Recent advances in herpesvirus genetics using bacterial artificial chromosomes.
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abstract
Although the members of the Herpesvirus family are responsible for a wide variety of human diseases, advances in the understanding of viral molecular mechanisms of pathogenesis have been hampered by the large size of herpesvirus genomes, rendering the viruses difficult to experimentally manipulate. Better techniques have been needed to facilitate mutagenesis of herpesvirus genomes, allowing for the assessment of the role of specific viral gene products in replication, immunity, and pathogenesis. Homologous recombination with plasmids containing genes of interest flanked by selectable markers has been a successful method for generating viral mutants, as has the generation of recombinant virus from transfection of cosmid clones. Although these efforts to generate recombinant viruses have met with modest success, the protocols have been cumbersome. More recently, a novel technique for the manipulation of herpesvirus genomes has been developed. This technology utilizes bacterial F plasmids, and allows for the stable cloning of herpesvirus genomes as bacterial artificial chromosomes (BACs) in Escherichia coli. Once cloned, such BACs are stable, and DNA purified from E. coli is infectious, fully capable of reproducing replication-competent virus. Manipulation of herpesvirus genomes is now feasible using the powerful techniques of bacterial genetics, and should facilitate a better understanding of the molecular pathogenesis of herpesvirus infections.
