Wageningen University, Laboratory of Virology, Binnenhaven 11, 6709 PD, Wageningen, The Netherlands
Correspondence
Just Vlak
just.vlak{at}wur.nl
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ABSTRACT |
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Present address: Sir Albert Sakzewski Virus Research, Royal Children's Hospital Centre, Herston Rd, Herston, 4029 QLD, Australian
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INTRODUCTION |
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Many reports have shown that genomic deletions and/or insertions of foreign DNA into the viral genome readily occur upon baculovirus infection in cell culture. For example, DIs with deletions of approximately 43 % of the Autographa californica multicapsid nucleopolyhedovirus (AcMNPV) genome (d43 DIs) are rapidly generated (Pijlman et al., 2001) and subsequently accumulate in cell culture (Kool et al., 1991
). Furthermore, DIs with reiterations of small viral sequences become abundant in later stages of passaging (Kool et al., 1993
; Lee & Krell, 1992
). These cis-acting sequences were subsequently identified as putative origins of viral DNA replication (oris) by transient replication assays. Ori activity in baculoviruses is associated with the homologous repeated regions (hrs) (Lu et al., 1997
), which are scattered throughout the viral genome and can also act as transcriptional enhancers (Friesen, 1997
). The presence of hrs is a common feature of baculoviruses, but they are also found in other large circular DNA viruses such as nimaviruses and ascoviruses (Van Hulten et al., 2001
; Bigot et al., 2000
), implying an important role for these interspersed repetitive sequences in viral DNA replication.
In a detailed study on DI formation following serial passage of AcMNPV in Spodoptera frugiperda (Sf21) insect cell culture (Lee & Krell, 1992, 1994
), a specific 2·8 kb AcMNPV sequence predominated in later passages. This fragment contained a viral sequence located on the HindIII-K restriction fragment of AcMNPV. In an independent study (Kool et al., 1994
), it was demonstrated that this AcMNPV HindIII-K fragment exhibited a strong ori activity in transient replication assays. The HindIII-K sequence was designated non-hr ori because it did not contain hr sequences. The AcMNPV non-hr ori is located within the open reading frame (ORF) encoding the p94 gene, which is an early gene of unknown function (Friesen & Miller, 1987
) and has probably co-evolved with the adjacent apoptosis-inhibiting gene, p35 (Clem et al., 1994
). A related baculovirus, Bombyx mori nucleopolyhedrovirus (BmNPV), lacks a p94-homologous ORF, but has retained 151 bp of the p94 gene containing the essential non-hr ori regions II and III as identified by Kool et al. (1994)
. This thus suggests that the non-hr ori is somehow involved in baculovirus replication. Non-hr oris are identified in many other baculoviruses and share structural similarities rather than sequence homology (Heldens et al., 1997
; Pearson et al., 1993
; Huang & Levin, 1999
; Luque et al., 2001
; Hu et al., 1998
; Jehle, 2002
). In Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV), the non-hr ori was shown to be non-essential for virus replication in vitro and in vivo. Deletion of the non-hr ori even led to enhanced genome stability by preventing DIs from becoming predominant upon passage (Pijlman et al., 2002
).
Since it has been clearly shown that the non-hr origin of DNA replication of AcMNPV accumulates in DIs upon serial passaging, the question is whether deletion of the non-hr ori can prevent the accumulation of DIs and therefore suppress the passage effect. A bacmid-mutagenesis approach was used to make site-specific deletions in the p94 coding sequence. On analysis of the AcMNPV mutants by serial undiluted passage in Sf21 insect cells, we observed that the viruses became more stable, but that the bacmid insertion containing the foreign genes was specifically lost.
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METHODS |
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Deletion mutagenesis by ET recombination in E. coli.
