An efficient way to introduce unique restriction endonuclease sites into a baculovirus genome

Journal of Virological Methods 76 (1998) 51 – 58

An efficient way to introduce unique restriction endonuclease sites into a baculovirus genome Song Yang a, Lois K. Miller a,b,* b

a Department of Genetics, Life Sciences Bldg., Uni6ersity of Georgia, Athens, GA 30602, USA Department of Entomology, Life Sciences Bldg., Uni6ersity of Georgia, Athens, GA 30602, USA

Received 11 June 1998; received in revised form 23 August 1998; accepted 23 August 1998

Abstract Recombinant baculoviruses which can be linearized at unique sites with restriction endonucleases can greatly facilitate the construction of other recombinants including baculovirus expression vectors and site-specific mutants. We designed a strategy to introduce unique restriction endonuclease sites at virtually any location in a baculovirus genome. The unique sites were first introduced onto a transfer plasmid which also contained in the vector portion of the plasmid an E. coli lacZ gene and a Sse8387I site, a sequence which is not found in the viral genome. Cotransfection of the transfer plasmid and circular viral DNA generated single-crossover recombinant viruses which could be distinguished as blue plaques in the presence of X-gal, a chromogenic indicator for lacZ. Single-crossover recombinants were purposefully isolated and propagated to generate double-crossover recombinants. Viral DNA isolated from the mixed virus population was digested with Sse8387I to linearize only the single-crossover viral DNA; double-crossover recombinants in the progeny viral population resulting from transfection with the Sse8387I-linearized viral DNA mixture were thus highly enriched, making the task of screening much easier. To demonstrate the feasibility of this approach, we introduced Bsu36I sites into the orf 24 and the 6lf-1 regions of Autographa californica multiple-nucleocapsid nuclear polyhedrosis virus (AcMNPV) to generate recombinant viruses vncBsuorf24 and vncBsuvlf1, respectively. Both recombinant viruses were obtained by screening only ten plaques. This method should also be applicable to other kinds of mutations and may be applicable to other double-stranded DNA viruses. © 1998 Elsevier Science B.V. All rights reserved. Keywords: AcMNPV; Homologous recombination; Recombinant viruses; Site-directed mutagenesis; Virus genetics; Baculovirus expression vectors

* Correponding author. Tel.: + 1 706 5422294; fax: + 1 706 5422279; e-mail: [email protected].. 0166-0934/98/$ - see front matter © 1998 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 0 9 3 4 ( 9 8 ) 0 0 1 2 3 - 2

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1. Introduction Autographa californica multiple-nucleocapsid nuclear polyhedrosis virus (AcMNPV) is an insect baculovirus which is used extensively as an eukaryotic gene expression vector (Jarvis, 1997) and is of interest as a biological pesticide (Black et al., 1997). In both basic and applied research on this virus, manipulation of the viral genome, such as introduction of mutations, is necessary. A conventional method used to modify baculoviral genomes is homologous recombination. A transfer plasmid is first constructed which carries the desired alteration flanked by viral sequences of the target site. Cotransfection of cultured insect cells with the transfer plasmid and viral DNA generates recombinant viruses bearing the alteration through homologous recombination. However, the proportion of recombinants in the progeny virus population is low (usually 1 – 2% or less) (Smith et al., 1983; Martens et al., 1995). Moreover, most of the recombinants are singlecrossovers in which the entire transfer plasmid is integrated into the viral genome (O’Reilly et al., 1992). Therefore, screening for double-crossover recombinants is laborious. An excellent way to overcome these problems is to use viral DNA which has been linearized at the target site before transfection (Kitts et al., 1990). This treatment greatly lowers the proportion of parental virus and single-crossover recombinants in the progeny virus population. The most widely used method to linearize AcMNPV DNA is to introduce Bsu36I restriction sites, which are not found in the wild-type (wt) AcMNPV sequence, into the viral genome at the site of interest. The recombinant viral DNA can then be linearized by digestion with Bsu36I. The presence of multiple Bsu36I sites increases the efficiency of generating recombinant (Kitts and Possee, 1993; Martens et al., 1995; Yang and Miller, 1998) probably because more complete linearization of the viral DNA is achieved when multiple Bsu36I sites are present. Positioning the Bsu36I sites to delete an essential gene greatly enhances the proportion of recombinants since virus viability depends on homologous recombination with the transfer plasmid (Kitts and Possee, 1993). Another way to

