Phosphorylation of Acyclovir in Vitro in Activated Burkitt Somatic Cell Hybrids ALOK K
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 1983, p. 10-14 Vol. 24, No. 1 0064804/83/070010-05$02.00/0 Copyright 0 1983, American Society for Microbiology Phosphorylation of Acyclovir In Vitro in Activated Burkitt Somatic Cell Hybrids ALOK K. DATTAl 2* AND JOSEPH S. PAGANO3'4 Cancer Research Centerl* and Departments ofBiochemistry and Nutrition,2 Medicine,3 and Bacteriology and Immunology,' School ofMedicine, University ofNorth Carolina, Chapel Hill, North Carolina 27514 Received 10 December 1982/Accepted 14 April 1983 Acyclovir [9-(2-hydroxyethoxymethyl)guanineI (ACV), a potent antiviral com- pound, was phosphorylated to the same extent by extracts from untreated and iododeoxyuridine-treated Epstein-Barr virus-containing latent D98/HR-1 somatic hybrid cells. ATP was the preferred phosphate donor over other nucleoside triphosphates. The cytosol extract from D98/HR-1 cells effected optimum phos- phorylation of thymidine at pH 8.0, whereas ACV was phosphorylated equally well over a wide pH range. Electrophoretic analysis of thymidine kinase-, deoxycytidine kinase-, and ACV-phosphorylating activities from both untreated and iododeQxyuridine-treated cell extracts displayed identical properties. A small part (5 to 10%o) of the loaded ACV-phosphorylating activity seemed to migrate with the deoxycytidine kinase activity from cytosol. dTTP and dCTP, at relatively high concentrations, partially inhibited ACV-phosphorylating activity. The results suggest that Epstein-Barr virus does not code for its own thymidine kinase and that phosphorylation of ACV in Epstein-Barr virus-producing cells is carried out by miultiple or as yet unidentified ATP-dependent nonspecific cellular phospho- transferases. The acyclic nucleoside analog acyclovir [9-(2- Treatment of D98/HR-1 somatic cell hybrids hydroxyethoxymethyl)guanine] (ACV) is cur- with iododeoxyuridine (IUdR) gives rise to the rently considered as one of the drugs for the induction of EBV-associated DNA polymerase topical treatment of certain herpes simplex virus with a subsequent induction of-viral DNA syn- (HSV) infections, such as primary genital herpes thesis and the production of EBV particles (16). and generalized cutaneous herpes in immuno- The induction of viral DNA synthesis in IUdR- compromised patients (10). Biochemical and ge- treated cells can be efficiently inhibited by ACV netic studies of its mechanism of action in HSV (15). This system was ideally suited for our systems have shown that the compound is con- purpose inasmuch as we could follow the induc- verted to its triphosphate form; the first step of tion of EBV-associated DNA polymerase activi- phosphorylation is carried out by virus-specified ty as an internal marker while analyzing the thymidine (TdR) kinase (4, 10). The monophos- extent of ACV phosphorylation and TdR kinase phate thus formed is subsequently converted to activity. This exercise was necessary in view of triphosphate by host enzymes (17). The triphos- the conflicting reports in favor of both existence phate form specifically inhibits the DNA poly- and nonexistence of EBV-associatod TdR ki- merase of HSV by competing with dGTP (10). nase (3, 19, 20). Moreover, in vaccinia virus and Cells infected with TdR kihase-negative virus herpesvirus saimiri systems, ACV is not at all were unable to phosphorylate ACV to a great effective despite the presence of virus-specified xtent, and hence the drug was ineffective. TdR kinase (10, 11). These findings raised the Reports from this laboratory (6, 7) show that question of whether the presence of virus-speci- ACV is effective in inhibiting Epstein-Barr virus fied TdR kinase is essential for ACV to be (EBV) replication. However, in contrast to effective. The studies reported in this paper are HSV, the amount of phosphorylated drug directed at answering this question. formed in vivo in EBV-infected cells is minimal and identical in both infected and noninfected MATERILUS AND METHODS cells (5). These observations led us to analyze Cels. Burkitt hybrid cells, D98/HR-1, clone 1, se- the process of ACV phosphorylation in vitro in a lected in hypoxanthine-aminopterin-ihymidine-con- latent EBV DNA-containing cell line (D98IHR- taining medium were a gift of Ronald Glaser, Ohio 1, lOa). State University, Columbus. 10 VOL. 24, 1983 ACYCLOVIR PHOSPHORYLATION IN CELL HYBRIDS 11 Chemicals. [14C]ACV (54 mCi/,Lmol) and unlabeled riboflavin for gel polymerization. Samples (50 ,ul each) ACV were gifts of G. Elion, Burroughs Wellcome Co., in 20%o glycerol-0.001% bromophenol blue were ap- Research Triangle Park, N.C. Nucleosides and their plied to the gel. After PAGE, the gels were sliced and triphosphates were obtained from P-L Biochemicals, assayed by dipping each slide in 100 ,ul of reaction Milwaukee, Wis. ATP was purchased from Sigma mixture. The incubations were for 3 at 37°C for ACV Chemical Co., St. Louis, Mo. Tetrahydrouridine was phosphorylation and 1 for TdR and CdR phosphoryla- provided by Y.