Members of the viral family Herpesviridae are characterised by double-stranded DNA genomes ranging in length from 125 to 240 Kbp. The genetic stability and potential for incorporating large amounts of foreign DNA have made the herpesviruses a promising target for the development of gene therapy and vaccine vectors. For these purposes, replication-limited herpesvirus vectors have been developed through the deletion of an essential viral gene, which can subsequently be provided in trans to permit virus replication in a controlled or limited manner in vivo (for a review see [1]). While many early studies based on these techniques were successful, the scope of determining viral gene function was limited by two factors. Firstly, genes could only be manipulated using homologous recombination in virus-susceptible cells. To successfully use this approach, the cells had to be amenable to a method of introducing foreign genetic material, to permit the introduction of the DNA transgene-material encoding the required modification. Secondly, it was difficult to efficiently generate mutant viruses with altered or deleted genes that are essential for virus replication. These types of mutations required the co-delivery of functional copies of the deleted genes to permit virus replication. This was generally achieved by the generation of stably transformed cell lines that constitutively expressed the gene of interest or by using helper viruses. As a result of these limitations, the development of novel vectors and gene function studies could be achieved, however it was a time consuming process. These requirements severely limited the capacity to efficiently generate viruses with the desired mutations and deletions. In contrast, the generation of BAC mutants is a much quicker process. However mutations affecting essential viral genes still require the gene product to be provided in trans to generate infectious virions.

A key step in the development of herpesviruses as biological vectors, and herpes biology in general, was the development of infectious clone technologies. Initial efforts to improve the efficiency for herpesvirus genomic manipulation involved the use of Escherichia coli plasmid replicons known as cosmids. As these cosmids can only accommodate up to 45Kbp of foreign DNA, a series of vectors that contained overlapping segments of the herpesvirus genome were required to facilitate the generation of recombinant viruses [2]. Typically modifications were made to one fragment, followed by the rescue of infectious virus by introducing the complementing cosmids into cells to generate infectious virus.

Luckow et al. [3] were the first to demonstrate the cloning of the complete genome of a large double-stranded DNA virus as a bacterial artificial chromosome (BAC) vector. This was achieved by cloning a baculovirus genome to permit propagation and manipulation of the viral genome in Escherichia coli and subsequent rescue of infectious virus. Messerle et al. (1997) subsequently extended this technique to the herpesvirus family by cloning the complete genome of murine cytomegalovirus into a BAC [4]. This facilitated the recovery of infectious virus once reintroduced into susceptible cells. Since this initial study the genomes of at least 20 herpesviruses of human and veterinary importance and their derivatives have been used to create infectious clones using BAC technologies (icBAC) [5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25]. More recently, BAC technology has also been extended to other DNA viruses including poxviruses [26,27], and to RNA viruses including coronaviruses and flaviviruses, through the development of infectious cDNA clones [28,29].

The cloning of large fragments of genomic DNA for the past three decades has underpinned advances made in genome sequencing, identification of the causative genetics of disease and the development of disease models. The three major vector types utilized in these studies are BACs, yeast artificial chromosomes (YAC) and mammalian artificial chromosomes. While each of these systems have their own advantages depending on the research question to be addressed, BACs are undoubtedly the system of choice for studying herpesviruses. BAC vectors are based on the E. coli fertility factor (F-factor) replicon which is maintained as a circular supercoiled extrachromosomal single copy plasmid in the bacterial host [2,30,31]. BACs can accept inserts up to 300 Kb in length, which is sufficient to allow the genomes of all known herpesviruses to be maintained as an icBAC. The principal advantage BACs have over the traditional YAC systems is stability of insert propagation over multiple generations. This is an essential property in the context of herpesvirus biology, as the genomes of many of these viruses contain a variety of repetitive sequence elements that could promote instability. However many studies have successfully propagated icBAC over multiple passages without detecting rearrangements [4,5,7,8,9,10,12,26,32,33]. Although YACs are capable of maintaining very large DNA inserts of up to 1Mb, they have numerous disadvantages, including instability, chimaerism and handling difficulties such as shearing of DNA [30,31].