construct recombinant baculoviruses is direct ligation in vitro (Ernst et al., 1994; Lu and Miller, 1996). With this strategy, circular recombinant viral DNAs are generated simply by ligating linearized viral DNA with a DNA fragment carrying the gene of interest and the proportion of recombinants could exceed 95% (Ernst et al., 1994; Lu and Miller, 1996). All of these strategies require the construction of recombinant viruses with unique restriction sites at suitable locations, a procedure which remains laborious. In this paper, we describe a method for introducing unique restriction sites at any locus in a baculovirus genome. This method reduces greatly the effort needed to identify double-crossover recombinants by effectively increasing their proportion in the progeny virus population. In principle, this approach can also be used for the introduction of non-lethal mutations anywhere in the viral genome.

2. Materials and methods

2.1. Cell line, plasmids and parental 6iruses The Spodoptera frugiperda (fall armyworm) IPLB-SF-21 (SF-21) cell line (Vaughn et al., 1977) was grown at 27 or 33°C in TC-100 medium (GIBCO/BRL) supplemented with 10% fetal bovine serum (Intergen, Purchase, NY) and 0.26% tryptose broth (O’Reilly et al., 1992). AcMNPV L1 wt variant (Lee and Miller, 1978) and mutant tsB837 (Lee and Miller, 1979; Mclachlin and Miller, 1994) were used as parental viruses in the construction of recombinant viruses vncBsuorf24 and vncBsuvlf1, respectively. Plasmid pPstNN2.6 contains a 2.6-kb fragment of AcMNPV DNA including the orf 24 (McLachlin et al., 1998). Plasmid porf24ncBsu was constructed based on pPstNN2.6 by site-directed mutagenesis (Deng and Nickoloff, 1992) to introduce two Bsu36I sites in the orf 24 region using oligomers ORF24cBsu (5%-AATAAAAAACATTTTCCTAAGGAATATATTTATTTC-3%) and ORF24nBsu (5%-GTTTTGTTTTTTACCTTAGGTCAAAATGTC-3%) as mutagenic primers. Plasmid pXA7Y335FLacZ, which contains a

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Sse8387I site and a lacZ gene in the multiple cloning site of pBluescript II KS+(Stratagene), was constructed by inserting a fragment containing the Drosophila melanogaster heat shock protein gene (hsp70 ) promoter-driven lacZ gene from plasmid pHSP70LacZ (Clem and Miller, 1994) into the BamHI site of plasmid pXA7Y335F, which also contains the 6lf-1 region of AcMNPV (Yang and Miller, 1998). The 6lf-1 containing region of AcMNPV in pXA7Y335FLacZ was replaced, using PstI and HindIII sites, by orf 24 sequences removed from porf24ncBsu to form plasmid pZorf24ncBsu, which would be used as the transfer plasmid to construct recombinant virus vncBsuorf24. The two Bsu36I sites on pZorf24ncBsu are flanked by 0.6 and 1.5-kb of viral sequences on either side and are 5-bp upstream and 14-bp downstream of the orf 24 translation start and termination codons, respectively. Plasmid pXA7ncBsu, which contains two Bsu36I sites flanked by 1.75 and 3.8-kb viral sequences was used as the transfer plasmid to construct recombinant vncBsuvlf1. Using the site-directed mutagenesis method (Deng and Nickoloff, 1992), one Bsu36I site was introduced 3-bp upstream of the 6lf-1 translation start codon and the other Bsu36I site was introduced 37-bp upstream of the 6lf-1 translation termination codon without changing the predicted amino acid sequence.