-C. Cheng, Cancer Research Center, tion. University of North Carolina, Chapel Hill. Treatment of cells with IUdR. Cells were seeded in RESULTS minimal essential medium and treated with IUdR for 3 ACV- and TdR-phosphorylating activities in days as described elsewhere (15). Preparation of phosphorylating activity. Mock-treat- IUdR-treated D981H1-1 cytosol extracts. ACV ed and IUdR-treated cells were processed as follows. was phosphorylated Equally by both untreated Cells were suspended in a buffer containing 10 mM and lUdR-treated cytosol extracts (Fig. 1). The KCI, 0.0015 M MgCl2, 10 mM Tris (pH 7.4), and 0.25 reaction was linear over a period of 120 min. M sucrose and kept suspended for 20 min at 0°C. However, the rate of ACV phosphorylation was Phenylmethylsulfonyl fluoride was then added to a 100 to 150 times lower than the rate of TdR final concentration of 1 mM, and the cells were phosphorylation. The phosphorylation was disrupted with a Dounce homogenizer. The suspen- mostly ATP dependent (Table 1) in both mock- sion was centrifuged at 800 x g for 5 min. The pellet formed was used as the source of nuclei. KCI was treated and IUdR-treated cytosol extkacts. added to the supernatant fluid to a final concentration To ascertain whether TdR kinase present in of 0.15 M, and ATP and P-mercaptoethanol were the cytosol extract plays any role in the phos- added to final concentrations of 10 and 5 mM, respec- phorylation, ACV and TdR kinase assays were tively. The supernatant fluid was centrifuged at carried out at different pHs. TdR was phosphor- 105,000 x g for 2 h. The clear supernatant fluid formed ylated maximally at pH 8.0 and had a: sharp pH was dialyzed for 8 to 12 h against a dialysis buffer (10 optimum (Fig. 2). In contrast, phosphorylation mM Tris [pH 7.4], 0.15 M KCI, 10 mM P-mercaptoeth- of ACV had very broad pH optitna in both anol, 10 mM ATP, 10 mM MgCl2, 20% glycerol, 1 mM phenylmethylsulfonyl fluoride). The dialyzed fraction IUdR-treated and untreated extracts. was clarified by centrifugation and was used as the PAGE analysis of TdR kinase-, CdR kinase-, cytosol extract. Total cell extract was prepared by and ACV-phosphorylating activity. For further suspending the cells in the same dialyzing buffer and insight into the different deoxynucleoside kinase then sonicating them. The rest of the procedure was activities in IUdR-treated D98/HR-1 cells, th- the same as for the cytosol prejparation. The washed whole-cell extracts from IUdR-treated and un- nuclear pellet used for DNA polymerase assays was processed as described before (8). Enzyme assays. Assays for TdR kinase were carried 12 T out as follows. The reaction volume of 100 ,u con- 6O tained 100 mM Tris (pH 7.5), 10 mM MgCl2, 10 mM C4cq 2 mM 100 IC ATP, 3 mM 3-mercaptoethanol, NaF, FM 10 10 [3H]TdR (10 cpm/pmol), and the required amount of 0 0- enzyme. The reactions were carried out at 37'C for 30 a. min. Samples (50 Fl each) of the reaction mixture were C removed and applied to DE-81 paper disks. The papers 8 were washed three times with 0.01 M ammonium acetate, followed by two rinses with water and with E' 'z 95% alcohol. Filters were dried, and radioactivity was a 6 '5,0 determined in toluene-based solvent. Q 0. For deoxycytidine (CdR) kinase assay the methods en 0 0 were the same except that [3H]CdR was used as the n- 4 substrate, and the reaction mixture contained 0.2 mM 0. tetrahydrouridine. 0 75 The reaction mixture for assay of ACV phosphor- 2 ylation was exactly the same as for the TdR kinase *0Z!In assay except that 500 ,uM [14C]ACV (54 cpm/pmol) was used as the acceptor nucleoside. Assays were E E carried out for 120 min unless otherwise stated. For 0. Q. determination of radioactivity, the DE-81 papers were rinsed three times with 0.001 M ammonium acetate, Minutes three times with 70%o alcohol containing 1 mM guano- FIG. 1. Kinetics of phosphorylation of ACV and sine and 0.001 M ammonium acetate, and three times TdR by the cytosol extracts from IUdR-treated and each by washing under pressure filtration with water untreated D98/HR-1 cells. ACV phosphorylation with and with 70%o alcohol. untreated (A) and IUdR-treated (A) cellular cytosol PAGE. Polyacrylamide gel electrophoresis (PAGE) extracts and TdR phosphorylation with untreated (0) was essentially the same as described elsewhere (13) and IUdR-treated (0) cellular cytosol extracts were except that ammonium persulfate was used instead of carried out as described in the text. 12 DATTA AND PAGANO ANTIMICROB. AGENTS CHEMOTHER. TABLE 1. Phosphate donor specificity for ACV 4 phosphorylationa pmol of ACV phosphorylated per mg of protein Phosphate 3 donor Untreated IUdR-treated D98/IIR-1 D98/HR-1 o cytosol cytosol x N,dhe E to AMLP 180 169 0.