Cloning of complete genomes of large dsDNA viruses ...

Journal of Virological Methods 167 (2010) 95–99

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Cloning of complete genomes of large dsDNA viruses by in vitro transposition of an F factor containing transposon ´ Johannes A. Jehle ∗∗ Yongjie Wang ∗ , Nina Stojiljkovic, Laboratory for Biotechnological Crop Protection, Department of Phytopathology, Agricultural Service Centre Palatinate (DLR Rheinpfalz), Breitenweg 71, 67435 Neustadt an der Weinstraße, Germany

a b s t r a c t Article history: Received 21 August 2009 Received in revised form 18 November 2009 Accepted 23 November 2009 Available online 3 December 2009 Keywords: Transposon Tn5 Whole genome cloning Mini-F replicon Bacterial artificial chromosome Transfection

An improved bacmid technology for cloning complete genomes of large dsDNA viruses with circular genomes has been developed and tested. The system, termed EZ::BAC, is based on Escherichia coli F factor replicon, a chloramphenicol resistant marker gene with the mosaic ends recognized specifically by the transposase of the Tn5. In vitro transposition was carried out for the baculovirus shuttle vector pMON14272 (136 kb) and the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genome (134 kb) as target DNAs. Transposon EZ::BAC was inserted randomly into the target DNAs, leading to 9 bp duplication of the flanking end at the insertion site. One of the obtained AcMNPV::BACs replicated in Sf21 cells after transfection. The random in vitro generation of viral bacmids using EZ::BAC facilitates the host-independent propagation of intact and functional viral genomes in E. coli cells and does not require sequence information of the target DNA as is necessary for the generation of bacmids in conventional systems. © 2009 Elsevier B.V. All rights reserved.

Comparative genomic studies are becoming an increasingly powerful and straightforward way towards inferring the evolutionary history as well as the biological features of large dsDNA viruses. With the improvements in methods of cloning and sequencing, many large eukaryotic dsDNA viruses have been sequenced completely. However, the availability of complete genome sequences is often challenged by the quality and quantity of viral DNA samples. For example, insect viruses normally have to be purified from either diseased hosts (larvae or adults) or infected tissue culture cells by homogenisation and centrifugation procedures. The rearing of insects in the laboratory, however, is rather time consuming and often hampered by contamination with other pathogens. To propagate the viruses in cell culture, specific cell lines are needed, which are not available in most cases. Hence, the yield of purified viruses is often too low to obtain adequate viral DNA for genetic analysis. An alternative to virus purification from an infected host or cell culture is to amplify genomic DNA by the whole genome amplification (WGA) technology using random or degenerate

∗ Corresponding author. Present address: Institut für Virologie, Universitätsmedizin Göttingen, Kreuzbergring 57, 37075 Göttingen, Germany. Tel.: +49 6321 671482; fax: +49 6321 671222. ∗∗ Co-corresponding author. Present address: Institute for Biological Control, Julius Kühn-Institute, Heinrichstr. 243, 64287 Darmstadt, Germany. E-mail addresses: [email protected], [email protected] (Y. Wang), [email protected], [email protected] (J.A. Jehle). 0166-0934/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2009.11.026

oligonucleotide-primed PCR (Cheung and Nelson, 1996; Telenius et al., 1992; Zhang et al., 1992). Although PCR-based WGA can generate a large amount of DNA directly from small samples, it can also generate non-specific amplification artefacts, give incomplete coverage of loci, and generate random DNA fragments less than 1 kb long that cannot be used in many downstream applications (Cheung and Nelson, 1996; Paunio et al., 1996; Telenius et al., 1992; Zhang et al., 1992). In contrast, multiple displacement amplification-based WGA provides a highly uniform representation across the genome (Dean et al., 2002). Recently, it was demonstrated that this method can be applied successfully to genome sequencing of the material-limited large dsDNA viruses, such as the Oryctes rhinoceros nudivirus (Wang et al., 2008). However, an amplification bias, albeit very low, cannot be ruled out. Additionally, all these methods cannot generate intact circular DNA molecules suitable for genetic manipulations in order to study gene function. For baculoviruses, the development of the bacmid technology allowing the propagation and manipulation of complete baculovirus genomes has revolutionized the functional studies of virus genes and allows ample amplification of viral genomes in Escherichia coli cells (Hilton et al., 2008; Luckow et al., 1993). However, this technique requires sequence information of the viral genomes to be cloned as well as suitable cell lines or hosts to generate the bacmids. The development and application are described of an EZ::TN based transposon, termed EZ::BAC, carrying an E. coli mini-F factor that enables cloning and propagating of the complete genomic

