IN-VITRO EXPRESSION OF THE HUMAN CYTOMEGALOVIRUS DNA-POLYMERASE GENE - EFFECTS OF SEQUENCE ALTERATIONS ON ENZYME-ACTIVITY

Genomic DNA of the Towne strain human cytomegalovirus polymerase (pol) gene (4.4-kb RsrII-NcoI segment of the EcoRI J fragment) was cloned i nto plasmids containing either the T3 or the T7 promoter for in vitro transcription-translation studies. The translation efficiency of unmod ified pol cRNA was poor in this system and could not be improved by ca pping. However, the efficiency could be enhanced by replacing the lead er sequence with a 40-bp AT-rich sequence derived from an alfalfa mosa ic virus, R4. pol cRNA directed the synthesis of a 140-kDa polypeptide in a rabbit reticulocyte translation system. The in vitro-translated wild-type enzyme possessed significant polymerization activity which c ould be stimulated by salt as could that of the authentic enzyme purif ied from virus-infected cells. To study the critical domains of this e nzyme, nine mutations were introduced into the pol gene around the con served domains of eukaryotic polymerase by oligonucleotide-directed mu tagenesis. Two constructs with mutations at amino acid residues 323 to 325 (M32QS) and 725 to 726 (M72II) remained active, with partial loss of enzyme activity, while the enzyme activities of other mutants with alterations at four domains located around amino acid residues 729 to 730 (M73HN), 804 to 807 (M80 and DE80), 910 to 913 (M91 and DE91), an d 962 to 964 (M96 and DE96) were abolished. DNA template and triphosph ate binding assays indicated that the mutation at 804 to 807 (conserve d region III) lost the ability to bind DNA template, and four mutants, M73HN (within conserved region II), M80 (in region III), M91 (in regi on I), and M96 (around region V [962 to 964; amino acid sequence KKR]) , failed to bind deoxyribonucleoside triphosphate. These data suggest that conserved region III is essential for DNA template binding, while residues between conserved region II and V (725 to 964) are involved in triphosphate binding.