Journal
JOURNAL OF VIROLOGY
Volume 76, Issue 11, Pages 5605-5611Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/JVI.76.11.5605-5611.2002
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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 homogenous bacterial clone of SeMNPV (bacmid). Sequences were identified at the junctions of the non-hr on units within the concatemers, which may be potentially involved in recombination events. Deletion of the SeMNPV non-hr on 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 on deletion mutant SeMNPV bacmid can be exploited as a stable eukaryotic heterologous protein expression vector in insect cells.
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