Recombinant Technology Approach

Jason S. Knight, Subhash C. Verma, Ke Lan, and Erle S. Robertson

Summary

Herpesvirus genetics have long been hindered by the large size of the typical herpesvirus genome and the consequent recalcitrance of these genomes to manipulation by standard molecular genetics techniques. However, two primary strategies have emerged that allow for the generation of targeted viral mutants. With these mutants, investigators can pursue critical questions regarding the relationship between specific viral genetic elements and the viral life cycle. The first strategy of viral genome manipulation utilizes the mammalian homologous recombination machinery to introduce specific changes into the native viral genome. This approach involves construction of a targeting vector containing both the desired mutation and a significant flanking viral sequence to permit efficient recombination. The targeting vector is then introduced into mammalian cells, along with viral DNA, and recombinant virus is subsequently selected, harvested, and purified. The second, and more recent, approach utilizes bacterial artificial chromosome (BAC) technology to reconstitute complete herpesvirus genomes in the context of a prokaryote, E. coli. This artificial genome is then manipulated in, and purified from, E. coli before introduction into a mammalian background in which viral phenotypes can be assessed. Both strategies are discussed in this review, with particular emphasis on the homologous recombination strategies that have continued to be a powerful genetic tool in many herpesvirus systems.

Key Words: Molecular genetics; herpesvirus; herpes simplex; Epstein-Barr virus; homologous recombination; bacterial artificial chromosome; BAC.

1. Introduction

Understanding the role of specific herpesvirus gene products in the context of intact virus has long been hampered by the large size of the herpesvirus genome. These genomes have been difficult to manipulate experimentally in vitro, limiting the ability to functionally characterize and create specifically targeted changes in the genetic material. The background mutation rate for DNA

From: Methods in Molecular Biology, vol. 292: DNA Viruses: Methods and Protocols Edited by: P. M. Lieberman © Humana Press Inc., Totowa, NJ

viruses is generally low; therefore, early characterization of herpesvirus genomes employed random mutagenesis to identify genomic elements that imparted either sensitivity or resistance to a variety of physical, chemical, and biological conditions; such selective conditions included host range properties, plaque morphology, drug resistance, and temperature sensitivity (1-4). These forward genetic approaches required eventual mapping and comparison of mutations by a variety of cumbersome techniques including cross-complementation, recombination analysis, and marker rescue assays.

With the advent of current molecular cloning technologies, progress in understanding small mammalian DNA tumor viruses advanced rapidly; however, progress in applying such technologies to more complex herpesvirus genomes has been a slower, stepwise process. Today, targeted manipulation of the herpesvirus genome generally employs one of three broad strategies. The first specifically introduces a selectable marker into the genome by co-expression of an intact wild-type viral genome along with a targeting vector containing the marker flanked by the herpesvirus sequence to be disrupted. The recombination machinery of the host cell then generates a small population of the desired mutant virus. This method has been particularly successful for herpes simplex virus (HSV), which propagates robustly in tissue culture (5-7). In contrast, this technology has been less useful for viruses such as cytomegalovirus (CMV) that are strongly cell-associated, that replicate slowly in tissue culture, or that enter the lytic cycle only under specific and limited culture conditions (8-11). Another disadvantage of this approach is that wild-type virus is invariably vastly overrepresented, and consequently selection and/or extensive purification of mutant virus are required.

A second genetic approach for generating viral mutants also utilizes the eukaryotic recombination machinery. A complete viral genome is generated in cells by cotransfection of a complete set of overlapping cosmid clones in the absence of viral infection. This method has several advantages including isolation of mutant virus in the absence of wild-type and abrogation of the need for a selectable marker (12). As this approach essentially utilizes the same technologies as for the aforementioned selective marker approaches, it is only discussed briefly here.

A third and more recent approach to herpesvirus genetics employs bacterial artificial chromosomes (BACs) to maintain and manipulate complete herpesvirus genomes in E. coli. This strategy has the obvious and powerful advantage of employing the recombinogenic activities of bacteria (E. coli) for manipulation of large viral genomes. However, the complex and highly repetitive nature of the herpesvirus genome has in some cases called into question the stability or integrity of the E. coli-manipulated viral genome. In this chapter we provide a detailed description of the traditional generation of mutant viruses utilizing homologous recombination and an overview of the more recent employment of BAC technologies for herpesvirus genetic manipulation.

2. Materials

2.1. Homologous Recombination

1. Viral cosmid DNA for targeting vector construction (frequently available from commercial sources).

2. Restriction enzymes for manipulation of cosmid DNA (e.g., New England Biolabs).

3. Plasmid DNA containing the green fluorescent protein (GFP) gene (e.g., EGFP-C1, BD Biosciences Clontech).

4. Cell line permissive for viral infection and propagation (e.g., BHK cells for herpes simplex, purified primary B cells for Epstein-Barr virus [EBV], and so on).

