HUMAN CYTOMEGALOVIRUS URACIL DNA GLYCOSYLASE IS REQUIRED FOR THE NORMAL TEMPORAL REGULATION OF BOTH DNA-SYNTHESIS AND VIRAL REPLICATION

Human cytomegalovirus (CMV) encodes a gene, UL114, whose product is ho mologous to the uraciI DNA glycosylase and is highly conserved in all herpesviruses. This DNA repair enzyme excises uracil residues in DNA t hat result from the misincorporation of dUTP or spontaneous deaminatio n of cytosine, We constructed a recombinant virus, RC2620, that contai ns a large deletion in the UL114 open reading frame and carries a 1,2- kb insert containing the Escherichia coli gpt gene, RC2620 retains the capacity to replicate in primary human fibroblasts and reaches titers that are similar to those produced by the parent virus but exhibits a significantly longer replication cycle. Although the rate of expressi on of alpha and beta gene products appears to be unaffected by the mut ation, DNA synthesis fails to proceed normally, Once initiated, DNA sy nthesis in mutant virus-infected cells proceeds at the same rate as wi th wild-type virus, but initiation is delayed by 48 h, The mutant viru s also exhibits two predicted phenotypes: (i) hypersensitivity to the nucleoside analog 5-bromode-oxyuridine and (ii) retention of more urac il residues in genomic DNA than the parental virus, Together, these da ta suggest UL114 is required for the proper excision of uracil residue s from viral DNA but in addition plays some role in establishing the c orrect temporal progression of DNA synthesis and viral replication, Al though such involvement has not been previously observed in herpesviru ses, a requirement for uracil DNA glycosylase in DNA replication has b een observed in poxviruses.