2.2. Site-directed mutagenesis Site-directed mutagenesis (Deng and Nickoloff, 1992) was performed using the Transformer™ Site-Directed Mutagenesis Kit (CLONTECH, Pal Alto, CA) to introduce Bsu36 I and Sse8387 I sites onto the transfer plasmids. The selection primer changes a unique SmaI site to a Sse8387 I site while the mutagenic primers contain Bsu36 I sites. Template plasmid DNA was denatured in 0.4 M NaOH at room temperature for 10 min, precipitated with ethanol and dried. 100 ng of phophorylated selection primer and 500 ng of each phophorylated mutagenic primer were annealed with 300 ng of the denatured template plasmid DNA in 20 ml of annealing buffer (20 mM Tris – HCl [pH 7.5], 10 mM MgCl2, 50 mM NaCl) by

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incubation at 95°C for 3 min and then chilling on ice for 5 min. Afterward, 5 ml of H2O, 3 ml of synthesis solution (100 mM Tris–HCl [pH 7.5], 5 mM dNTP, 10 mM ATP and 20 mM DTT), 1 ml of T4 DNA polymerase and 1 ml of T4 DNA ligase were added. This reaction mixture was incubated at 37°C for 2 h. The DNA was precipitated, digested with 1.5 ml of SmaI for 2 h and then used to transform competent E. coli mutS cells. DNA (80 ng) isolated from the mixed transformation overnight culture was digested again with 3 ml SmaI and used to transform competent E. coli XL-1 Blue cells. Individual transformants were screened by PCR for the presence of Bsu36 I and Sse8387 I sites the plasmid.

2.3. Generation of recombinant 6iruses One microgram of virus DNA and 5 mg of transfer plasmid DNA were cotransfected into 106 SF-21 cells using Lipofectin (GIBCO/BRL) (O’Reilly et al., 1992). Budded virions (BVs) were harvested after growth at 27°C for three days and plaque purified (O’Reilly et al., 1992) at 27°C (for vncBsuorf24) or 33°C (for vncBsuvlf1). Singlecrossover recombinants (either blue plaques in the presence of 120 mg/ml X-gal (5-bromo-4-chloro-3indolyl-b-D-galactopyranoside) (Sigma) or occlusion body positive plaques at 33°C) were isolated and plaque-purified three times. Several such single-crossover recombinants were propagated individually in 35-mm dishes three times and combined by mixing 1 ml of tissue culture supernatant from each amplified virus. Viral DNA was isolated from the BV mixture (O’Reilly et al., 1992) and digested extensively with Sse8387I. Digested viral DNA (1 mg) was used to transfect 106 SF-21 cells. Viral isolates were plaque-purified twice from BVs collected 3 days after the transfection and amplified for examination by PCR.

2.4. PCR analysis of 6iral DNA Viral DNA for PCR analysis was prepared by the miniprep method (O’Reilly et al., 1992). 0.1 mg of viral DNA and 0.1 mg of each primer were used in a 20-ml reaction. The PCR reactions were carried out by denaturing the DNA templates at

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95°C for 1 min, annealing primers at 48°C for 1 min and extending primers at 72°C for 1 min in a total of 20 cycles.

3. Results

3.1. Description of the strategy to introduce new sites into the 6iral genome We chose to introduce two Bsu36I sites at the locus of interest in the AcMNPV because they would be unique in the genome. The Bsu36I sites were first introduced onto a transfer plasmid containing the target sequence of AcMNPV and the mutations creating the Bsu36I sites were designed in a way that sequences of putative gene products or other functional elements such as promoters were not affected so that the resulting recombinant virus would be viable. The transfer plasmid also contained an hsp70 promoter-driven-lacZ gene and a Sse8387I site, which is not found in AcMNPV genome, for the purpose of identification and restriction of single-crossover recombinants, respectively (Fig. 1). Cotransfection of circular viral DNA and the transfer plasmid generates both single- and double-crossover recombinants at low frequency. While double-crossover recombinants may not be phenotypically distinguishable from parental virus and thus difficult to identify, single-crossover recombinants are readily identified by their blue plaques due to the integrated transfer plasmid containing the lacZ gene. At this step, singlecrossover recombinants are purposefully isolated and amplified by serial passage during which double-crossover recombinants also arise. These double-crossover recombinants may include those containing both Bsu36I sites, those containing only one Bsu36I site and parental virus (Fig. 1). It should be noted that the yield of double-crossover recombinants containing both Bsu36I sites would be significantly lowered if the first crossover occurs between the two Bsu36I sites. Therefore, to minimize this problem, seve15 ral amplified single-crossover recombinants are combined to increase the chances that at least one