The capacity to continually propagate a viral genome with high fidelity also provides the opportunity to explore other avenues of herpesvirus biology, compared to tradition cell-based methodologies. Each BAC clone represents a single replicative template that has been cloned during the replication process, termed clonal selection. As such, BAC clones permit an assessment to be made not only of the genomic variation that exists within a virus isolate, but also how these differences impact on viral biology. Although the concept of quasispecies is used almost exclusively to describe RNA virus populations because of the relatively high error rate of RNA polymerase [34], sequence heterogeneity has recently been identified for BAC clones of murine cytomegalovirus and Gallid herpesvirus 2 and for plaque-purified pseudorabies virus stocks [35,36,37,38].

Further, a BAC clone can help ameliorate the effects of in vitro passage of virus isolates. There are numerous examples where attenuation of the virulence of a herpesvirus can be achieved by repeated passage in susceptible cells [39,40,41]. While this is sometimes the aim of passage, for example in the development of an attenuated strain for use as a vaccine, the apparent accumulation of mutations can make it difficult to effectively study important viral properties such as virulence. An icBAC not only allows the efficient production of many copies of virus genome, it also provides the mechanism to return to the first passage without the need for in vivo back-passaging in the natural host and subsequent re-isolation.

The primary methodology for developing icBAC is dependent on some knowledge of the viral genome sequence. This approach was first described by Messerle et al. (1997) who developed a transfer vector to facilitate the use of homologous recombination to transfer a BAC vector into the genome of murine cytomegalovirus. In order to increase the likelihood of successful transfer, a selection cassette was included in the vector. This approach has been widely used to generate the majority of icBAC [9,11].

Other methods of icBAC construction have also been reported, although the utility of these techniques across a broader range of viruses has yet to be demonstrated.

Smith and Enquist (1999) generated an icBAC of pseudorabies virus by first using homologous recombination in eukaryotic cells to insert a single loxP site into the viral genome. This construct then recombined at this site with a plasmid containing the BAC vector [9].

Where available, the complete genome sequence of a virus can be used to identify unique restriction endonuclease sites that can be used to increase the efficiency of homologous recombination in host cells. This approach was used by Mahony et al. (2002) to facilitate the construction of an icBAC for Bovine herpesvirus 1 (BoHV-1) [12].

Recently Ooi and coworkers [42] have also demonstrated BACs maintained as linear replication-competent constructs. This was achieved by inserting a bacteriophage N15 tos site into a circular BAC that was then resolved in vivo to produce hairpin telomeres that capped the ends of the linearized BAC [42]. These linear BACs have been shown to be as amenable to recombination techniques as more traditional circular BACs [43]. This technique has not yet been applied to viral icBACs.

Due to these handling features, it is not surprising that BAC vectors have dominated the development of infectious herpesvirus clones.

The limitation of availability of genomic sequence in the construction of herpesvirus clones has potentially been overcome by two recent reports that described the insertion of BAC vectors into viral genomes using transposon-mediated methodologies [44,45]. However, so far this method has only been reported for a murine herpesvirus-68 strain and a baculovirus shuttle vector.

The generation of recombinant viruses prior to the application of icBAC was reliant on low frequency homologous recombination in virus-susceptible eukaryotic cells [3]. Typically this would require the development of a transfer or shuttle plasmid containing the transgene of interest flanked by regions of virus sequence upstream and downstream of where the transgene was to be inserted. The transfer vector was then introduced into virus-susceptible cells followed by virus infection, and the recombinant virus was plaque-purified. Various strategies were used to increase the efficiency of this process, including the addition of selectable markers or the co-transfection of infectious viral DNA and shuttle plasmid DNA [46]. Overall these were time-consuming and labour intensive methods, and due to these limitations, the focus turned to utilizing the powerful recombination machinery of bacteria.