6. Reagents for standard calcium phosphate transfection.

7. Electroporation unit (e.g., Bio-Rad Gene Pulser II).

8. Fluorescence microscope for visualization of GFP virus (e.g., Olympus X170).

1. Virus and cells: viral cosmid DNA or virally infected cells for producing viral DNA.

2. pBAC vector for generation of shuttle vector (commercially available from various molecular biology companies).

3. Restriction endonucleases for the manipulation of shuttle vectors and viral DNA (e.g., New England Biolabs and Invitrogen).

4. Cell line permissive for viral latent and lytic replication (e.g., HEK293 and BJAB cells).

5. E. coli DH10B for transformation of pBAC containing viral DNA.

6. Reagents and equipment for standard transformation and electroporation into E.

coli.

7. Antibiotics, available commercially (e.g., Sigma).

8. 12-O-Tetradecanoyl phorbol-13-acetate (TPA, e.g., Sigma) for the induction of lytic replication

3. Methods

3.1. Homologous Recombination Approaches to Herpesvirus Genetics

3.1.1. Design and Generation of Targeting Vector

Here we describe the preparation of a targeting vector for production of a general GFP herpesvirus that can facilitate the identification of herpesvirus-infected cells or trace dissemination of virus from a peripheral site of inoculation to different tissues.

Biochem Biophys Res Commun Herpesvirus

Fig. 1. Schematic representation of targeting vector for homologous recombination. A cassette containing the puromycin and green fluorescent protein (GFP) genes under the control of the CMV promoter is inserted into a unique PmeI restriction site in a commercially available herpesvirus cosmid. a, b, c, and d represent primers for differ-entiai determination of wild-type and recombinant virus. ab amplifies wild-type virus. ac and db amplify recombinant virus. IRES, internal ribosome entry; LUR, long unique region; TR, tandem repeat.

Fig. 1. Schematic representation of targeting vector for homologous recombination. A cassette containing the puromycin and green fluorescent protein (GFP) genes under the control of the CMV promoter is inserted into a unique PmeI restriction site in a commercially available herpesvirus cosmid. a, b, c, and d represent primers for differ-entiai determination of wild-type and recombinant virus. ab amplifies wild-type virus. ac and db amplify recombinant virus. IRES, internal ribosome entry; LUR, long unique region; TR, tandem repeat.

3.1.2. Construction of GFP Expression Cassette

This cassette potentially contains several elements including the GFP gene, a eukaryotic promoter to drive GFP gene expression, a drug resistance gene (such as puromycin or neomycin), and a eukaryotic promoter to drive expression of the drug-resistance gene. In some cases a polycistronic gene may be constructed using an internal ribosome entry site ([IRES] such as that of the hepatitis C virus [HCV]) to facilitate expression of GFP and drug resistance from a common promoter. This cassette should be constructed in a vector background with unique flanking restriction sites such that the cassette can be excised for insertion into the targeting vector (Fig. 1).

For viruses such as herpes simplex, with a tissue culture system that robustly supports lytic infection, the insertion of a drug resistance gene into the aforementioned GFP cassette may be unnecessary. However, for viruses such as EBV and Kaposi's sarcoma-associated herpesvirus (KSHV), which are primarily maintained in tissue culture as latently infecting genomes, drug selection may allow the establishment of latently infected producer cell lines that can be used to generate free virus by induction of lytic replication with chemical inducers or viral transactivating genes. This distinction will be discussed further below.

3.1.3. Construction of Targeting Vector for Generation of Recombinant Virus

Ideally the GFP cassette should be inserted into a unique restriction site in a region of the genome that does not encode a gene product, eliminating the possibility that the cassette will disrupt a viral open reading frame (ORF) and minimizing the potential for disruption of viral gene transcription. Careful consideration of this site is important, as the end goal of this strategy is to generate a recombinant GFP herpesvirus that mimics the functionality of the wild-type virus. Ideally, the aforementioned restriction site should be chosen with careful consideration of available cosmid sequences such that digestion with the appropriate enzyme results in a single cut and linearization of the cosmid. The GFP/drug resistance cassette can then be excised from its plasmid and inserted into the digested cosmid by a standard molecular ligation reaction. A restriction site can be chosen such that the insert is flanked by at least 3 kb of viral DNA sequence on the 5' and 3' termini. Again, flanking sequence should be chosen to minimize the dysregulation of the gene expression pattern of viral genes in the adjacent areas.

An example of such a strategy utilizes the PmeI site that lies downstream of ORF 18 and ORF 19 of the KSHV genome (13). Because ORF 18 is transcribed to the right and ORF 19 to the left, this site is downstream of both transcripts, minimizing the likelihood that insertion of the GFP cassette will disrupt viral gene expression. Importantly, this is the only PmeI restriction site in the available cosmid fragment, permitting unique insertion of the GFP cassette. This site has previously been used in the construction of a GFP KSHV BAC (13), but the same advantages apply in the construction of a targeting vector for homologous recombination.