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Y. Wang et al. / Journal of Virological Methods 167 (2010) 95–99

DNA of large circular dsDNA viruses as a bacterial artificial chromosome (BAC). Since the BAC is generated by an in vitro transposition, only a tiny amount of viral DNA needs to be available originally. This PCR-independent method overcomes the shortfalls of the PCR-based WGA and conventional bacmid technology. It yields sufficient amounts of high quality viral genomic DNA for sequencing, gene functional studies and genetic modification. To construct transposon EZ::BAC, the transposon construction vector EZ::TN pMOD-3 < R6K␥ori/MCS > (Epicentre, Hess. Oldendorf, Germany), termed pMOD in the following, was used. This vector contains a multiple cloning site (MCS) and an E. coli conditional origin of replication (R6K␥ori) flanked by the hyperactive 19 bp mosaic ends (ME) of transposon Tn5 that are recognized specifically by Tn5 transposase (Fig. 1) (Goryshin and Reznikoff, 1998). In order to release the constructed transposon from the recombinant plasmid, two endonucleotide restriction sites, AatII and NheI, were introduced into the pMOD vector by PCRbased site-directed mutation. First, the primer pair AatII-292-FP 5 -GCGATCGGTGCGGACGTCTTCGCTATTACG-3 and AatII-292-RP 5 -CGTAATAGCGAAGACGTCCGCACCGATCGC-3 were designed to mutate the pMOD plasmid for an additional AatII site (GACGT↓C,

underlined above) at nt position 292. The PCR master mix and reaction were as follows: 2.5 U of Pfx DNA polymerase with high fidelity (Invitrogen, Karlsruhe, Germany) and 30 ng of template pMOD DNA were added to a 50 ␮l of PCR reaction containing 1× Pfx buffer, 0.25 ␮M each primer and 0.3 mM dNTPs. PCR was carried out as follows: 95 ◦ C, 30 s; 12 cycles of 95 ◦ C, 30 s, 55 ◦ C, 1 min, 68 ◦ C, 3 min; 68 ◦ C, 5 min. 10 U of DpnI (Fermentas, St. Leon-Rot, Germany) was directly added to the PCR products, which were incubated at 37 ◦ C for 1 h to digest the parental supercoiled dsDNA. After purification (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany), 1 ␮l of the digested PCR amplicons was eletrotransformed into competent E. coli DH10B cells (Invitrogen, Karlsruhe, Germany) following the protocols described in MicroPulser Electroporation Apparatus Operating Instructions and Applications Guide (BIO-RAD, Muenchen, Germany). The generated AatII site was verified by AatII digestion (Fermentas, St. Leon-Rot, Germany) of the plasmid extracted from ampicillin-resistant colonies, in which the nucleotides G288C and C290G of the pMOD were replaced successfully with A288T and G290C, respectively. Second, the primer pair NheI-819-FP 5 -CCGCTTCCTCGCTAGCTGACTCGCTGCGC-3 and NheI-819-RP 5 -GCGCAGCGAGTCAGCTAGCGAGGAAGCGG-3 were

Fig. 1. Schematic diagram of construction of the transposon EZ::BAC. Two restriction sites AatII and NheI were introduced into pMOD-AN using site-directed mutation. The plasmids pMOD-AN and pCC1BAC were ligated via a BamHI site resulting in pEZBAC, which contains the transposon EZ::BAC. ME, mosaic end; MCS, Multiple cloning sites; SqFP-C, the reverse complement sequence of the primer SqFP (Epicentre, Hess. Oldendorf, Germany).


