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. 2002 Jun;76(11):5605-11.
doi: 10.1128/jvi.76.11.5605-5611.2002.

Pivotal role of the non-hr origin of DNA replication in the genesis of defective interfering baculoviruses

Gorben P Pijlman  1 , Jos C F M Dortmans, Angela M G Vermeesch, Kai Yang, Dirk E Martens, Rob W Goldbach, Just M Vlak
Affiliations

Pivotal role of the non-hr origin of DNA replication in the genesis of defective interfering baculoviruses

Gorben P Pijlman et al. J Virol. 2002 Jun.

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.

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Figures

FIG. 1.
FIG. 1.
Restriction profile of intracellular DNA of wild-type SeMNPV-US1 upon passaging (P1 to 25) in Se301 insect cells. (A) DNA digested with XbaI and run in a 0.6% agarose gel. Passage numbers are indicated above the lanes, and the viral genomic XbaI-A and -F fragments are indicated on the left. Lane M contains a λ/EcoRI/BamHI/HindIII DNA size marker. Sizes (in kilobases) of the hypermolar novel bands (2.6 to 7.0) and the novel 9-kb fragment are indicated on the right. (B) Southern blot using the SeMNPV non-hr ori (nt 83122 to 84048) as a probe. The viral genomic 6.6-kb XbaI-F (containing the non-hr ori) and an additional hybridizing 5.3-kb band are indicated on the left.
FIG. 2.
FIG. 2.
Schematic overview of the genetic organization of hypermolar and other non-hr ori hybridizing bands compared to the complete SeMNPV genome. (A) Genetic organization of the genomic DNA with nucleotide positions according to the complete SeMNPV genome (16). Arrows represent the respective ORFs. Solid and light-grey boxes refer to sequences on either side of XbaI (Xb) 83132, containing SspI (S), PstI (P), EcoRI (E), and XhoI (Xh) sites. The non-hr ori is presented as a hatched box between the two SspI sites (11). (B) Genetic arrangement of hypermolar 2.6-, 3.0-, and 4.1-kb fragments of SeMNPV-US1 (Sewt) and a nonhypermolar cohybridizing 5.3-kb fragment (genomic fragment of a SeMNPV deletion mutant) in the Southern blots. Nucleotide positions and sequence overlaps and insertions are indicated at the junction sites. (C) Genetic arrangement of the hypermolar 3.0-kb fragment of SeBAC10ph, containing two junctions.
FIG. 3.
FIG. 3.
Replicative form of the hypermolar 2.6- and 3.0-kb XbaI fragments by partial digestion of ICV SeMNPV-US1 DNA of P10, using increasing amounts of XbaI. On the right the genomic 6.6-kb XbaI-F and the additional 5.3-kb band as well as the hypermolar XbaI bands of 2.6 and 3.0 kb are indicated. On the left the multimers of the 2.6- and 3.0-kb XbaI fragments are indicated by arrows.
FIG. 4.
FIG. 4.
Restriction profile (PstI) of parental SeMNPV bacmid SeBAC10 and the non-hr ori deletion mutant SeBAC10Δnonhr. The genomic PstI-I fragment containing the non-hr ori (7,017 bp) and the PstI fragment with the Cmr gene insertion (7,303 bp) are indicated.
FIG. 5.
FIG. 5.
Restriction profile (XbaI) of ICV DNA of serially passaged SeMNPV bacmids SeBAC10ph (A) and SeBAC10phΔnonhr (B) in Se301 insect cells. Passage numbers (top) and the hypermolar band of 3.0 kb (SeBAC10ph) are indicated.
FIG. 6.
FIG. 6.
(A) Titers of serially passaged BV of SeMNPV-US1 (⧫), SeBAC10ph (▵), and SeBAC10phΔnonhr (○). TCID50, 50% tissue culture infective dose. (B) Pictures of infected Se301 insect cells with SeBAC10ph and SeBAC10phΔnonhr at P2 and P20, respectively.
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