Mutagenesis of the Repeat Regions of Herpesviruses Cloned as Bacterial Artificial Chromosomes

Abstract

Cloning of infectious and pathogenic herpesvirus genomes in a bacterial artificial chromosome (BAC) vector greatly facilitates genetic manipulation of their genomes. BAC-based mutagenesis strategies of viruses can advance our understanding of the viral gene functions and determinants of pathogenicity, and can ultimately help to develop molecularly defined improved vaccines against virus diseases. Unlike the virus stocks, where continuous passage in tissue culture can lead to phenotypic alterations such as loss of virulence or immunogenicity, viral genomes can be stably maintained with high fidelity as BAC clones in bacteria. Thanks to the “RecA” or the inducible phage “λ Red” homologous recombination systems and a variety of positive and negative selection strategies, viral genomes cloned as BAC can be efficiently manipulated in E. coli. All the manipulations, including DNA fragment deletion or insertion, point mutations, or even multiple modifications in repeat regions can be carried out accurately in E. coli, and the mutated DNA can be used directly to reconstitute mutant viruses in transfected host cells. Furthermore, using self-excision strategies, the non-viral bacterial replicon sequence can be excised automatically during virus reconstitution, thus generating recombinant viruses virtually identical to the wild-type parent viruses. Here, we describe the various technologies of manipulating the infectious BAC clones of a group E herpesvirus as an example through a combination of different approaches.