3.1.4. Mutagenesis Strategies

The desired recombinant virus may possess either a manipulated or disrupted viral gene, in contrast to the general introduction of a foreign gene, GFP, as described just above. This usually depends on the specific application or genetic question. We will briefly outline approaches for introducing such mutations.

As mentioned above, GFP provides a signal that marks GFP recombinant virus-infected cells. However, for addressing specific gene functions in the context of the whole virus, genes can be knocked out and the resultant virus can be assayed for viral replication, packaging, infection, and so on. Based on the GFP

recombinant virus backbone, another cassette containing a second selectable marker can be introduced into a specific viral ORF by homologous recombination, disrupting the coding region of the targeted gene. The advantage of introduction of another selection marker into the viral genome is to facilitate double selection or selection of cloned infected populations, which include the GFP (initial selection marker) "wild-type" recombinants with a green color in infected cells and those that can be positively selected by antibiotics as "mutant" recombinants. This strategy might also employ one of the many fluorescent color proteins available as the second selection marker.

The strategy for construction of this second targeting vector is very similar to that described above for GFP. The primary difference is that the cassette containing the second selection marker should be introduced into a unique restriction site within the coding region of the viral gene of interest. A cassette encoding neomycin/puromycin/red fluorescent protein (RFP) under control of the CMV promoter can be digested from a commercially available construct. For generation of mutant virus, this cassette should be introduced by restriction digest into a cos-mid vector that contains a large fragment of viral genome including the target gene of interest. Cells stably infected with GFP recombinant virus can then be trans-fected with the mutant targeting vector. With induction of lytic replication, the targeting vector can recombine with GFP recombinant virus. To get a pure population of mutant virus, the supernatant containing GFP "wild-type" virus and GFP "mutant" virus can be used to infect permissive cells. By serial dilution of supernatant, and by employing the selection principle described above, pure mutant virus may be obtained.

3.1.5. Transfection and Induction of Lytic Replication

Cell type and transfection method are clearly dependent on the specific her-pesvirus being manipulated. For HSV, BHK cells are generally cotransfected with intact viral DNA and the targeting vector by a standard calcium phosphate transfection protocol. A similar approach is taken for varicella-zoster virus (VZV) and CMV with melanoma and fibroblast cell lines, respectively.

As no robust system for EBV or KSHV lytic infection exists in vitro, the targeting vector is generally transfected into a virus-infected cell line (typically a B cell) by electroporation. Successful electroporation of B cells can be performed with the Bio-Rad Gene Pulser at 200 V and 960 ^F (see Note 1). Lytic infection is induced either by cotransfection of a lytic transactivator (BZLF1 for EBV and Rta for KSHV) or by treatment with chemical agents such as phorbol esters or butyric acid (14,15). Although treatment with chemical agents produces a more robust overall lytic response, cotransfection of the transactivator has the advantage of enriching for lytic replication only in the transfected population of cells, potentially reducing the background of wild-type virus with no potential to recombine. Similarly, cotransfection of lytic gene transactivators has proved useful for other viruses such as the use of VZV ORF62 in the generation of intact virus from overlapping cosmid clones (16).

The efficiency of lytic virus induction can be monitored by immunofluorescence of a lytic gene (e.g., gB) and also by specific polymerase chain reaction (PCR) analysis of the cell supernatant for viral DNA (see Note 2). PCR analysis may differentiate wild-type from recombinant virus by specific PCR primers that will detect and distinguish the wild-type from recombinant virus DNA. It should, however, be noted that these methods only indicate the production of viral progeny and not the infectious nature of these progeny virions. Returning to the example of the unique PmeI site in the viral cosmid construct, PCR primers for distinguishing wild-type and recombinant virus might be designed as indicated in Fig. 1.

3.1.6. Selection and Isolation of Recombinant Virus

For herpesviruses such as HSV, which lytically replicate to high titer and form robust plaques in tissue culture, permissive cells are simply transfected with targeting vector and virion DNA, and plaques of GFP-expressing virus are purified from wild-type by limiting dilution strategies. For lytic viruses this strategy has proved highly efficient in genetic analysis of virus-encoded genes with mutation of these ORFs determining functional domains required for infection and replication (17).

For viruses with tissue culture systems that less robustly support lytic infection, induction of latently infected cells may be necessary, as discussed previously. Ideally, upon induction recombinant virus will be produced and will egress from the cell into the supernatant, where it can be collected for further purification and study. In some cases, recovery of recombinant virus may be enhanced by transfecting latently infected cells with the targeting vector followed by drug selection. Selection will enrich for cells transfected with the targeting vector and should increase the ratio of recombinant to wild-type virus in the cell supernatant. To obtain a pure recombinant virus population, the supernatant can be diluted and used to infect permissive cells at varying dilutions. The infected cells are again drug-selected, and, under continuous selection, stable recombinant virus-infected cells can be obtained. These latently infected cell lines can then be induced to generate recombinant virus. After several cycles of limiting supernatant dilution, cell infection, and selection, a pure recombinant virus-producer cell line can be obtained (see Note 3).

