstract: The generation of deletion mutants, including defective interfering viruses, upon serial passage of Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) in insect cell culture has been studied. Sequences containing the non-homologous region origin of DNA replication (non-hr ori) became hypermolar in intracellular viral DNA within 10 passages in Se301 insect cells, concurrent with a dramatic drop in budded virus and polyhedron production. These predominant non-hr ori-containing sequences accumulated in larger concatenated forms and were generated de novo as demonstrated by their appearance and accumulation upon infection with a genetically homogeneous bacterial clone of SeMNPV (bacmid). Sequences were identified at the junctions of the non-hr ori units within the concatemers, which may be potentially involved in recombination events. Deletion of the SeMNPV non-hr ori using RecE/RecT-mediated homologous ET recombination in Escherichia coli resulted in a recombinant bacmid with strongly enhanced stability of virus and polyhedron production upon serial passage in insect cells. This suggests that the accumulation of non-hr oris upon passage is due to the replication advantage of these sequences. The non-hr ori deletion mutant SeMNPV bacmid can be exploited as a stable eukaryotic heterologous protein expression vector in insect cells.
authors: Laboratory of Virology, Wageningen University, Wageningen, The Netherlands.
authors: Gorben P
authors: GP
authors: Pijlman
authors: Jos C F M
authors: JC
authors: Dortmans
authors: Angela M G
authors: AM
authors: Vermeesch
authors: Kai
authors: K
authors: Yang
authors: Dirk E
authors: DE
authors: Martens
authors: Rob W
authors: RW
authors: Goldbach
authors: Just M
authors: JM
authors: Vlak
mesh_terms: D000818:Animals; D002460:Cell Line; D004261:DNA Replication; D004279:DNA, Viral / Q000502:physiology*; D003673:Defective Viruses / Q000235:genetics / Q000502:physiology*; D017924:Nucleopolyhedroviruses / Q000235:genetics / Q000502:physiology*; D018741:Replication Origin / Q000502:physiology*; D018411:Spodoptera / Q000821:virology; D014779:Virus Replication / Q000502:physiology*
chemical_list: D004279:DNA, Viral