designed to mutate the pMOD plasmid containing the AatII mutation for a NheI site (G↓CTAGC, underlined above) at nt position 819. Based on the same procedures described above, the mutations of C821A and A822G were introduced and confirmed by NheI digestion (Fermentas, St. Leon-Rot, Germany). The mutated pMOD was named pMOD-AN (Fig. 1). For cloning E. coli mini-F replicon into pMOD-AN, the copy control plasmid pCC1BAC (Epicentre, Hess. Oldendorf, Germany) was used. It contains F factor based partitioning and single copy number regulation system (Fig. 1). The schematic diagram for generation of the desired transposon EZ::BAC is shown in Fig. 1. The pMOD-NA and pCC1BAC were digested with BamHI (Fermentas, St. Leon-Rot, Germany) and ligated according to the procedure provided by the supplier (Invitrogen, Karsruhe, Germany). One micro-liter of the ligation reaction was transformed into E. coli DH10B. Recombinant colonies were selected on the LB plates supplied with 50 ␮g/ml ampicillin and 170 ␮g/ml chloramphenicol and subject to determining of the insertion orientation based on various restriction endonuclease (REN) digestion such as BamHI, BgII (Fermentas, St. Leon-Rot, Germany), AatII and NheI (data not shown). Consequently, the recombinant pEZBAC with clockwiseoriented insertion of pCC1BAC was obtained, which contains the constructed transposon EZ::BAC (Fig. 1). Transposon EZ::BAC is 8568 bp in length and characterised by a mini-F gene cassette, a chloramphenicol resistance marker gene and the transposon Tn5 specific mosaic ends (Fig. 1). For efficient in vitro transposition reaction, transposon EZ::BAC was released from the pEZBAC vector by AatII and NheI double digestion and purified by gel separation. To test whether EZ::BAC is able to mediate in vitro generation of bacmid DNA, the baculovirus shuttle vector (pMON14272) was first considered as target DNA (Luckow et al., 1993). It is 136 kb in length similar to the genome sizes of most large eukaryotic dsDNA viruses and has a kanamycin resistance marker that simplified greatly the initial screening process after EZ::BAC insertion. The in vitro transposition reaction was carried out according to the protocols provided for the EZ::TN system (Epicentre, Hess. Oldendorf, Germany). Briefly, 0.2 ␮g target DNA and an equimolar amount of the transposon EZ::BAC were added in a 10 ␮l reaction volume containing of 1 ␮l buffer and 1 U Tn5 transposase (Epicentre, Hess. Oldendorf, Germany). One micro-liter of the reactions was electroporated into competent E. coli DH10B cells. Putatively positive clones containing pMON14272 inserted with EZ::BAC (termed pMON14272::BAC) were screened for kanamycin (50 ␮g/ml LB agar) and chloramphenicol (12.5 ␮g/ml LB agar) double resistance and by PCR amplification specific for EZ::BAC sequence (primers pIB FP and the reverse complement sequence of SqFP, Epicentre) (Fig. 1) as well as the baculovirus lef-8 gene (Jehle et al., 2006) (data not shown). The successful generation of pMON14272::BAC was further confirmed by PstI digestion analysis. Bacmid DNA was isolated from one of the positive clones using QIAGEN Large-Construct Kit. About 0.8 ␮g DNA and 10 U PstI (Fermentas, St. Leon-Rot, Germany) were added in a 10 ␮l reaction and incubated at 37 ◦ C for 2 h. The digested DNAs were loaded onto a 0.7% agarose gel in 1× TAE buffer and electrophoresed at 80 V for 3 h. According to the predicted sequence, EZ::BAC contains four PstI sites that should yield three large fragments of 3638, 2947 and 1540 bp, and two smaller fragments of 110 and 333 bp flanking both ends of the EZ::BAC transposon (Fig. 1). Upon PstI digestion, therefore, pMON14272::BAC will principally reveal five novel fragments compared to pMON14272. Three of them originate from EZ::BAC; two result from the split of one of the PstI fragments of pMON14272 inserted with EZ::BAC. As shown in Fig. 2, the >23 kb PstI fragment of pMON14272 disappeared and gave rise to two additional fragments of about 23 and 7 kb in pMON14272::BAC; the EZ::BAC specific three large PstI fragments of 3.6, 2.9 and 1.5 kb are also visible. Hence,

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Fig. 2. PstI restriction profiles of pMON14272 and pMON1472::BAC DNAs. The PstI fragment marked with arrow in lane pMON14272 was inserted with EZ::BAC after in vitro transposition and splitted into two smaller PstI fragments after digestion (labelled with arrow in lane pMON14272::BAC). These three large PstI fragments originated from EZ::BAC are marked with arrowheads. The ␭ HindIII and 1 kb DNA markers (Fermentas, St. Leon-Rot, Germany) are given on the left side.

