DNA polymerase in phorbol ester-treated Epstein-Barr virus-carrying cells

A heat-labile Epstein-Barr virus-specific DNA polymerase stimulatory protein having a molecular mass of 45 kDa was purified from phorbol 12-myristate 13-acetatetreated P3HR-1 cells by column chromatography. The virus DNA polymerase stimulatory protein was precipitated by sera from patients with nasopharyngeal carcinoma but not by sera from healthy donors. The interaction of the stimulatory protein with DNA polymerase was stoichiometric. Furthermore, this protein stimulated Epstein-Barr virus DNA polymerase but not herpes simplex virus type 1 or type 2 or human DNA polymerase a. The stimulatory protein did not alter the Km value of dTTP or DNA but did increase the Vn. of DNA polymerase. Salt concentrations between 100mM and 150mM KCI were optimal for this protein-induced stimulation of Epstein-Barr virus DNA polymerase activity. The presence of the stimulatory protein in the reaction mixture enhanced the sensitivity of virus DNA polymerase to phosphonoformate. There are five types of herpesviruses that induce human disease. Among these, Epstein-Barr virus (EBV) is known to have close association with Burkitt lymphoma and nasopharyngeal carcinoma (NPC) (1). The EBV genomes present in both virus-infected and virus-carrying cells (2) are able to induce several virus-specified proteins. It has been shown that EBV induces a virus-specified DNA polymerase and DNase (3-6). The activities of both enzymes increase when EBV-carrying cells, P3HR-1 cells, are treated with the tumor-promoting ester phorbol 12-myristate 13-acetate (PMA) (3, 7). Procedures to highly purify EBV DNA polymerase by various column chromatographic techniques have been described (3, 7-10). Based on our experience, recovery ofthe enzyme activity is poor and quite inconsistent throughout the purification. This raises the question of whether the loss of enzyme activity during purification is the result of the separation of a stimulatory protein from the enzyme or is due to instability of the enzyme. This communication describes the purification and some properties ofan EBV-specific DNA polymerase stimulatory protein. MATERIALS AND METHODS Chemicals. All chemicals were of reagent grade or better. Unlabeled dNTPs, calf thymus DNA, dithiothreitol, bovine serum albumin, phenylmethylsulfonyl fluoride, and PMA were obtained from Sigma. ICN was the source of [3H]dTTP. Phosphonoformate was a gift from Astra A.B. (Sodertalje, Sweden). NPC patient sera used for immunostaining were provided by Tseng-Chou Lynn from the National Taiwan University Hospital. Treatment of P3HR-1 Cells with PMA. The EBV-producer cell line P3HR-1 was maintained at 370C in humidified 5% C02/95% air. The P3HR-1 cells were grown as a stationaryphase suspension culture in RPMI 1640 medium containing 10% fetal calf serum. For the induction of virus-specified proteins, cells seeded at a density of 5 x 105/ml were grown as suspension cultures for 6 days at 34TC. The cells were then pelleted and suspended in fresh medium containing PMA at 40 ng/ml. After a 4-day incubation, the cells were collected by centrifugation and stored at -70'C until use. Preparation of Viral and Human DNA Polymerases. EBVspecified DNA polymerase from PMA-treated P3HR-1 cells was purified as described (3). This involved DEAE-cellulose, phosphocellulose, and DNA-cellulose column chromatography. The final specific activity ofEBV DNA polymerase was >4.0 x 105 units (defined below) per mg of protein. Herpes simplex virus (HSV) type 1 and type 2 DNA polymerases were purified from HSV-1 (KOS strain)and HSV-2 (333 strain)-infected HeLa cells, respectively, as described (11, 12). Human DNA polymerase a was purified from HeLa cells by a published procedure (12). DNA Polymerase Assay. The reaction mixture for the assay of different types of DNA polymerase was the same except for the concentration of KCl. The mixture (100 ILI) was composed of 50 mM Tris-HCl, pH 8.0/4 mM MgCl2/0.5 mM dithiothreitol/0. 1 mM dATP/0. 1 mM dGTP/0.1 mM dCTP/5 ,uM [3H]dTTP (40 mCi/,tmol; 1 Ci = 37 GBq) containing bovine serum albumin at 0.2 mg/ml, 15 ,ug of activated calf thymus DNA, and different amounts of KCl. For EBV, HSV-1, HSV-2, and human DNA polymerase a, the concentrations of KCl were 150, 200, 200, and 0 mM, respectively. One unit of enzyme activity is defined as the amount of enzyme that catalyzes the incorporation of 1 pmol of dTMP into activated DNA per min at 370C as described (12). Electrophoresis, Blotting, and Immunostaining. NaDodS04/polyacrylamide gel electrophoresis was carried out according to Laemmli (13) using 10% gels. After electrophoresis, slab gels were silver stained following the method of Merril et al. (14). Proteins were transferred to nitrocellulose paper according to the method of Bowen et al. (15). Immunostaining of proteins on nitrocellulose paper was carried out according to Legochi and Verma (16). Serological Test. Immunoprecipitation of EBV DNA polymerase stimulatory protein was accomplished by adding diluted (1:5) NPC patient serum or control serum from healthy individuals. After incubation at room temperature for 20 min, 10 ,lI of previously swollen protein A-conjugated Sepharose (Pharmacia) was added. After further incubation for 1 hr at 4°C, the immunocomplexes were pelleted by Abbreviations: EBV, Epstein-Barr virus; PMA, phorbol 12myristate 13-acetate; HSV, herpes simplex virus. *To whom reprint requests should be addressed. 5728 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Proc. Natl. Acad. Sci. USA 82 (1985) 5729 centrifugation and the supernatant was examined for DNA polymerase stimulatory protein activity using purified EBV DNA polymerase.