Identification of mutation sites of a temperature-sensitive mutant of HCMV DNA polymerase activity

The human cytomegalovirus (strain Towne) temperature-sensitive mutant ts 256 exhibits a virus specific DNA polymerase-negative phenotype. The position of the mutation of ts 256 was determined by three step marker-rescue assays to be within a 5.1 kb XbaI-BamHI fragment between map unit 0.33 and 0.35 in a HindIII-D fragment located in a long unique region. Nucleotide sequencing showed that the 5.1 kb fragment contained three open reading frames corresponding to those of the genes for UL52, UL53 and UL54 (DNA polymerase gene), respectively, of strain AD169. The functions of UL52 and UL53 are unknown. Comparison of the DNA sequences of the 5.1 kb fragments of the wild-type and ts 256 mutant revealed two base changes within UL53 and UL54, respectively, which result in amino acid substitutions. The mutation in the UL54 gene was located within a distinct conserved region VI common to alpha-like DNA polymerases, suggesting that this base change would be responsible for DNA negative phenotype.