Cloning and Mutagenesis of a Herpesvirus Genome as an Infectious Bacterial Artificial Chromosome

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 94; no. 26; pp. 14759 - 14763
• Main Authors: Messerle, M, Crnkovic, I, Hammerschmidt, W, Ziegler, H, Koszinowski, U H
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences of the United States of America 23.12.1997; National Acad Sciences; National Academy of Sciences; The National Academy of Sciences of the USA
• Subjects: Animals; Biological Sciences; Chromosomes, Bacterial; Cloning; Cloning, Molecular; Cytomegalovirus; Deoxyribonucleic acid; DNA; Escherichia coli - genetics; Eukaryotic cells; Genetic vectors; Genetics; Genome, Viral; Genomes; Herpesviridae - genetics; Mice; Microbiology; Mutagenesis; Mutation; Plasmids; Proteins; Transfection; Viruses
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.94.26.14759

A strategy for cloning and mutagenesis of an infectious herpesvirus genome is described. The mouse cytomegalovirus genome was cloned and maintained as a 230 kb bacterial artificial chromosome (BAC) in E. coli. Transfection of the BAC plasmid into eukaryotic cells led to a productive virus infection. The feasibility to introduce targeted mutations into the BAC cloned virus genome was shown by mutation of the immediate-early 1 gene and generation of a mutant virus. Thus, the complete construction of a mutant herpesvirus genome can now be carried out in a controlled manner prior to the reconstitution of infectious progeny. The described approach should be generally applicable to the mutagenesis of genomes of other large DNA viruses.