3.1.7. Characterization

To determine whether the desired recombinant herpesvirus was successfully generated, it is necessary to complete genetic and phenotypic characterization of the virus (see Notes 4 and 5). The first line of characterization involves restriction analysis using enzymes that cut with moderate frequency in herpesvirus genomes such as BamHl, EcoRl, Notl, and ¿all. It is recommended that analysis of at least three restriction profiles be determined. Any discord between wild-type and recombinant profiles is probably attributable to the desired recombination event. The identity of bands modified by the recombination event should then be confirmed by Southern hybridization with probe/probes specific to the sequence immediately flanking the recombination event as well as any genetic material inserted in the site (selectable marker). Once the alteration of the genetic structure of the genome has been confirmed, the integrity of the recombination event should be confirmed by PCR amplification and sequencing of any junctions between the viral and foreign DNA (see Note 6).

Phenotypically, the recombinant virus can be characterized by gene expression profiling of genes immediately flanking the recombination event. Such analysis can be done by standard methods such as Northern blotting or realtime PCR. For viruses with a cell culture system that supports lytic infection, a one-step growth curve can be performed to compare the in vitro growth kinetics of the recombinant virus with that of the wild-type virus. For viruses lacking a system for characterizing lytic infection, other phenotypic properties should be considered, such as the efficiency of recombinant EBV to immortalize B lymphocytes in vitro in comparison with the efficiency of wild-type EBV. A final, and costly, means of characterization involves putting the recombinant virus into an animal system for consideration of pathogenicity. Such characterization is undoubtedly warranted when the recombinant virus, or subsequent derivatives, will ultimately be employed in animal pathogenesis experiments.

3.2. BAC Approaches to Herpesvirus Genetics

3.2.1. Background

A BAC is a DNA molecule of plasmid origin into which another large DNA fragment (l00-300-kb insert size; average, 150 kb) can be integrated without loss of the plasmid's capacity for self-replication. Yeast artificial chromosomes (YACs) were the first vectors used for cloning large genomic DNA fragments; however, YACs have numerous problems including difficulty in purifying cloned DNA from contaminating yeast DNA and frequent spontaneous rearrangements of the foreign DNA (18,19). In contrast, BAC clones show surprising stability of the foreign DNA (20) and can accommodate DNAs larger than 300 kb. One of the first technologies employed by Shizuya et al. in 1992 (21) to clone large DNA fragments was based on the E. coli F factor. Strict control of the F-factor replicon maintains a single copy of the plasmid DNA per cell, reducing the risk of recombination events via repetitive DNA elements present in the foreign DNA. Interestingly, cosmid clones have also been shown to maintain foreign DNA more stably under the control of F-derived replicons than the common origin of plasmid replication (21).

3.2.2. Cloning of Herpesvirus Genomes Into BACs

The cloning limit of BACs exceeds the size of the largest known her-pesviruses (hCMV); therefore, BACs can theoretically support and maintain complete herpesvirus genomes. In 1997, the first infectious herpesvirus BAC, the murine CMV, was constructed and was subsequently followed by other viruses (22-28). The large size and the recalcitrance to in vitro manipulation of the herpesvirus genome prevented simple ligation of the viral genome into a BAC by standard molecular cloning procedures. Efficient cloning required a preliminary step in which the BAC plasmid cassette is itself introduced into the viral genome, essentially through the homologous recombination strategy described above. Insertion of the BAC sequence is followed by isolation of a BAC recombinant virus (Fig. 2). Frequently, a selection marker (antibiotic resistance) or reporter gene such as GFP/lacZ is employed to facilitate recovery of recombinant virus.

3.2.3. Purification and Recovery of BAC Recombinant Virus

Before genetic manipulation, the BAC viral recombinant must be purified, followed by transformation into E. coli competent cells. For successful transformation, an intact, circularized form of the recombinant virus must be isolated prior to transformation into E. coli. Two approaches are available for isolation and purification of the viral genome. The most common approach takes advantage of the herpesvirus replication cycle, which results in circular replica-tive intermediates of viral DNA prior to cleavage into unit-length linearized forms for packaging in the virus particle. DNA harvested by Hirt extraction early after transfection gives a significant amount of circular replicative intermediate (29). Alternatively, linear DNA can be harvested and ligated in vitro (Fig. 3). This procedure can potentially give both circular and concatamaric version of the genome, but only circular versions will propagate.

3.2.4. BAC Stability in E. coli

Herpesvirus genomes contain highly repetitive sequences that are potential targets for recombination. Therefore, a herpesvirus BAC can be maintained in a bacterial strain of E. coli that is devoid of the recABCD gene (20). However, for mutagenesis and gene manipulation, the recombination system may need to be re-expressed. This can be achieved by shuttling the BAC recombinant virus

Fig. 2. Construction of bacterial artificial chromosome (BAC) herpesvirus by homologous recombination. Efficient cloning of the BAC herpesvirus requires a preliminary step in which the BAC plasmid cassette is introduced into the viral genome through homologous recombination. Insertion of the BAC sequence is followed by isolation of a BAC recombinant virus. A selection marker (antibiotic resistance) may be employed to facilitate efficient recovery of recombinant virus.