has the first crossover outside the two Bsu36I sites. Viral DNA is isolated from the mixture of virions and digested with Sse8387I, linearizing only the DNA of single-crossover recombinants because the double-crossover recombinants have lost the vector portion of the transfer plasmid as well as the Sse8387I site (Fig. 1). Transfection of cells with the Sse8387I-digested viral DNA amplifies the circular double-crossover viral DNA but not the linearized single-crossover viral DNA. Consequently, the percentage of double-crossover recombinants in the progeny virus population is greatly increased, making the screening for double-crossover recombinants much more efficient. We employed polymerase chain reaction (PCR) to identify isolated virus clones containing both Bsu36I sites. The primers used in the PCR were designed in such a way that their 3%-ends match the mutant sequences but differ from the wt sequences (Fig. 2B). Therefore, only when both Bsu36I sites are incorporated into the viral genome can the primers generate the expected PCR product.

3.2. Construction of two recombinant 6iruses, 6ncBsuorf 24 and 6ncBsu6lf 1, with two Bsu36I sites Recombinant virus vncBsuorf24 was constructed by first cotransfecting wt virus DNA and transfer plasmid pZorf24ncBsu (Fig. 2A). This plasmid has an hsp70 promoter-driven lacZ gene, a Sse8387I site in the multiple cloning site and a 2.6-bp region of AcMNPV encompassing the 0.5kb orf 24 which had been modified by site-directed mutagenesis to contain two Bsu36I sites immediately flanking the orf 24 open reading frame. Eight blue plaques, representing single-crossover recombinants, were plaque-purified three times and then passed three times in SF-21 cells to generate double-crossover recombinants. The BV stocks were then combined. Viral DNA was isolated from this virus pool, digested extensively with Sse8387I and transfected into cells. Ten random virus clones were purified from the transfection supernatant by plaque assay. Their DNAs were screened by PCR using primers Porf24nBsu and Porf24cBsu (Fig. 2B). PCR products of the expected 0.5-kb size

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were generated from DNAs of two virus clones (Fig. 2C). The presence of two Bsu36I sites in both these clones was confirmed by restriction endonuclease digestion (data not shown). We also used PCR primers T3 and pBSH3, which were specific for the multiple cloning site of the pBluescript-based transfer plasmid, to detect possible single-crossover recombinants. None of the screened virus clones generated the corresponding 69-bp PCR product with T3 and pBSH3, suggesting they did not carry the vector portion

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of the transfer plasmid (Fig. 2C). Clone 4 was designated vncBsuorf24. Recombinant virus vncBsuvlf1 was generated in a similar way with a few differences. The transfer plasmid, pXA7ncBsu, contains two Bsu36I site and a Sse8387I site but does not contain the lacZ as the marker gene (Fig. 2A). Instead of using wt virus as the parental virus, we used tsB837, which is defective in occlusion body (OB) production at 33°C due to its temperature sensitive mutation in the 6lf-1 gene (Lee and Miller, 1979; McLachlin and Miller,

Fig. 1. Strategy to introduce two Bsu36I sites into AcMNPV genome. Double lines indicate viral DNA and a single line represents plasmid vector DNA. The filled box represents a hsp70 promoter-driven lacZ gene.

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Fig. 2.