the experimental data indicated that the restriction patterns of the cloned pMON14272::BAC are in good agreement with the predicted results, confirming the insertion of EZ::BAC into pMON14272. After the successful in vitro generation of pMON14272::BAC, it was tested whether EZ::BAC is able to mediate cloning of a viral genome. For this purpose genomic DNA of AcMNPV (133,894 bp) was used as target DNA (Ayres et al., 1994). The in vitro insertion reaction and transformation of E. coli DH10B cells were the same as described above. Putatively positive clones were first selected on LB plates containing chloramphenicol (12.5 ␮g/ml) and then screened using PCR amplification specific for the viral genes of lef-8, lef-9 and polh (Jehle et al., 2006). Out of 504 tested colonies, two positive clones #55 and #360 were obtained and further verified using BamHI digestion of the isolated bacmid DNA. It showed that the transposon was inserted randomly into the 25.6 kb (clone #55) and 8.5 kb (clone #360) BamHI fragments of AcMNPV genomic DNA, respectively (Fig. 3). AcMNPV::BACs revealed three novel fragments compared to AcMNPV. The 8.1 kb fragment marked with arrowhead originated from EZ::BAC; the rest two labelled with arrow resulted from the split of the BamHI fragment of AcMNPV inserted with EZ::BAC (Fig. 3). The insertion sites of EZ::BAC in both clones were sequenced using transposon specific SqFP and T7 primers (Epicentre, Hess. Oldendorf, Germany) (Fig. 1). Sequence analyses of clone #55 revealed the EZ::BAC insertion at nt 129,403 into AcORF148 encoding the occlusion-derived virus envelope protein 56 (ODV-E56) (Braunagel et al., 1996; Theilmann et al., 1996) (Supplementary Data, Fig. S1. A). As for clone #360, in vitro transposon insertion occurred at nt 9,007 into AcORF12, a gene with unknown function (Supplementary Data, Fig. S1. B). In both cases, the insertion of EZ::BAC resulted in the generation of a 9 bp target site sequence duplication flanking each side of the inserted transposon (Supplementary Data, Fig. S1). No other mutations caused by the insertion of the transposon were observed

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Fig. 3. BamHI restriciton profiles of AcMNPV::BAC clones #55 and #360. The BamHI fragments marked with arrow in lane AcMNPV were inserted with EZ::BAC after in vitro transposition and splitted into two BamHI fragments in clone #360 (labelled with solid arrows) and clone #55 (labelled with dotted arrows). The 8.1 kb BamHI fragment of EZ::BAC is marked with arrowhead. The ␭ HindIII and 1 kb DNA markers (Fermentas, St. Leon-Rot, Germany) are given on the left and right sides.

within the sequenced upstream and downstream of the insertion site. Whereas AcORF148 (ODV-E56) is considered as an essential baculovirus gene (Herniou et al., 2003), the function of AcORF12 is unknown. Therefore, we checked whether the insertion of EZ::BAC into AcORF12 produces viable viruses. AcMNPV::BAC DNA was extracted from clone #360 and transfected into Sf21 insect cells by using FuGENE HD Transfection Reagent according to the supplier’s protocols (Roche, Mannheim, Germany). The cells were grown in Grace’s insect media. After incubation at 27 ◦ C for 5 days, numerous infected cells showing viral occlusion body formation were observed (Supplementary Data, Fig. S2). This indicated that the AcMNPV::BAC was able to successfully replicate in Sf21 cells. This result also suggests that AcORF12 may be non-essential for AcMNPV replication in cell culture. E. coli mini-F replicon cassette has been used to clone viral whole genomic DNA based on homologous recombination between wildtype viral DNA and recombinant plasmid containing F factor (Hilton et al., 2008; Luckow et al., 1993; Schumacher et al., 2000; Wang et al., 2003). However, the prerequisites of their methods were that the viral genomic DNAs have been sequenced and characterised, and that specific cell lines are available for co-transfecting and screening. This procedure also leads to the deletion mutation of the viral genomic DNA. In contrast, the mini-F based transposon cloning system developed in our study principally works on any large circular dsDNA viral genomes. Since the Tn5 attachment site of EZ::BAC is non-specific, it confers insertion of the mini-F replicon randomly on its target DNA. As shown by sequencing of the target sites and the transfection experiments with Sf21 cells it does not give rise to any DNA sequence deletion and may produce viable viruses after transfection into susceptible cells. Depending on the insertion site, the resulting mutated genomes may or may not be infective. Given that the EZ::BAC needs as few as 0.2 ␮g viral genomic DNA for in vitro insertion reaction, it overcomes the limitation of the availability of viral genomic DNA, and opens the door to amplify functional genomes of uncharacterised viruses. This technique will be especially useful for generating whole virus genomes, when cell culture or host rearing is unavailable for virus propagation.

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