Fig. 2. Construction of bacterial artificial chromosome (BAC) herpesvirus by homologous recombination. Efficient cloning of the BAC herpesvirus requires a preliminary step in which the BAC plasmid cassette is introduced into the viral genome through homologous recombination. Insertion of the BAC sequence is followed by isolation of a BAC recombinant virus. A selection marker (antibiotic resistance) may be employed to facilitate efficient recovery of recombinant virus.

Fig. 3. Alternative construction of bacterial artificial chromosome (BAC) herpesvirus by in vitro ligation. A unique restriction site within the viral genome may permit direct ligation of the BAC plasmid into the viral genome.

from a recA- to a recA+ strain (22,30,31) or by using a bacterial strain in which expression is controlled by an inducible promoter (22,32).

3.2.5. Alternatives to Homologous Recombination for Generation of BAC Clones

Although BAC cloning typically requires homologous recombination between viral sequence flanking the BAC plasmid and viral DNA, this is not always the case. If unique restriction sites are present in the viral genome, BAC vector sequence can potentially be ligated in vitro. An example of ligation of the BAC plasmid is shown in Fig. 3. The viral DNA is digested at unique restriction sites and ligated with the BAC plasmid similarly digested with the same restriction enzyme. In one example, transfection of the ligation mix generated a recombinant BAC-containing virus. Circular viral DNA was isolated and used to transform into E. coli. Transformed colonies screened for the BAC contained full-length viral genomes (33).

3.2.6. Manipulation of the Herpesvirus-BAC Genome

Once the viral genome is cloned into a BAC system, a variety of techniques are available for the manipulation of viral genome following transformation into E. coli. Some commonly used methods are described here.

3.2.6.1. Shuttle Plasmids

Shuttle plasmids are used for the mutagenesis of the herpesvirus genome in E. coli via homologous recombination. This method is believed to be one of the simplest methods for the introduction of various kinds of mutations (e.g., point mutation, deletions, insertions, or sequence replacement) into viral BAC. A schematic of this strategy is shown in Fig. 4, in which the desired mutations are cloned into a suicide plasmid (unable to replicate in E. coli) that contains a viral sequence homologous to the target site in the viral BAC. The resulting shuttle vector is then cotransformed along with the viral BAC into an E. coli strain that conditionally expresses RecA. Homologous recombination via RecA activity potentially leads to the insertion of a mutated copy of the gene into BAC viral DNA.

In this method the mutations are introduced into linear DNA with the use of recET from prophage Rac or redaP from bacteriophage X. A linear DNA fragment containing a selectable marker and a homologous sequence flanking the target mutated sequence is transferred into a recombination-positive E. coli strain. By a double crossover event, the target gene (with selectable marker) is introduced into the BAC vector (Fig. 5). This method has advantages over shuttle plasmid-based recombination because the RecET recombination is more efficient and requires homologies of only 25-50 nucleotides for crossover. These homologous sequences can be provided by the synthetic oligonucleotide primers used to amplify the liner target DNA sequence.

3.2.6.3. Random Transposon Mutagensis

Transposons (Tn) are mobile genetic elements that insert themselves into DNA at random sequences. Transformation of a Tn donor plasmid into E. coli-containing viral BAC can lead to mutation in the BAC sequence, as the transposon element has the ability to integrate randomly on DNA (Fig. 6). By using

Shuttle vector

Shuttle vector

mut mut

Fig. 4. Introduction of mutations into the viral genome by a shuttle vector and recombinant A (recA)-dependent recombination in E. coli. BAC, bacterial artificial chromosome; ORF, open reading frame.

Fig. 4. Introduction of mutations into the viral genome by a shuttle vector and recombinant A (recA)-dependent recombination in E. coli. BAC, bacterial artificial chromosome; ORF, open reading frame.

a suicide origin of replication on the Tn donor vector, propagation of the vector can be eliminated. The Tn insertion site can be easily mapped and sequenced from primer sites within the Tn element; this is a major advantage over chemical mutagenesis when one is screening large BAC genomes.

3.2.7. Limitations of BAC Herpesviruses

One major concern of BAC-derived technology is the long-term stability of eukaryotic DNA in E. coli. To date, even complex sequences such as inverted and direct repeats appear to be extremely stable in E. coli. However, Smith and Enquist (30) reported that F-plasmid sequences were unstable following passage of virus in experimental animals. Additionally, it is not well understood how many heterologous genes can be packaged into the BAC-derived virus

Fig. 5. Mutations can be introduced into linear DNA with the use of recET from prophage or redap from bacteriophage X. A linear DNA fragment containing selectable marker and homologous sequence flanking the target mutated sequence is transferred into a recombination-positive E. coli strain. By a double crossover event, the target gene (with selectable marker) is introduced into the bacterial artificial chromosome (BAC) vector. Recombination requires only 25-50 nucleotides for crossover, and these homologous sequences can be provided by the synthetic oligonucleotide primers used to amplify the linear target DNA sequence. ORF, open reading frame; RecA, recombinant A; TR, tandem repeat.

