Epstein-Barr virus genetics: talking about the BAC generation

Abstract

Genetic mutant organisms pervade all areas of Biology. Early on, herpesviruses (HV) were found to be amenable to genetic analysis using homologous recombination techniques in eukaryotic cells. More recently, HV genomes cloned onto a bacterial artificial chromosome (BAC) have become available. HV BACs can be easily modified in E.coli and reintroduced in eukaryotic cells to produce infectious viruses. Mutants derived from HV BACs have been used both to understand the functions of all types of genetic elements present on the virus genome, but also to generate mutants with potentially medically relevant properties such as preventative vaccines. Here we retrace the development of the BAC technology applied to the Epstein-Barr virus (EBV) and review the strategies available for the construction of mutants. We expand on the appropriate controls required for proper use of the EBV BACs, and on the technical hurdles researchers face in working with these recombinants. We then discuss how further technological developments might successfully overcome these difficulties. Finally, we catalog the EBV BAC mutants that are currently available and illustrate their contributions to the field using a few representative examples.

Figure 1

The EBV BAC system: an overview. The cloned EBV-BAC can be manipulated in E.coli cells using multiple techniques that rely on homologous recombination. The mutated EBV BAC is then introduced into 293 cells and selected with an antibiotic to create a producer cell line from which infectious particles that contain the mutant EBV BAC can be produced. The episomes present in the producer cell line can be extracted and reintroduced in E.coli where multiple controls such as restriction analyses, Southern blotting and sequencing, can be readily performed.

Figure 2

EBV BAC mutagenesis in E. coli. Recombination with linearized targeting vectors. This method allows deletions or exchanges of genetic material from the EBV DNA against foreign sequences. The latter can be mutated versions of an EBV gene, or DNA fragments of cellular or bacterial origin. Selection of successfully recombined BACs requires the introduction of an antibiotic resistance cassette flanked by Flp-recombinase target (FRT) sites. Transient introduction of the FLP recombinase allows excision of the antibiotic resistance cassette.

Figure 3

Chromosomal building in E. coli. Recombination with circular targeting vectors. Chromosomal building is one of several techniques that allow seamless mutagenesis. It is based on a targeting vector that carries: i) an antibiotic resistance cassette e.g. ampicillin (amp); ii) the sequence to be introduced into the EBV-BAC (represented by the grey shading and star) flanked by EBV-specific sequences (designated as A and B on the EBV BAC and A' and B' on the targeting vector) that will determine its site of insertion; iii) the gene that encodes the lacZ enzyme; and iv) a temperature-sensitive origin of replication that is operative only at 30°C. The targeting vector is introduced into E.coli cells that carry the EBV BAC. Recombination between both prokaryotic episomes is performed by a recA recombinase present on the targeting vector, whose expression is driven by an arabinose-inducible promoter. Homologous recombination can be initiated anywhere within the regions of homology (indicated by an arrow). The antibiotic resistance cassettes present on the targeting vector (amp) and on the EBV BAC (cam) allow the selection of co-integrates, which are a fusion vector comprising the targeting vector and the EBV BAC. Propagation at 42°C (non-permissive temperature) forces the loss of free targeting vectors. A second round of recombination resolves the co-integrates and reconstitutes both the EBV BAC and the targeting vector. Depending on which flanking region initiates resolution of the co-integrate, a recombinant EBV BAC containing either the foreign sequence (a) or the wild type (b) will be generated. Reconstituted targeting vectors are eliminated by culture at non-permissive temperature. Candidate clones are assessed for their sensitivity to ampicillin and expression of the lacZ gene. LacZ-negative and ampicillin-sensitive clones, indicative of reconstituted EBV BACs, are then submitted to restriction enzyme analysis, colony PCR or any other appropriate technique. goi: gene of interest.

Figure 4

Assessing the EBV BAC structure by restriction enzyme analysis. (A) Schematic overview of the EBV genome indicating the position of the various DNA repeats. IR: internal repeats, TR: terminal repeats, FR: family of repeats. (B) Restriction analysis of EBV BACs stably transfected into 293 cells. The left panel shows the predicted position of viral fragments after BamHI restriction. The right panel shows actual examples of abnormal EBV BACs rescued from producer cell lines. One EBV BAC carried fewer NotI repeats than the control (lane 1, purple arrow), another EBV BAC contained fewer TRs but more FRs (lane 2 red and green arrows, respectively), and a third recombinant carried fewer BamHI-W repeats (lane 5, blue arrow). Examples of large deletions (lane 3 and 4) are also shown. All of these abnormal clones were discarded. wt: wild type.