Construction of HSV-1 BACs and Their Use for Packaging of HSV-1-Based Amplicon Vectors

Viruses are parasites that are strictly dependent on the host cell for replication. In general, the more complex a virus, the more complicated the network of interactions between the virus and the host. Many of these interactions are still unrevealed or not understood in detail. The generation of mutant viruses is an important strategy to obtain insights into the functional role of individual virus genes. A classical genetic approach used chemical mutagenesis to produce mutant viruses, however, identifying the mutation responsible for an observed phenotype and excluding second-site mutations is difficult. Development of site-directed mutagenesis of the herpes simplex virus type 1 (HSV-1) genome by homologous recombination allowed targeted mutation of individual HSV-1 genes (1 ,2 ). This technique remains laborious and, in some cases, impossible, because null mutants of genes that are essential for HSV-1 replication can only be produced on complementing cell lines (3 ). A more-efficient strategy for mutating HSV-1 was achieved by cloning the entire virus genome as a set of overlapping clones in cosmids. This allows the manipulation of specific virus genes on individual cosmid clones in Escherichia coli (E. coli) and their subsequent characterization. Cotransfection of this modified cosmid set into permissive cells leads to the reconstitution of recombinant HSV-1 genomes by homologous recombination (4 ). However, one problem of the cosmids is their instability, in particular those clones that contain repeated sequences. Therefore, alternative cloning vectors, which permit the amplification, manipulation, and characterization of large DNA fragments are used (5 ).