For deletion mutagenesis of the p94 coding sequence of the AcMNPV bacmid, 7476 bp primers were designed with 50 bp 5' ends flanking the deletion target region on the AcMNPV genome (Table 1). The 3' ends of the primers annealed to the chloramphenicol gene of pBeloBAC11 (Shizuya et al., 1992
) from nt 735 to 1671. PCR on pBeloBAC11 was performed using the Expand long-template PCR system (Roche) according to the manufacturer's protocol, giving a product of 1048 bp. The PCR product was purified using the High pure PCR purification kit (Roche), digested with DpnI to eliminate residual pBeloBAC11 template, phenol/chloroform extracted and ethanol precipitated. For ET recombination, 70 ml LB medium was inoculated with 0·7 ml of an overnight culture of E. coli DH10
containing the AcMNPV bacmid and homologous recombination helper plasmid pBAD-
(Muyrers et al., 1999
). At an OD600 of 0·10·15, ET protein expression from pBAD-
was induced by the addition of 0·7 ml 10 % L-arabinose. The cells were harvested at an OD600 of 0·30·4 and made electrocompetent by three washes with ice-cold 10 % glycerol. The cells were transformed with
0·5 µg purified PCR product in 2 mm electroporation cuvettes (Eurogentec) using a Biorad Gene Pulser (2·3 kV, 25 µF, 200
). The cells were resuspended in 1 ml LB medium and incubated for 1 h at 37 °C and subsequently spread on agar plates containing kanamycin and chloramphenicol. Colonies were picked and screened by restriction analysis and PCR.
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Viral DNA isolation, Southern hybridization, molecular cloning and sequencing.
Intracellular viral DNA was isolated as previously described (Summers & Smith, 1987). Digested viral DNA was run overnight in ethidium bromide-stained 0·6 % agarose gels and Southern blotting was performed by standard capillary upward blotting (Sambrook et al., 1989
) using Hybond-N (Amersham Pharmacia) filters. As a DNA size marker,
DNA digested with EcoRI/HindIII/BamHI was used. Random-primed DNA probes for Southern hybridization were made using the DIG non-radioactive nucleic acid labelling and detection system (Roche). For the non-hr ori probe, a PCR product (1036 bp) of the AcMNPV non-hr ori was made with primers DZ123 (5'-TGCGGCCAGGTTTTGTAGAATG-3'; nt 114056114077; Ayres et al., 1994
) and DZ124 (5'-GCATGGAACGCGTTTGTCAC-3'; nt 115072115091), purified using the High pure PCR purification kit (Roche) and DIG-labelled overnight. For the BAC vector probe, BAC-Bsu36I (Pijlman et al., 2002
) was DIG-labelled overnight. Hybridization and colorimetric detection with NBT/BCIP (Roche) were performed according to the manufacturer's recommendations. Hypermolar viral HindIII bands were cut from the gel, purified with the Matrix gel extraction system (Marlingen) and cloned into pBluescript by electrotransformation of E. coli DH5
using standard methods (Sambrook et al., 1989
). Automatic sequencing was performed at Baseclear, The Netherlands. Sequence analyses were performed using BLAST (Altschul et al., 1997
). In silico reassembling of bacmid sequences and computational predictions of restriction digests were done using the Lasergene DNASTAR package.
SDS-PAGE and immunodetection.
Protein samples were analysed in 12 % SDS-PAGE gels as described in Sambrook et al. (1989). Protein masses were determined using the low molecular mass protein marker (Amersham Pharmacia Biotech). Semi-dry blotting was performed onto an Immobilon-P membrane (Millipore) using a Tris/glycine buffer (25 mM Tris base, 192 mM glycine, 10 %, v/v, methanol, pH 8·3). Immobilon-P membranes were blocked in 2 % low-fat milk powder (Campina, The Netherlands) in TBS (0·2 M NaCl, 50 mM Tris/HCl, pH 7·4). The marker was visualized on the membrane by Ponceau-red staining (Sambrook et al., 1989
). Immunodetection of CSFV-E2 was performed by incubation with a monoclonal antibody (mAb A18, Intervet International B.V.) diluted 1 : 10 000 in TBS with 1 % low-fat milk powder for 1 h at room temperature. Subsequently, anti-mouse antibody conjugated with horseradish peroxidase (Amersham) was used at a concentration of 1 : 10 000 and detection was performed with an Enhanced Chemiluminescent-light (ECL) Detection Kit (Amersham).
