FUNCTIONAL EXPRESSION AND CHARACTERIZATION OF THE EPSTEIN-BARR-VIRUS DNA-POLYMERASE CATALYTIC SUBUNIT

A recombinant baculovirus containing the complete sequence for the Eps tein-Barr virus (EBV) DNA polymerase catalytic subunit, BALF5 gene pro duct, under the control of the baculovirus polyhedrin promoter was con structed. Insect cells infected with the recombinant virus produced a protein of 110 kDa, recognized by anti-BALF5 protein-specific polyclon al antibody. The expressed EBV DNA polymerase catalytic polypeptide wa s purified from the cytosolic fraction of the recombinant virus-infect ed insect cells. The purified protein exhibited both DNA polymerase an d 3'-to-5' exonuclease activities, which were neutralized by the anti- BALFS protein-specific antibody. These results indicate that the 3'-to -5' exonuclease activity associated with the EBV DNA polymerase (T. Ts urumi, Virology 182:376-381, 1991) is an inherent feature of the polym erase catalytic polypeptide. The DNA polymerase and the exonuclease ac tivities of the EBV DNA polymerase catalytic subunit were sensitive to ammonium sulfate in contrast to those of the polymerase complex purif ied from EBV-producing lymphoblastoid cells, which were stimulated by salt. Furthermore, the template-primer preference for the polymerase c atalytic subunit was different from that for the polymerase complex. T hese observations strongly suggest that the presence of EBV DNA polyme rase accessory protein, BMRF1 gene product, does influence the enzymat ic properties of EBV DNA polymerase catalytic subunit.