Abstract
Herpesviruses are large DNA viruses and include important human and veterinary pathogens. Their genomes can be cloned as bacterial artificial chromosomes (BACs) and genetically engineered in Escherichia coli using BAC recombineering methods. While the recombineering methods are efficient, the initial BAC-cloning step remains laborious. To overcome this limitation, we have developed a simple, rapid, and efficient BAC-cloning method based on single-step transformation-associated recombination (STAR) in Saccharomyces cerevisiae. The linear viral genome is directly integrated into a vector comprising a yeast centromeric plasmid and a BAC replicon. Following transfer into E. coli, the viral genome can be modified using standard BAC recombineering techniques. We demonstrate the speed, fidelity, and broad applicability of STAR by cloning two strains of both rat cytomegalovirus (a betaherpesvirus) and Kaposi's sarcoma-associated herpesvirus (a gammaherpesvirus). STAR cloning facilitates the functional genetic analysis of herpesviruses and other large DNA viruses and their use as vaccines and therapeutic vectors.