EXPRESSION OF THE CATALYTIC SUBUNIT (UL54) AND THE ACCESSORY PROTEIN (UL44) OF HUMAN CYTOMEGALOVIRUS DNA-POLYMERASE IN A COUPLED IN-VITRO TRANSCRIPTION TRANSLATION SYSTEM/

The catalytic subunit (UL54) and accessory protein (UL44) of human cyt omegalovirus (HCMV) DNA polymerase have been cloned and expressed in a n in vitro-coupled transcription/translation reticulocyte lysate syste m, The influence of the 5'-untranslated region (5'-UTR) on the efficie ncy of expression from the circular plasmids has been investigated. Fo r expression of both UL54 and UL44, a truncated form of the alfalfa mo saic virus (AMV) RNA4 5'-UTR was found to be superior to the full-leng th AMV 5'-UTR or the original HCMV 5'-UTRs of different lengths. Prote in products with M-r approximate to 140 and 55 kDa were detected by so dium dodecyl sulfate-polyacrylamide gel electrophoresis analysis in th e UL54 and UL44 in vitro expression reactions, respectively. The prope rties of the expressed enzyme were compared with those of native HCMV DNA polymerase purified from HCMV-infected cells. DNA polymerase and 3 '-5' exonuclease activities of the expressed UL54/UL44 complex were fo und to be dependent on salt concentration in the same manner as the ac tivities of the native enzyme. The in vitro-expressed enzyme resembles the purified HCMV DNA polymerase in its affinity for deoxynucleoside triphosphates as well as in its sensitivity to known inhibitors (cidof ovir diphosphate, ganciclovir triphosphate, and foscarnet). This strai ghtforward method for protein expression may also be applicable to oth er enzymes where reproducible generation of fully functional products is desirable. (C) 1997 Academic Press.