Jc. Lin et al., FUNCTIONAL-CHARACTERIZATION OF PARTIALLY PURIFIED EPSTEIN-BARR-VIRUS DNA-POLYMERASE EXPRESSED IN THE BACULOVIRUS SYSTEM, Virus genes, 8(3), 1994, pp. 231-241
The DNA polymerase gene of Epstein-Barr virus (EBV) was cloned into ba
culovirus transfer vector (pBlueBac). The recombinant baculovirus (AcE
BP-15) was obtained by cotransfection of Spodoptera frugiperda (Sf9) c
ells with infectious DNA from Autographa californica multiple nuclear
polyhedrin virus (AcMNPV) and pBlueBac plasmid carrying EBV polymerase
gene. Infection of Sf9 cells with the recombinant virus produced subs
tantial quantities of the EBV DNA polymerase protein of the expected s
ize (110 kD). The identity of the EBV polymerase 110-kD polypeptide wa
s determined by (a) immunoprecipitation and Western blot analyses with
rabbit polyclonal antiserum specific for a synthetic peptide derived
from the coding sequence of the polymerase gene; (b) identification of
a polypeptide of identical size (110 kD) from EBV-infected cells; (c)
measurement of DNA polymerase activity similar to that of the enzyme
induced in EBV-infected cells; and (d) neutralization of the enzymatic
activity by the rabbit antiserum and inhibition by phosphonoacetic ac
id. Our results indicate that the baculovirus expression system provid
es large quantities of functional polymerase suitable for biochemical
and structural analyses, thereby furthering our understanding of the m
echanism of viral DNA replication and its inhibition by antiviral drug
s.