The large size of DNA inserts in BACs limits the efficient application of traditional restriction endonuclease and ligation techniques to introduce mutations, however some methods involving restriction enzymes have been successfully utilized. RecA-assisted restriction endonuclease (RARE) cleavage involved protecting a specific restriction site by using a complementary oligonucleotide that prevented methylation when bound to the complementary sequence [47]. After dissociation of the complex, the restriction site was amenable to routine restriction and ligation procedures. This technique was used to generate a chimaeric BAC and then introduce deletions in a BAC containing the full-length LRP-1 gene [47]. NotI sites have been used to retrofit selectable markers into mammalian BACs due to the infrequent occurrence of NotI sites in mammalian genomic DNA [48]. These applications were limited by the location of pre-existing restriction sites and successful manipulation was laborious.

The application of BAC clones in the development of human disease models facilitated the development of strategies to apply bacterial genetics to increase the efficiency and precision of BAC manipulation using sequence-dependent and independent technologies. BAC engineering of viral icBACs is now common practice both to elucidate gene function and for the generation of recombinant viruses. Methods have been established for both site-specific changes, such as point mutations, insertions, deletions or gene fusions, and for random mutagenesis utilizing transposable elements. In contrast to the use of homologous recombination to generate recombinant viral progeny in eukaryotic cells, mutated genomes within icBAC can be fully characterized before attempting to recover infectious virus, avoiding time-consuming selection steps [4]. Following transfection of the modified icBAC into permissive eukaryotic cells, infectious recombinant virus may be rescued. Existing methodologies continue to be improved to enhance the efficiency of recombinant virus production and to remove marker and BAC vector sequences for use in transgenic studies.

Mutagenesis of BACs within E. coli was originally mediated by RecA, part of the DNA repair machinery of the bacterial cell [49]. In E. coli, double-strand breaks of the bacterial chromosome are repaired mainly through the RecBCD pathway. During this process, bacterial RecA identifies double-strand homologous sequences and strand invasion occurs, whereby RecA unwinds the dsDNA and assimilates it to its complementary sequence. The displaced single-stranded DNA is digested by exonuclease activity and DNA ligase completes the repair process (reviewed in [50,51]). Utilising constitutive bacterial recombination pathways for BAC mutagenesis is limited because the majority of laboratory strains of E. coli have been engineered to be recombination negative to maintain stability of DNA inserts by preventing unwanted recombination, deletions or rearrangements. Also, linear double-stranded DNA molecules are degraded due to exonuclease activity in RecBCD E. coli strains. Thus for BAC manipulation, RecA-mediated homologous recombination has to occur in a temporally-controlled manner. This can be achieved by either transferring the BAC construct to a recombination positive bacterial strain for manipulation, or by transiently inducing recombination functions that are encoded by either the host chromosome or on a helper plasmid. Homologous recombination techniques allow any type of mutation, point, insertion, deletion or gene fusion, to be efficiently introduced. The probability of a recombination event occurring is dependent on the length of the homology arms, and is increased by the presence of Chi sequences within the homology arms [52]. These Chi sequences signal RecBCD exonuclease to begin the process of DNA repair that facilitates recombination.

Site-specific recombinases (SSRs) have been used in conjunction with homologous recombination techniques in bacteria to further manipulate and produce recombinant BACs. SSRs have been used to delete sequences such as prokaryotic regulatory sequences, selection markers and the BAC vector backbone. This is discussed in further detail in Section 7Section 7. SSRs have also been used to insert sequences into BACs (see Figure 4) [101]. Deletions occur at higher efficiencies than insertions, as insertions are kinetically unfavourable, however SSRs are still very useful tools and large insertions have been achieved [102]. The most common SSRs used are Cre recombinase and Flp/Flpe recombinase. These recombinases target specific loxP or FRT sites respectively, and efficiencies are extremely high [67,76,103]. By flanking sequences to be removed, such as BAC vector sequences, with recognition sites in the same orientation, upon expression of recombinase functions, intervening sequences are looped out and excised [9,26,78,104,105].

