Cloning of β-Herpesvirus Genomes as Bacterial Artificial Chromosomes

Herpesviruses form a family of DNA viruses that are of considerable medical importance (1 ). Although the genomes of many members of the herpesviruses have been completely sequenced, our knowledge on the function of the majority of herpesvirus genes is still quite insufficient. This is especially true for the members of the β-herpesviruses, i.e., the cytomegaloviruses (CMVs), because their slow replication kinetics, cell association, and large genome size (230 kb) made the construction of viral mutants a difficult and tedious procedure (2 ). Conventional mutagenesis protocols for herpesviruses are based on the insertion of marker genes into the viral genome, which allows to disrupt or delete viral genes (3 –5 ). Unfortunately, the method has certain limitations. The protocols rely on recombination events in eukaryotic cells that are relatively rare and difficult to control. Accordingly, adventitious deletions, rearrangements, and illegitimate recombination events in the viral genomes have frequently been observed. It is especially cumbersome that the verification of the mutant genome structure can only be done at the very end of the lengthy isolation procedures of the mutant virus. Generation of viral mutants requires an obligatory selection process against the wild-type virus. In the end, the recombinant virus has to be plaque-purified and separated from the wild-type virus. This makes the isolation of viral mutants with growth disadvantages a difficult task.