SELECTABLE INSERTION AND DELETION MUTAGENESIS OF THE HUMAN CYTOMEGALOVIRUS GENOME USING THE ESCHERICHIA-COLI GUANOSINE PHOSPHORIBOSYL TRANSFERASE (GPT) GENE

We describe the mutagenesis of the IRS1-US5 region of the human cytome galovirus genome, demonstrating the potential of the E. coli guanosine phosphoribosyl transferase (gpt) gene as a selectable marker for inse rtion and deletion mutagenesis of high passage (AD169, Towne) as well as low passage (Toledo) strains of virus. Despite evidence suggesting that the US3 gene product may play a regulatory role, disruption of th is gene with a gpt insert had no effect on growth of any of these stra ins of virus in resting or dividing human fibroblasts, or in human thy mus plus liver implants in SCID-hu mice. Transcripts of the gpt gene, under control of the herpes simplex virus thymidine kinase promoter ad jacent to the US3 enhancer in the viral genome, accumulated with delay ed early (beta) kinetics. Mutants with deletions in the IRS1 and US3-U S5 regions were isolated by back-selection against gpt with the drug 6 -thioguanine by growing virus in human Lesch-Nyhan (hypoxanthineguanin e phosphoribosyl transferase deficient) skin fibroblasts immortalized with human papillomavirus oncogenes. Thus, we demonstrate a dependable method for insertion and deletion mutagenesis that can be applied to any region of the viral genome.