Insertions can be created by co-integrating a shuttle plasmid such as pRETRObac into the BAC clone via a single loxP site, however this can only occur at one location on the BAC vector and thus is not useful for specific gene modifications [101]. The ‘flip-flop’ and HSVQuik systems for herpes simplex virus have also used site-specific recombination for the creation of BAC vectors and insertion mutagenesis [106,107]. The flip-flop system developed by Kuroda et al. utilized both Cre and Flpe recombinase to first integrate the BAC vector and mutation of interest into HSV and then to remove the BAC vector backbone [106]. It was specifically designed so that non-recombinants exceeded the maximum packaging capacity of the virus and were therefore unable to grow, simplifying the screening process [106]. The HSVQuik system from Terada et al. [107] similarly used sequential site-specific recombination to first insert the transgene into the BAC in bacteria and then to remove the BAC backbone in eukaryotic cells [107]. In this system, typically 90% of resultant virions correctly had the BAC backbone removed [107].

A major potential concern of BAC engineering, particularly for in vivo transgenic or vaccine applications, is the presence of residual BAC and marker sequences in the final recombinant. Various methods have been developed for markerless BAC mutagenesis, including the use of site-specific recombinases as detailed above [4,9,15,17,19,20,22,26,32,66,83,104,108,109,110]. One of the disadvantages of using SSRs, particularly in transgenic studies, is the persistence of a single 34 bp restriction site or ‘scar’ that remains after recombination has occurred [108]. However the presence of this small scar in viral vectors or engineered vaccines is generally not a concern. Wagner et al. [111] and Tischer and coworkers [112] utilized sequence duplications on the insertion DNA to delete BAC sequences by stimulating recombination once reconstituted in eukaryotic cells. Markerless constructs have also been created through co-targeting of a selectable marker to the E. coli chromosome [113]. This was achieved by using an excess of the BAC targeting cassette, which resulted in a high probability that those colonies containing the selectable marker in their chromosome would also contain a mutation in the BAC they harbor. Pofsai et al. [114] co-integrated the mutation of interest into the host chromosome and subsequently utilized the homing endonuclease I-SceI to create a double-strand break at a pre-engineered restriction site. This stimulated intramolecular recombination, and recombinants were screened for by allele-specific PCR, eliminating the need for selection markers. Recombination efficiencies varied, but were low for large deletions, and high efficiencies were only achieved with repetitive colony selection [114]. Herring and coworkers [115] used a similar technique they termed “gene gorging”. This required a donor plasmid carrying the mutation of interest to be linearised with I-SceI in vivo to stimulate Red recombination functions. These were provided on separate mutagenesis plasmids. Again no selection was required and screening for recombinants was by PCR [115]. Tischer et al. [116] refined this method, later termed “en passant” recombineering, by using I-SceI cleavage to provide the substrate for additional recombination steps. Subsequent intramolecular recombination of sequence duplications resulted in removal of the selectable marker. Assisted large fragment insertion by Red/ET-recombination (ALFIRE) also utilized I-SceI [117]. Other homing endonucleases such as I-CeuI have been used for BAC mutagenesis, for example in the Homingbac baculovirus cloning system [118]. Parent Homingbac baculoviruses were pre-engineered to contain a eukaryotic GFP fluorescent marker gene. Digestion with I-CeuI resulted in excision of this GFP marker and transgene DNA with compatible end sequences could then be ligated into the parent baculovirus [118]. Although unlikely to be 100% efficient, background parental virus could not be detected in this study.

