IDENTIFICATION AND CHARACTERIZATION OF PSEUDORABIES VIRUS DUTPASE

Sequence analysis within the long segment of the pseudorabies virus (P rV) genome identified an open reading frame of 804 bp whose deduced pr otein product of 268 amino acids exhibited homology to dUTPases of oth er herpesviruses. The gene was designated UL50 because of its colinear ity with the homologous gene of herpes simplex virus type 1. An antise rum raised against a bacterially expressed fragment of PrV UL50 specif ically detected a 33-kDa protein in lysates of infected cells, which i s in agreement with the predicted molecular mass of the PrV UL50 prote in. A UL50 negative PrV mutant (PrV UL50(-)) was constructed by the in sertion of a beta-galactosidase expression cassette into the UL50 codi ng sequence, A corresponding rescuant (PrV UL50resc) was also isolated . The interruption of the UL50 gene led to the disappearance of the 33 -kDa protein, whereas restoration of UL50 gene expression restored det ection of the 33-kDa protein. Enzyme activity assays confirmed that UL 50 of PrV codes for a dUTPase which copurifies with nuclei of infected cells, PrV UL50(-) replicated with an only slightly reduced efficienc y in epithelial cells in culture compared with that of its parental wi ld-type virus strain, Our results thus demonstrate that UL50 of PrV en codes a protein of 33 kDa with dUTPase activity which copurifies with nuclei of infected cells and is dispensable for replication in culture d epithelial cells.