A recombinant human cytomegalovirus with a large deletion in UL97 has a severe replication deficiency

Human cytomegalovirus encodes a protein kinase (UL97) that confers sensitiv ity to ganciclovir by phosphorylating it to the monophosphate. The function of this unusual kinase in viral replication is unknown. We constructed two independent isolates of a recombinant virus, RC Delta 97, that contain lar ge deletions in this gene and carry a 4.8-kb insertion containing a selecta ble genetic marker. These mutant viruses were isolated by using a populatio n of primary cells (HEL97) that express this gene from integrated copies of a defective retroviral vector. The recombinant viruses were severely impai red in their ability to replicate in primary fibroblasts, attaining virus t iters that were 2 to 3 orders of magnitude lower than those produced by the parent virus. Despite the severe replication deficit, both of these viruse s retained the ability to form small, slowly growing plaques in primary fib roblasts, demonstrating that UL97 is not absolutely essential for replicati on in cell culture. The replication deficit was relieved when UL97 was prov ided in trans in the complementing cell line, showing that the phenotype wa s due to a deficiency in UL97. Thus, the UL97 gene product plays a very imp ortant role in viral replication in tissue culture and may be a good target for antiviral chemotherapy.