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RESULTS |
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Analysis of genomic alterations upon serial passage
To investigate the putative formation of DIs and to find the molecular basis for the unexpected rapid decrease in protein production upon serial passage of the parental virus and the three mutants, intracellular viral DNA of P1, P10 and P20 was subjected to digestion with HindIII (Fig. 2). For all viruses, two bands of 10·9 and 9·2 kb were submolar in P20 with respect to the other fragments. The genetic content of these bands was investigated in more detail (see Fig. 4
). Typically, novel fragments of 3·2, 1·8 and 1·6 kb appeared in P20 of AcGFPE2 (Fig. 2A
), but not in the DNA preparations of the other (mutant) viruses (Fig. 2B
D). These fragments accumulated relative to the genomic HindIII fragments of the parental virus, suggesting that these sequences were part of DI molecules. According to the hypothesis, we investigated whether these novel fragments contained the non-hr ori. Therefore, the viral DNA was digested and transferred to a membrane and Southern hybridized with a DIG-labelled non-hr ori probe (Fig. 2
). The original non-hr ori-containing HindIII-K fragment in AcGFPE2 of 2971 bp hybridized to the non-hr ori probe (Fig. 2A
). Moreover, the results showed that the novel 3·2, 1·8 and 1·6 kb bands in AcGFPE2 hybridized strongly with the non-hr ori probe (Fig. 2A
), indicating that the accumulated sequences indeed contained the non-hr ori. As expected, the non-hr ori probe did not hybridize to Ac
Cp94 (Fig. 2B
) and Ac
p94 (Fig. 2D
), but did hybridize to the fragment with the CmR insertion in Ac
Np94 (Fig. 2C
). Thus, deletion of the non-hr ori (in Ac
Cp94 and Ac
p94) or a non-hr ori flanking sequence (in Ac
Np94) prevented the generation and accumulation of DIs, but apparently did not prevent the decrease in recombinant protein production.
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To investigate whether, in addition to a deletion of the BAC vector parts, the expression cassette containing GFP and CSFV-E2 genes was also deleted, restriction digests with XhoI and KpnI were carried out (Fig. 4C). Again, the results showed that all XhoI and KpnI fragments derived from the BAC vector as well as from the GFP and CSFV-E2 genes (sizes of fragments on the left in Fig. 4C
correspond to sizes on the physical map in Fig. 4B
) were deleted upon passage in AcGFPE2, Ac
Cp94 and Ac
p94 and were submolar in Ac
Np94 (Fig. 4C
). In contrast to the deletions in the BAC vector and the expression cassette, the genomic XhoI-J, -K, -L and KpnI-E fragments of viral origin (named after digests of the complete AcMNPV genome sequence; Ayres et al., 1994
) remained at nearly equimolar levels in the three mutants. In AcGFPE2, however, all fragments became submolar as a consequence of predominating non-hr ori concatemers with 3·2, 1·8 and 1·6 kb units (arrows, Fig. 4C
). The results clearly indicated that, irrespective of the presence or absence of the non-hr ori, sequences from the BAC vector and the expression cassette were systematically deleted and that sequences of viral origin were retained upon passage in insect cells.
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DISCUSSION |
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Passaging of the parental AcMNPV bacmid (AcGFPE2) resulted in the predominance of DI molecules containing reiterated non-hr oris (Figs 2 and 3). Sequence overlaps on the junction sites of the concatenated non-hr ori molecules suggested that they were generated by homologous recombination during viral DNA replication (Pijlman et al., 2002
). The generation of non-hr ori DIs was also expected to occur for Ac
Np94, because this mutant only lacks the p94 N-terminal part but still contains the five non-hr ori subdomains, which are required for optimal ori activity (Kool et al., 1994
; Fig. 1A
). However, this was not the case. It may be that auxiliary sequences located in the adjacent N-terminal part of p94 are necessary either for the excision of non-hr oris from the genome, or are responsible for a higher ori activity, thereby giving the DIs a stronger replicative advantage. The latter hypothesis is supported by data from transient replication assays by Kool et al. (1994)
, who showed that plasmids containing the entire sequence from HindIII to EcoRV (see Fig. 1A
) had a slightly greater replication ability than the smaller HindIIIXhoI region, which is retained in the Ac
Np94 mutant. Thus, the AcMNPV non-hr ori is not essential for virus replication in vitro, and deletion of the non-hr ori and/or flanking auxiliary sequences prevent the accumulation of DIs enriched in non-hr oris upon serial passage in insect cells.
In spite of the fact that the formation of non-hr ori DIs did not occur in the three recombinants, BV titres (based on GFP expression) and CSFV-E2 production decreased rapidly upon passaging of all viruses (Fig. 1B, C). We demonstrated that the non-essential BAC vector including the expression cassette was spontaneously deleted from the viral genome upon passage in insect cells (Fig. 4
). This cassette comprises a bacterial mini-F replicon (or BAC vector), two antibiotic resistance genes and two foreign genes (CSFV-E2 and GFP) under the control of baculovirus p10 and polyhedrin promoters. Spontaneous baculovirus mutants carrying this deletion quickly became predominant upon passage, which explained the drop in foreign protein production levels.
