Epstein-Barr virus genetics: talking about the BAC generation

Genetics became an integral part of the Epstein-Barr virus (EBV) research field at an early stage. Identification of viral strains with unusual properties, e.g. incapable of initiating lytic replication, such as Raji, or of transforming B cells, such as P3HR1, later coupled to sequencing allowed the identification of genes or of a group of genes likely to be involved in these functions [1-3]. Although these early EBV mutants appeared spontaneously, they provided an important tool for EBV research. More recently, strategies have been developed to allow researchers to direct mutagenesis of the EBV genome in order to design specific mutants of interest.The ability to associate specific genes with unique mutant phenotypes was an important step, however, definitive evidence that such phenotypes are associated with specific genes required the construction of revertants. For example, proof that the P3HR1 phenotype was caused by the loss of EBNA2 required the reintroduction of this gene back into the mutant genome through transfection of an EBV DNA fragment that spans the EBNA2 region and the observation that a successfully recombined virus had regained its transforming ability [4,5]. Not only did this observation define EBNA2 as a key transforming gene, it also provided an elegant method to select for recombinants from the background of defective P3HR1 viruses. Indeed, lymphoblastoid cell lines (LCL) generated with supernatants from EBNA-2 transfected P3HR1 cells contained predominantly, if not exclusively, recombinant viruses [4,5]. Therefore, the introduction of EBNA2 provided a potent selection method that could be used to construct mutant viruses. Recombination with a combination of cosmid that contained EBNA2 and of overlapping cosmids that carried a mutated version of another EBV gene, e.g. EBNA3, allowed the generation of EBV mutants that had both re-acquired EBNA2 and incorporated the mutated gene [6]. This technology, based on homologous recombination in eukaryoti