Saturating Mutagenesis and Characterization of a Herpesvirus Genome Using In Vivo Reconstitution of Virus from Cloned Subgenomic Regions

The study of genome structure and gene function is pivotal in understanding the mechanisms of replication, pathogenesis, and virulence of herpesviruses. In this respect, mutagenesis and sequence analysis of genes encoded by the virus are of great importance. However, the herpesvirus genomes are large, with sizes ranging between 120 and over 200 kbp and encoding between 70 and 200 genes (see ref. 1 for a review). This large size hampers handling and systematic mutagenesis of the virus genome using standard modern molecular biology techniques. Most current methods of mutagenesis therefore do not rely on direct modification of the viral genome in vitro but depend on exchange in vivo, by homologous recombination, of a viral gene by a copy of the latter gene that is truncated in vitro by insertion of a marker gene. Mutant virus progeny can be screened or selected for, depending on the marker gene that is used. Commonly used marker genes are thymidine kinase and lacZ . This procedure is generally used, reliable, and has yielded a wealth of information on the function of herpers simplex virus type 1 (HSV-1) encoded genes. However, it requires prior mapping and cloning of every gene to be mutagenized and is therefore less feasible if the virus is a novel or less-well-known herpesvirus.