Functional characterization of partially purified Epstein-Barr virus DNA polymerase expressed in the baculovirus system.
The DNA polymerase gene of Epstein-Barr virus (EBV) was cloned into baculovirus transfer vector (pBlueBac). The recombinant baculovirus (AcEBP-15) was obtained by cotransfection of Spodoptera frugiperda (Sf9) cells 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 substantial quantities of the EBV DNA polymerase protein of the expected size (110 kD). The identity of the EBV polymerase 110-kD polypeptide was 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 acid. Our results indicate that the baculovirus expression system provides large quantities of functional polymerase suitable for biochemical and structural analyses, thereby furthering our understanding of the mechanism of viral DNA replication and its inhibition by antiviral drugs.
Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333.
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D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D015153:Blotting, Western; D002478:Cells, Cultured; D002848:Chromatography, DEAE-Cellulose; D003001:Cloning, Molecular / Q000379:methods; D004268:DNA-Binding Proteins; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis* / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D015967:Gene Expression Regulation, Viral; D015751:Genes, pol / Q000235:genetics*; D004854:Herpesvirus 4, Human / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012995:Solubility; D018411:Spodoptera / Q000821:virology; D014162:Transfection; D014764:Viral Proteins; D015678:Viral Structural Proteins / Q000096:biosynthesis* / Q000235:genetics; D014779:Virus Replication / Q000235:genetics*
D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C083202:BALF5 protein, Epstein-barr virus; D004259:DNA-Directed DNA Polymerase