While site-specific mutagenesis techniques are necessary to target specific genetic loci, these techniques are not suited for global genome analysis. In contrast, random mutagenesis is particularly useful for the rapid generation of libraries of gene disruptions. These libraries can then be screened to identify mutations in specific genes and to elucidate the effect of the mutation on virus function. The most widely used random mutagenesis methods rely on transposon technologies to achieve gene disruption. Transposons are mobile genetic elements originally discovered in maize by McClintock [119] and multiple transposon families have since been characterized . These elements can be activated by a transposase to induce movement and can be engineered to contain SSR recognition sites, foreign DNA, primer binding sites, regulatory elements and antibiotic selection markers between their end sequences [120]. Although many transposon families exist, the most useful and efficient for BAC modification are those that preferentially insert into negatively supercoiled plasmid DNA [121,122]. The major advantages of transposon mutagenesis are that the target sequence does not have to be known, only very small amounts of DNA are required and a large number of mutants can be rapidly generated, which can then be screened to identify the insertion site of the transposon [123]. A disadvantage of this method is that multiple insertions can occur leading to deletions and rearrangements of the BAC construct, however this can be controlled by varying the insert to vector ratio [123]. Because of the advantages of this technology, transposable elements occupy a unique niche in BAC mutagenesis.

Although transposons were used extensively preceding the development of BACs, including for mutagenesis of herpes simplex virus 1 [124], the technology was first applied to BAC constructs in 1999. Brune and coworkers [125] utilized Tn1721, a member of the Tn3 transposon family, in a mini-transposon system called TnMax, originally developed in 1993 [121], to rapidly identify essential and non-essential genes of murine cytomegalovirus. The transposon construct was delivered into E. coli harbouring the BAC construct on a temperature-sensitive plasmid, and at permissive temperatures transposition occurred [125]. Simultaneously, Smith et al. [9] used a mini-Tn5 transposon on a delivery plasmid for mutagenesis of a pseudorabies virus BAC clone and this technology was later also applied to a human cytomegalovirus BAC clone [126]. These systems relied on transposition occurring within bacteria, and required further characterization to ensure the transposon had inserted into the BAC and not into the bacterial chromosome. In 2004, an in vitro Tn5 transposition system was used to create a gene disruption library of a bovine herpesvirus-1 BAC clone [12]. This utilized a hyperactive Tn5 system developed for mutagenesis of plasmids by Goryshin in 1998 [127]. Because the reaction is performed in vitro, transposition only occurs into target BAC DNA and mutants can then be easily isolated after transformation into bacteria. Other transposition systems used for random mutagenesis of herpesvirus BACs have included a Tn3-based system for mutagenesis of a murine cytomegalovirus BAC and the use of MuA transposition for a bovine herpesvirus-4 BAC [128,129]. Transposition methods have been used to produce nested deletions and small insertions, however the major use of these transposon systems is the rapid creation of gene disruption libraries [130].

Transposon technology can also be applied to targeted BAC mutagenesis. The Tn7 transposon is site-specific, targeting attTn7 sites when transposition is activated [131]. In initial studies when applied to cytomegalovirus BACs, partial and complete deletions were reported to occur frequently [132]. However, others have used Tn7 transposition systems with great success [3,133,134]. Recently, Tn7 has again been used for site-specific transposition by introducing a Tn7 transposon target site into a varicella vaccine virus BAC by RecA-mediated recombination and subsequently introducing foreign sequences by site-directed transposition [135]. Currently, transposition-based technologies are the system of choice for global mutagenesis studies of herpesviral genes due to the rapid speed in which a large number of mutants can be generated.

The development of BACs has revolutionized molecular cloning of large DNA molecules and they are now widely used in many applications, including investigations into herpesvirus biology. The stability, large insert size and ease of manipulation of the BAC constructs within the E. coli host has rapidly led to the preference for BACs over YACs and cosmids. Protocols for cloning viral genomes as BACs are now widely published. The use of homologous recombination, using both λ Red recombination and ET cloning, allows precise site-specific mutagenesis for characterization of viral genes and genetic engineering of recombinant virus strains. This has widespread implications in delivery of therapeutics, particularly in the area of DNA vaccine development. Alternatively, BACs can support rapid characterization of viral genes through random transposon mutagenesis. This is particularly useful in initial studies into newly isolated, unstudied viruses where genomic sequence data is still unknown. BACs are currently widely used in transgenic and knock out studies and are being used to monitor gene expression and target specific tissues in vivo. Due to the diverse applications for BACs, they will continue to be a cornerstone of virological research.