Fig. 5. Mutations can be introduced into linear DNA with the use of recET from prophage or redap from bacteriophage X. A linear DNA fragment containing selectable marker and homologous sequence flanking the target mutated sequence is transferred into a recombination-positive E. coli strain. By a double crossover event, the target gene (with selectable marker) is introduced into the bacterial artificial chromosome (BAC) vector. Recombination requires only 25-50 nucleotides for crossover, and these homologous sequences can be provided by the synthetic oligonucleotide primers used to amplify the linear target DNA sequence. ORF, open reading frame; RecA, recombinant A; TR, tandem repeat.

before size constraints becomes problematic. For HSV, it has been reported that up to 30 kb in addition to the viral genome can be packaged. Other viruses remain to be more thoroughly explored by this approach.

The second major problem with BAC-based technology is in developing homologous recombination strategies for shuttle insertion of novel genes into BAC viruses. This strategy is limited because of the lack of unique and suitable restriction sites in most viral genomes. However, this drawback can be partially addressed by the diverse mutagenesis techniques available in E. coli. Transposon mutagenesis has been most useful to date in allowing rapid generation of herpesvirus BAC mutants by insertion of Tn5 across the viral genome.

3.2.8. Further Application of Herpesvirus-BAC Technology

BAC technology has significantly advanced the analysis and molecular genetics of the herpesvirus genomes. BAC is probably the most flexible and efficient technology for generating mutant viruses, especially when the specif-

Fig. 6. Random mutagenesis of the bacterial artificial chromosome (BAC) herpesvirus genome by insertion of a transposable element. Transformation of a transpo-son (Tn) donor plasmid into E. coli containing viral BAC can lead to mutation in the BAC sequence as the transposon element has the ability to integrate randomly on DNA.

Fig. 6. Random mutagenesis of the bacterial artificial chromosome (BAC) herpesvirus genome by insertion of a transposable element. Transformation of a transpo-son (Tn) donor plasmid into E. coli containing viral BAC can lead to mutation in the BAC sequence as the transposon element has the ability to integrate randomly on DNA.

ic herpesvirus BAC of interest has already been developed. This has been particularly true for a-herpesviruses, as, to date, such viruses have aided greatly in defining the roles of specific viral gene products in pathogenesis. Random mutagenesis strategies have been developed for insertional, deletional, or trans-poson-mediated approaches that can quickly establish the roles of specific genes in the viral life cycle (26,34-36). BAC technology may also prove to be a powerful tool for generating recombinant viruses expressing heterologous therapeutic genes utilized as herpesvirus gene therapy vectors (37). Additionally, herpesvirus BACs can function as helper-virus-free reagents in the propagation of gene therapy vectors such as HSV amplicons and adeno-associated virus vectors. Moreover, BACs can also be useful in vaccine design, by not only providing a substrate to generate safe live attenuated vaccine but also as immunogens for DNA vaccines.

The use of BAC technology for generating y-herpesvirus recombinants is still somewhat in its infancy. The stability and successful generation of mutants in the y-herpesviruses EBV and KSHV will further enhance the capability of this technology in analysis of the large DNA virus genomes. However, studies utilizing this technology for EBV and KSHV have not produced consistent results in targeting specific viral genes. Success in generation of specific mutations in a reproducible and stable fashion will further increase the utility of this technology.

4. Notes

1. For transfection of cells to introduce the recombinant plasmids, one must be sure that the cells are greater than 98% viable to ensure effective transfection and sufficient recombination efficiency. Cells should be fed 24 h prior to transfection and growing exponentially. DNA should be mixed thoroughly in buffer to ensure proper solubility before transfection. Usually DNA stocks for transfection should be kept at 4°C and at a concentration of no greater than 0.5 pg/mL. It is important to note that your DNA preparation should be of the highest purity, and we recommend using CsCl-purified DNA, prepared with two spins before collecting for use. Once these DNA preparations are prepared, we have found they can be stored and used indefinitely with little loss in activity.

2. Transfected cells can be monitored for production of virion particles. Samples of the supernatant can be collected and spun at maximum speed in a microcentrifuge for 20 min to collect possible virus particles. The resulting pellet can then be heated to 95°C for 15 min, followed by treatment with proteinase K (followed by killing of the protease). This crude lysate can then be used for PCR analysis of the viral DNA or a specific sequence introduced into the virus. PCR analysis should always include the appropriate positive and negative controls as transfected DNA can sometimes be stable and carried forward in the supernatant.

3. The use of GFP as a means of tracking recombinant virus production should be carefully monitored. In some cases we have seen GFP transferred to virion particles and introduced into infected cells without recombinant virus. This false trans-duction can be misleading and lead to incorrect conclusions as to the presence of a recombinant virus.

