Replication, integration, and packaging of plasmid DNA following cotransfection with baculovirus viral DNA

Abstract

Infection-dependent replication assays have been used to identify numerous putative origins of baculovirus replication. However, plasmid DNA, when cotransfected into insect cells with Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) DNA, replicates independently of any viral sequence in cis (11). Cotransfection of transfer plasmids and baculovirus DNA is a common procedure used in generating recombinant viruses and in measuring the level of gene expression in transient-expression assays. We have examined the fate of a series of vector plasmids in cotransfection experiments. The data reveal that these plasmids replicate following cotransfection and the replication of plasmid DNA is not due to acquisition of viral putative origin sequences. The conformation of plasmid DNA replicating in the cotransfected cells was analyzed and found to exist as high-molecular-weight concatemers. Ten to 25% of the replicated plasmid DNA was integrated into multiple locations on the viral genome and was present in progeny virions following serial passage. Sequence analysis of plasmid-viral DNA junction sites revealed no homologous or conserved sequences in the proximity of the integration sites, suggesting that nonhomologous recombination was involved during the integration process. These data suggest that while a rolling-circle mechanism could be used for baculovirus DNA replication, recombination may also be involved in this process. Plasmid integration may generate large deletions of the viral genome, suggesting that the process of DNA replication in baculovirus may be prone to generation of defective genomes.

FIG. 1

Relative replication efficiency of plasmid DNA in cotransfected insect cells. Sf21 cells (10⁶) were transfected with 0.5 μg of AcMNPV DNA (lanes 1 and 2) or 0.5 μg of pUC19 DNA (lanes 3 and 4) or were cotransfected with 0.5 μg of AcMNPV DNA plus equal molar equivalents of pAchr5 (lanes 5 and 6), pUC19 (lanes 7 and 8), pBSK⁻ (lanes 9 and 10), or pBR322 (lanes 11 and 12) DNA. Total intracellular DNA was purified at 48 h posttransfection and digested with EcoRI (DpnI−) or EcoRI plus DpnI (DpnI+). After electrophoresis, the DNA fragments were transferred to a Qiagen nylon membrane and hybridized with ³²P-labeled pUC18 DNA. The positions of marker DNA (sizes in kilobases) are indicated on the left of the blot.

FIG. 2

Replicated plasmid in cotransfected Sf21 cells exists as high-molecular-weight DNA. (A) Sf21 cells were transfected with 0.5 μg of AcMNPV DNA (lane 1), 0.5 μg of pUC19 DNA (lanes 2 and 3), or cotransfected with 0.5 μg of AcMNPV and pUC19 DNA (lanes 4 to 9). Total intracellular DNA was purified at 48 h postcotransfection and digested with DpnI plus HindIII (D+H, lane 4), DpnI plus PstI (D+P, lane 5), DpnI alone (D, lane 6), DpnI plus NotI (D+N, lane 7), DpnI plus EagI (D+Ea, lane 8), or DpnI plus MluI (D+M, lane 9). One microgram of purified, undigested input AcMNPV DNA (U, lane 1), 200 pg of undigested pUC19 DNA (U, lane 2), or 200 pg of pUC19 DNA digested with EcoRI (E, lane 3) were included as controls. After electrophoresis, DNA samples were blotted and probed with ³²P-labeled pUC18 DNA (left). After the exposure shown on the left, the membrane was stripped and reprobed with ³²P-labeled AcMNPV DNA to confirm the positions of viral DNA fragments (right). The positions of marker DNA (sizes in kilobases) are indicated between the blots. (B) Total intracellular DNA, purified at 48 h posttransfection from cells cotransfected with 0.5 μg of AcMNPV DNA and 0.5 μg of pUC19 DNA, was completely digested with DpnI (lane 1) and then partially digested with increasing amounts of SmaI (lanes 2 to 9). The fragments were separated by electrophoresis and then blotted and probed as described in the legend for Fig. 1. The positions of size markers (kilobases) are indicated on the left of the blot while the positions of various forms of concatenated plasmid DNA are indicated on the right.

