MUTATIONS IN ACTIVE-SITE RESIDUES OF THE URACIL-DNA GLYCOSYLASE ENCODED BY VACCINIA VIRUS ARE INCOMPATIBLE WITH VIRUS VIABILITY

The D4R gene of vaccinia virus encodes a functional uracil-DNA glycosy lase that is essential for viral viability (D. T. Stuart, C. Upton, M. A. Eligman, E. G. Niles, and G. McFadden, J. Virol. 67:2503-2513, 199 3), and a D4R mutant, ts4149, confers a conditional lethal defect in v iral DNA replication (A. K. Millns, M. S. Carpenter, and A. M. DeLange , Virology 198:504-513, 1994). The mutant ts4149 protein was expressed in vitro and assayed for uracil-DNA glycosylase activity. Less than 6 % of wild-type activity was observed at permissive temperatures, but t he ts4149 protein was completely inactive at the nonpermissive tempera ture. Mutagenesis of the ts4149 gene back to wild type (Arg-179-->Gly) restored full activity. The ts4149 protein was considerably reduced i n lysates of cells infected at the permissive temperature, and its act ivity was undetectable, even in the presence of the uracil glycosylase inhibitor protein, which inhibits the host uracil-DNA glycosylases bu t not that of vaccinia virus. Thus the ts4149 protein is thermolabile, correlating uracil removal with vaccinia virus DNA replication. Three active-site amino acids of the vaccinia virus uracil-DNA glycosylase were mutated (Asp-68-->Asn, Asn-120-->Val, and His-181-->Leu), produci ng proteins that were completely defective in uracil excision but stil l retained the ability to bind DNA. Each mutated D4R gene was transfec ted into vaccinia virus ts4149-infected cells in order to assess the r ecombination events that allowed virus survival at 40 degrees C. Genet ic analysis and sequencing studies revealed that the only viruses to s urvive were those in which recombination eliminated the mutant locus. We conclude that the uracil cleavage activity of the D4R protein is es sential for its function in vaccinia virus DNA replication, suggesting that the removal of uracil residues plays an obligatory role.