4. The production of a recombinant virus is very time-consuming, and viruses typically have a tendency to acquire their most stable genomic state. Therefore, if there are small amounts of wild-type virus in the viral preparation, over time this can become the dominant genome if the selective pressures are absent. This potential problem has to be carefully considered, as continued passage in culture can quickly lead to loss of your recombinant virus.

5. Another common pitfall is the assumption that the presence of either GFP DNA or selective marker plasmid DNA confirms the presence of the recombinant virus. This should always be checked with an induction and passage of the virus into fresh uninfected cells. Additionally, the resulting virus genome should be characterized fully to be sure that deletions and rearrangements have not occurred that might impair viral viability. Herpesviruses have a strong propensity for recombination and rearrangements. There have been many occasions on which an investigator has identified a recombinant virus by GFP or selective marker analysis, but the remaining viral genome was deleted or rearranged. This rearranged or deleted virus then becomes the predominant genome, as it is usually selected for by the investigator.

6. One highly efficient approach to determination of a stable recombinant virus genome is to select a number of unique sites spanning the genome that can be amplified by PCR analysis. This can be performed with minimal effort and confirms the presence of a relatively complete genome. The presence of specific mutations can also be determined for stability over multiple passages by this method.

Acknowledgments

J. S. K. is supported by the Lady Tata Memorial Trust. E. S. R. is funded by NIH grants NCI CA72150-07, NCI CA91792-01, and DCR DE14136-01 and is also a scholar of the Leukemia and Lymphoma Society of America.

References

1 Aurelian, L. and Roizman, B. (1964) The host range of herpes simplex virus. Virology 22, 452-461.

2 Ejercito, P. M., Kieff, E. D., and Roizman, B. (1968) Characterization of herpes simplex virus strains differing in their effects on social behaviour of infected cells. J. Gen. Virol. 2, 357-364.

3. Kit, S. and Dubbs, D. R. (1963) Nonfunctional thymidine kinase cistron in bro-modeoxyuridine resistant strains of herpes simplex virus. Biochem. Biophys. Res. Commun. 13, 500-504.

4 Schaffer, P., Vonka, V., Lewis, R., and Benyesh-Melnick, M. (1970) Termperature-sensitive mutants of herpes simplex virus. Virology 42, 1144-1146.

5 Goldstein, D. J. and Weller, S. K. (1988) An ICP6::lacZ insertional mutagen is used to demonstrate that the UL52 gene of herpes simplex virus type 1 is required for virus growth and DNA synthesis. J. Virol. 62, 2970-2977.

6 Neidhardt, H., Schroder, C. H., and Kaerner, H. C. (1987) Herpes simplex virus type 1 glycoprotein E is not indispensable for viral infectivity. J. Virol. 61, 600-603.

7 Post, L. E., Mackem, S., and Roizman, B. (1981) Regulation of alpha genes of herpes simplex virus: expression of chimeric genes produced by fusion of thymidine kinase with alpha gene promoters. Cell 24, 555-565.

8 Jones, T. R., Muzithras, V. P., and Gluzman, Y. (1991) Replacement mutagenesis of the human cytomegalovirus genome: US10 and US11 gene products are nonessential. J. Virol. 65, 5860-5872.

9 Kemble, G. W. and Mocarski, E. S. (1989) A host cell protein binds to a highly conserved sequence element (pac-2) within the cytomegalovirus A sequence. J. Virol. 63,4715-4728.

10 Spaete, R. R. and Mocarski, E. S. (1987) Insertion and deletion mutagenesis of the human cytomegalovirus genome. Proc. Natl. Acad. Sci. USA 84, 7213-7217.

11 Vieira, J., Farrell, H. E., Rawlinson, W. D., and Mocarski, E. S. (1994) Genes in the HindIII J fragment of the murine cytomegalovirus genome are dispensable for growth in cultured cells: insertion mutagenesis with a lacZ/gpt cassette. J. Virol. 68, 4837-4846

12 Wagner, M., Ruzsics, Z., and Koszinowski, U. H. (2002) Herpesvirus genetics has come of age. Trends Microbiol. 10, 318-324.

13 Zhou, F. C., Zhang, Y. J., Deng, J. H., Wang, X. P., Pan, H. Y., Hettler, E., and Gao, S. J. (2002) Efficient infection by a recombinant Kaposi's sarcoma-associated her-pesvirus cloned in a bacterial artificial chromosome: application for genetic analysis. J. Virol. 76, 6185-6196.

14 Nakamura, H., Lu, M., Gwack, Y., Souvlis, J., Zeichner, S. L., and Jung, J. U. (2003) Global changes in Kaposi's sarcoma-associated virus gene expression patterns following expression of a tetracycline-inducible Rta transactivator. J. Virol. 77,4205-4220.

15 Tomkinson, B., Robertson, E., and Kieff, E. (1993) Epstein-Barr virus nuclear proteins EBNA-3A and EBNA-3C are essential for B-lymphocyte growth transformation. J. Virol. 67, 2014-2025.