FIG. 3

Detection of replicated plasmid DNA in progeny budded virus particles. Sf21 cells (10⁶) were cotransfected with 0.5 μg of AcMNPV DNA plus 0.5 μg of pUC19 DNA (lanes 3 to 11) or pAchr5 (lane 12). Progeny virions were harvested at 72 h postcotransfection (lanes 3 and 12). The supernatant from the pUC19-transfected cells was serially passaged undiluted four times (lanes 4 to 7) or passaged with different amounts of viruses (lanes 8 to 11). Budded virions from each passage supernatant were purified by sucrose gradient centrifugation, and the virion DNA was purified and doubly digested with SmaI plus DpnI (lanes 3 to 12). Following agarose gel electrophoresis, the fragments were blotted onto a Qiagen nylon membrane and hybridized with ³²P-labeled pUC18 probe. The positions (in kilobases) of SmaI-digested pUC18 (lanes 1) and pAchr5 (lane 2) DNA, included as the molecular weight markers and hybridization controls, are indicated on the left. The arrows on the right indicate the positions of SmaI-linearized plasmid DNA (pUC19 or pAchr5) contained in the viral DNA and the fragments of the digested pUC19 or pAchr5 DNA likely covalently linked to viral DNA (high-molecular-weight DNA).

FIG. 4

Conformation of plasmid DNA packaged into budded virions. P3 budded virions from serially passaged virus stocks originally obtained from cells cotransfected with pUC19 and AcMNPV DNA were purified, and the virus DNA was isolated. Samples of undigested virion DNA (U, lane 6) or virion DNA digested with EcoRI (E, lane 4) HindIII (H, lane 5), PstI (P, lane 6) NotI (N, lane 8), EagI (Ea, lane 9), MluI (M, lane 10), Sse8387I (Se, lane 11), or Sse8387I plus MluI (Se+M, lane 12) were resolved on agarose gels and Southern blots were prepared. Controls included undigested purified AcMNPV DNA (AcMNPV, lane 1) and undigested pUC19 DNA (U, lane 2) or pUC19 DNA digested with EcoRI (E, lane 3). The blots were first probed with pUC19 DNA (left) and then stripped and reprobed with AcMNPV DNA (right).

FIG. 5

Restriction mapping of recombinant plasmids with HpaII digestion. Purified P3 viral DNA carrying integrated plasmid DNA was digested with MluI, treated with T4 DNA ligase, and transformed into DH5α cells to selectively amplify viral sequences carrying pUC19 DNA. Recombinant clones were selected in the presence of ampicillin, and purified plasmid DNA from these clones was digested with HpaII and separated on 5% nondenaturing polyacrylamide gels. The migration positions and the relative sizes of pUC19 HpaII fragments are indicated on the left and right, respectively. The missing HpaII fragment(s) detected in each lane, indicating the location of an insertion of viral DNA, is shown at the bottom of each figure for certain clones.

FIG. 6

DNA sequence analysis of the junction sites between pUC19 and AcMNPV DNA. (A) The HpaII map of pUC19 is shown at the top, and below are maps of recombinant plasmids that were predicted to carry viral inserts. Downward arrowheads indicate the approximate locations of viral inserts on these plasmids. The dashed lines indicate regions that were sequenced to determine the junction sites between pUC19 and viral DNA. Arrows indicate the specific primers and directions of the sequencing reactions. (B) Each nucleotide sequence is presented as a continuous sequence in the direction of 5′ to 3′ from either end of a specific insert. The name of each plasmid and the corresponding primers used for sequencing are indicated at the beginning of the sequence. pUC19 sequences are in italics, and viral DNA sequences are underlined. The numbers above the sequences indicate the nucleotide positions on pUC19 (43) or the AcMNPV genome (2). The bold letters represent insertion elements that do not align with either pUC19 or viral DNA sequences. The numbers between slashes represent numbers of continuous nucleotides which are not shown due to space limits.

FIG. 6

DNA sequence analysis of the junction sites between pUC19 and AcMNPV DNA. (A) The HpaII map of pUC19 is shown at the top, and below are maps of recombinant plasmids that were predicted to carry viral inserts. Downward arrowheads indicate the approximate locations of viral inserts on these plasmids. The dashed lines indicate regions that were sequenced to determine the junction sites between pUC19 and viral DNA. Arrows indicate the specific primers and directions of the sequencing reactions. (B) Each nucleotide sequence is presented as a continuous sequence in the direction of 5′ to 3′ from either end of a specific insert. The name of each plasmid and the corresponding primers used for sequencing are indicated at the beginning of the sequence. pUC19 sequences are in italics, and viral DNA sequences are underlined. The numbers above the sequences indicate the nucleotide positions on pUC19 (43) or the AcMNPV genome (2). The bold letters represent insertion elements that do not align with either pUC19 or viral DNA sequences. The numbers between slashes represent numbers of continuous nucleotides which are not shown due to space limits.