Pivotal role of the non-hr origin of DNA replication in the genesis of defective interfering baculoviruses.
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.
Pijlman
Gorben P
GP
Laboratory of Virology, Wageningen University, Wageningen, The Netherlands.
Dortmans
Jos C F M
JC
Vermeesch
Angela M G
AM
Yang
Kai
K
Martens
Dirk E
DE
Goldbach
Rob W
RW
Vlak
Just M
JM
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*
D004279:DNA, Viral