16 Cohen, J. I. and Seidel, K. E. (1993) Generation of varicella-zoster virus (VZV) and viral mutants from cosmid DNAs: VZV thymidylate synthetase is not essential for replication in vitro. Proc. Natl. Acad. Sci. USA 90, 7376-7380.

17. Roizman, B. and Knipe, D. M. (2002) Herpes simplex viruses and their replication, in Fields Virology, vol. 2 (Knipe, D. and Howley, P., eds.), Lippincott Williams & Wilkins, pp. 2399-2459.

18 Ramsay, M. (1994) Yeast artificial chromosome cloning. Mol. Biotechnol. 1,181-201.

19 Schalkwyk, L. C., Francis, F., and Lehrach, H. (1995) Techniques in mammalian genome mapping. Curr. Opin. Biotechnol. 6, 37-43.

20 Shizuya, H., Birren, B., Kim, U. J., Mancino, V., Slepak, T., Tachiiri, Y., and Simon, M. (1992) Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc. Natl. Acad. Sci. USA 89, 8794-8797.

21 Kim, U. J., Shizuya, H., de Jong, P. J., Birren, B., and Simon, M. I. (1992) Stable propagation of cosmid sized human DNA inserts in an F factor based vector. Nucleic Acids Res. 20,1083-1085.

22 Messerle, M., Crnkovic, I., Hammerschmidt, W., Ziegler, H., and Koszinowski, U. H. (1997) Cloning and mutagenesis of a herpesvirus genome as an infectious bacterial artificial chromosome. Proc. Natl. Acad. Sci. USA 94, 14759-14763.

23 Stavropoulos, T. A. and Strathdee, C. A. (1998) An enhanced packaging system for helper-dependent herpes simplex virus vectors. J. Virol. 72, 7137-7143.

24! Delecluse, H. J., Hilsendegen, T., Pich, D., Zeidler, R., and Hammerschmidt, W.

(1998) Propagation and recovery of intact, infectious Epstein-Barr virus from prokaryotic to human cells. Proc. Natl. Acad. Sci. USA 95, 8245-8250.

25 Saeki, Y., Ichikawa, T., Saeki, A., et al. (1998) Herpes simplex virus type 1 DNA amplified as bacterial artificial chromosome in Escherichia coli: rescue of replication-competent virus progeny and packaging of amplicon vectors. Hum. Gene Ther. 9, 2787-2794.

26 Wagner, M., Jonjic, S., Koszinowski, U. H., and Messerle, M. (1999) Systematic excision of vector sequences from the BAC-cloned herpesvirus genome during virus reconstitution. J. Virol. 73, 7056-7060.

27 Adler, H., Messerle, M., and Koszinowski, U. H. (2003) Cloning of herpesviral genomes as bacterial artificial chromosomes. Rev. Med. Virol. 13, 111-121.

28! Delecluse, H. J., Kost, M., Feederle, R., Wilson, L., and Hammerschmidt, W. (2001) Spontaneous activation of the lytic cycle in cells infected with a recombinant Kaposi's sarcoma-associated virus. J. Virol. 75, 2921-2928.

29 Hirt, B. (1967) Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26, 365-369.

30 Smith, G. A. and Enquist, L. W. (1999) Construction and transposon mutagenesis in Escherichia coli of a full-length infectious clone of pseudorabies virus, an alpha-herpesvirus. J. Virol. 73, 6405-6414.

31 Horsburgh, B. C., Hubinette, M. M., Qiang, D., MacDonald, M. L., and Tufaro, F.

(1999) Allele replacement: an application that permits rapid manipulation of herpes simplex virus type 1 genomes. Gene Ther. 6, 922-930.

32 Kempkes, B., Pich, D., Zeidler, R., and Hammerschmidt, W. (1995) Immortalization of human primary B lymphocytes in vitro with DNA. Proc. Natl. Acad. Sci. USA 92, 5875-5879.

33 McGregor, A. and Schleiss, M. R. (2001) Molecular cloning of the guinea pig cytomegalovirus (GPCMV) genome as an infectious bacterial artificial chromosome (BAC) in Escherichia coli. Mol. Genet. Metab. 72, 15-26.

34 Brune, W., Messerle, M., and Koszinowski, U. H. (2000) Forward with BACs: new tools for herpesvirus genomics. Trends Genet. 16, 254-259.

35 Muyrers, J. P., Zhang, Y., Testa, G., and Stewart, A. F. (1999) Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res. 27, 1555-1557.

36. Brune, W., Menard, C., Hobom, U., Odenbreit, S., Messerle, M., and Koszinowski, U. H. (1999) Rapid identification of essential and nonessential herpesvirus genes by direct transposon mutagenesis. Nat. Biotechnol. 17, 360-364.

37. Borst, E., and Messerle, M. (2000) Development of a cytomegalovirus vector for somatic gene therapy. Bone Marrow Transplant 25 Suppl 2, S80-82.

0 0

Post a comment