Instability of mini-F plasmids, which are also known as bacterial artificial chromosomes (BACs), in eukaryotic cells has been reported to occur in several other cases. An infectious clone of the pseudorabies virus was maintained as a stable BAC in E. coli, but reconstitution of the virus led to the spontaneous deletion of the BAC vector insertion upon transfection of mammalian cells (Smith & Enquist, 1999). Approximately 56 kb of flanking viral sequence was deleted along with the BAC vector sequence. In contrast, when the BAC vector was inserted at a different locus, the virus was stable, suggesting that the location of BAC vector insertion might also be important (Smith & Enquist, 2000
). Wagner et al. (1999)
showed that during construction of a mouse cytomegalovirus (CMV) BAC, overlength genomes were not stable in mammalian cells. To overcome this problem, they designed duplicated viral sequences flanking the BAC vector insertion, allowing spontaneous excision by homologous recombination. Adler et al. (2001)
further showed that excision of BAC vector sequences (by Cre-lox recombination) from cloned MHV-68 genomes was critical for reconstitution of wild-type properties. Similarly, insertion of the BAC vector in CMV requires deletion of non-essential genes, because CMVs only tolerate 5 kb of additional sequence in their genomes (Brune et al., 2000
).
Most likely, these properties of herpesvirus BACs are the result of physical limitations of the virus capsid, which can only package a genome of a defined (maximum) size. Maximum packaging capacity is also observed for other DNA viruses. An overlength of only 5 % leads to unstable adenovirus and EpsteinBarr virus genomes (Bett et al., 1993; Bloss & Sugden, 1994
). For baculoviruses, a maximum packaging capacity may also exist, although the rod-shaped baculovirus nucleocapsids are presumed to be more flexible with respect to DNA content as they contain genomes of up to almost 180 kb (e.g. Xestia c-nigrum granulovirus; Hayakawa et al., 1999
) and allow inserts of up to 25 kb (Roosien et al., 1986
). Still, the occurrence of spontaneous (major) deletions in baculoviruses is a general phenomenon. In SeMNPV, deletions of up to 25 kb of non-essential sequences are routinely observed upon infection of cultured insect cells (Heldens et al., 1996
; Dai et al., 2000
; Pijlman et al., 2002
) and are located in the largest region between two adjacent hrs (SeMNPV hr1 and hr2; IJkel et al., 1999
). In AcMNPV, deletions are frequently found in the EGT/DA26 locus (Kumar & Miller, 1987
), which is located in the middle of AcMNPV inter-hr region hr12 (Ayres et al., 1994
). Since hrs are believed to be involved as ori in viral DNA replication, we hypothesize that the occurrence of genomic deletions is more likely in regions with a low ori density. This may explain why deletions in the BAC vector sequence are likely to occur. Yet the BAC vector itself may also display a certain intrinsic genetic instability. Alternatively, the heterologous gene may confer a certain level of toxicity to the infected cells, thereby creating an added selection pressure against intact bacmids. However, toxicity in insect cells has never been observed with CSFV-E2, which is a commercialized baculovirus expression product used as the major constituent of a marker vaccine against classical swine fever (Van Rijn et al., 1999
). In addition, bacmids equipped with an expression cassette not containing CSFV-E2 also showed specific loss of BAC vector sequences (G. P. Pijlman, unpublished results).
In this paper we have shown that reconstitution in insect cells of infectious baculovirus from a bacmid is accompanied by genetic instability of BAC vector sequences. Recently we have obtained similar results with SeMNPV bacmid-derived viruses in cultured insect cells (G. P. Pijlman, unpublished results). Once the instability is removed by spontaneous deletion of the (non-essential) BAC vector during viral DNA replication, a more stable virus is generated, which predominates subsequent passages. The present observations may constitute a major concern for the utilization of bacmid-derived baculoviruses for the large-scale production of heterologous proteins, especially in insect-cell bioreactors involving many virus passages. Although the generation of mutant baculoviruses by the classical method is more time-consuming than the generation of a recombinant bacmid, it may yield a virus with greater stability. An improvement for the bacmid strategy would be to introduce the heterologous gene(s) at a more stable locus remote from the BAC vector insertion. Alternatively, a bacmid could be developed in which the BAC vector is deliberately excised (using Cre-lox recombination) upon replication in insect cells, while leaving the introduced heterologous gene(s) intact.
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ACKNOWLEDGEMENTS |
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Received 18 June 2003;
accepted 24 June 2003.
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