Bernhard Neuhierl and Henri-Jacques Delecluse
Recombinant viral genomes cloned onto BAC vectors can be subjected to extensive molecular genetic analysis in the context of E. coli. Thus, the recombinant virus technology exploits the power of prokaryotic genetics to introduce all kinds of mutations into the recombinant genome. All available techniques are based on homologous recombination between a targeting vector carrying the mutated version of the gene of interest and the recombinant virus. After modification, the mutant viral genome is stably introduced into eukaryotic cells permissive for viral lytic replication. In these cells, mutant viral genomes can be packaged into infectious particles to evaluate the effect of these mutations in the context of the complete genome.
Key Words: Herpesviruses; Epstein-Barr virus; viral recombinants; genetic analysis; viral mutants.
1.1. Construction of Mutants by Recombination Using Linearized Targeting Vectors
Molecular genetic analysis of viruses offers a direct approach for the analysis of viral gene function and is becoming a standard tool in many laboratories. The scope of introduced modifications ranges from knockout mutations that render viral genes or groups of viral genes nonfunctional to more subtle changes such as point mutations. Insertion of conditional expression systems within the complete virus genome even allows sequential activation and repression of viral gene expression, allowing a more thorough understanding of how viruses interact with their host cells. Small virus genomes (<20 kb) can be modified by standard recombinant DNA technology, i.e., cloning using restriction enzyme cleavage
From: Methods in Molecular Biology, vol. 292: DNA Viruses: Methods and Protocols Edited by: P. M. Lieberman © Humana Press Inc., Totowa, NJ
and ligation (1). With increasing size, virus genomes become more difficult to manipulate and propagate. Herpesviruses, for example, possess large genomes (up to 200 kb) that are not amenable to standard cloning techniques, and modification of these genomes relies on homologous recombination.
Classically, homologous recombination was performed in eukaryotic cells carrying the virus to be modified (2-4). A major difficulty of this kind of approach is that the mutant virus coexists with the wild-type virus in the infected cell and therefore must first be purified. More recently, single-copy F-plasmid replicons (also known as bacterial artificial chromosomes [BACs]) were used to clone up to 300 kb of genomic DNA (5). Introduction of these replicons into large viral DNAs has allowed cloning and manipulation of these genomes in the context of a prokaryotic host (6-10). Genetic analysis using viruses cloned onto BACs is less time-consuming than the classical methods. Sequential introduction of multiple mutations into viral genomes cloned onto BACs is possible within a reasonable amount of time. In this review, we focus on a few techniques commonly performed in our laboratories to construct and propagate viral mutants. Our own experience is strongly focused on y-herpesviruses, in particular the Epstein-Barr virus (EBV), but all large DNA viruses cloned onto BACs should be amenable to this technology. In fact, many large DNA viruses, including most herpesviruses, have now been cloned onto BACs and are already available for further manipulations (6-10). This review makes no pretence of being exhaustive, and many alternative protocols can be found in the literature available on BACs.
In all methods presented, viral mutants are constructed by homologous recombination between a targeting vector containing the mutated version of the gene of interest and the cloned viral genome. The type of recombinase (recA or X-phage recombinase) and the structure of the targeting vector (linear or circular) vary in the different methods used. Some characteristics of these techniques, all of which exclusively make use of viral recombinant genomes cloned onto BACs, are given in Table 1. The first type of method reported here makes use of linearized vectors and a selectable marker gene, which is either left on the virus genome or only partially removed in a further step. This method will therefore not be applicable when it is imperative to avoid the presence of foreign sequences on the viral genome. The targeting vector includes the gene of interest in its mutated version as well as its left and right viral flanking sequences that will provide targets for homologous recombination (Fig. 1). The viral sequences cloned onto the targeting vector are therefore in the same configuration as in the wild-type viral genome with the exception of the gene of interest that carries mutations (or deletions). The minimal size of the flanking regions varies with the recombination system used. If an E. coli recA-depend-ent system is used, recombination frequencies are usually good when targeting vector and viral genomes share more than 1 kb of homologous sequences on
Methods for Homologous Recombination on Large Plasmids
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