S. Yang, L.K. Miller / Journal of Virological Methods 76 (1998) 51–58

1994). The budded BVs resulting from the initial cotransfection of tsB837 and pXA7ncBsu DNAs were screened for single cross-over recombinants by plaque assay at 33°C. Single-crossover recombinants were occlusion positive because the acquired 6lf-1 gene produced wt VLF-1. Four occlusion positive virus clones were purified, propagated and combined. Their DNAs were digested with Sse8387I and used for transfection of SF-21 cells. Occlusion body positive plaques were isolated from the progeny virus population and screened with primers 14J-vlf1 and 15J-vlf1 in PCR reactions (Fig. 2B). Six of the ten screened recombinants generated the expected 1.1-kb PCR product (Fig. 2D), suggesting that they had acquired both Bsu36I sites. All of the ten clones appeared to be double-crossover recombinants since PCR analysis with primers Sse \ SmaI-pBS and M13-20 FORWARD, which anneal to the multiple cloning site of pBluescript and thereby generate a 110-bp fragment, failed to produce PCR products corresponding to the vector part (Fig. 2D). Clone 1 was later confirmed by restriction endonuclease digestion to have the two Bsu36I sites (data not show) and was designated vncBsuvlf1.

4. Discussion We have developed a strategy based on homologous recombination to introduce restriction sites at virtually any locus in the genome of the baculovirus AcMNPV. Our approach includes the additional steps of identification of singlecrossover recombinants, generation of doublecrossover recombinants from single-crossover recombinants and then selection against singlecrossover recombinants to increase the percentage

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of double-crossover recombinants in the final virus stock. Although more steps means a longer procedure, the overall personal time and effort required to obtain double-crossover recombinants is greatly reduced. The effectiveness of this method was demonstrated by introducing two unique Bsu36I sites into AcMNPV genome to construct recombinants vncBsuorf24 and vncBsuvlf1. In each case, desired recombinants were obtained by screening only ten plaques. The same strategy could also be used to introduce other mutations including deletions and insertions. However, if a region is to be manipulated extensively, it is more practical to first introduce unique restriction sites at the locus of interest and then use the linearized recombinant viral DNA to construct other recombinants with modifications at that locus. To do this, a procedure involving direct cloning of PCR products into baculoviruses may be used to replace a non-essential viral gene with a marker gene containing unique restriction sites or insert the marker gene close to an essential viral gene (Gritsun et al., 1997). Alternatively, our method makes it convenient to introduce unique restriction sites into any locus, including essential genes, with minimal change of the viral genome. Recombinant viruses with two appropriately positioned sites are especially useful for subsequent introduction of mutations between the unique restriction sites. To use this method successfully, it is important not to contaminate the single-crossover isolates with parental virus after the first cotransfection because the parental virus DNA can not be restricted with the single-crossover recombinant DNA, thereby elevating the background level of parental virus. For this reason, we purified the single-crossover isolates three times but careful

Fig. 2. Identification of recombinants vncBsuorf24 and vncBsuvlf1 by PCR. (A) Structures of transfer plasmids pZorf24ncBsu and pXA7ncBsu, which were used to construct vncBsuorf24 and vncBsuvlf1, respectively (not drawn to scale). (B) Primers used to screen vncBsuorf24 and vncBsuvlf1. Nucleotides different from wt sequence are indicated by lower case letters. The primers match exactly the mutant sequences. (C) and (D) vncBsuorf24 and vncBsuvlf1, respectively, were screened by PCR using primers indicated above each gel. Ten clones were examined for each recombinant. The combined single-crossover recombinants (S) in each case were analyzed in parallel as controls. The sizes (in nucleotides) of 1-kb DNA ladders are marked at left of each gel. Oligomers T3 (5%-AATTAACCCTCACTAAAGGG-3%), pBSH3 (5%-GCTTATCGATACCGTCG-3%), Sse\SmaI-pBS (5%-CCTGCAGCCCGGGGG-3%) and M13-20FORWARD (5%-TGTAAAACGACGGCCAGT-3%) were used to amplify the vector portion of the transfer plasmids to detect single-crossover recombinants.

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selection of well-isolated plaques may be sufficient in practice. Another concern is that resolution of a single-crossover recombinant can result in not only a recombinant containing the expected mutations but also parental virus. If the viability of the expected recombinant is suspected to be lower than that of the parental virus, it may be desirable to amplify the single-crossover recombinants a minimal number of times to obtain only sufficient viral DNA for restriction and transfection.

Acknowledgements We thank Jeanne McLachlin and Jeff Rapp for advice. This work was supported in part by Public Health Service grant AI23719 from the National Institute of Allergy and Infectious Diseases.

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