Epstein-Barr Virus-Associated Thymidine Kinase

Transcription

1 JOURNAL OF VIROLOGY, Apr. 1978, p Vol. 26, No X/78/ $02.00/0 Copyright 1978 American Society for Microbiology Printed in U.S.A. Epstein-Barr Virus-Associated Thymidine Kinase SHEN-TU CHEN, JOHN E. ESTES, ENG-SHANG HUANG, AND JOSEPH S. PAGANO* Cancer Research Center and the Department ofmedicine, School ofmedicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina Received for publication 8 December 1977 Superinfection of Raji cells with Epstein-Barr virus induced a new thymidine kinase that was distinguishable from both adult and fetal kinases of the host cell by discontinuous electrophoresis on polyacrylamide gels and glycerol gradients. At least two distinct human thymidine kinase same pattern of TK activities as other cell cul- (TK) activities have been demonstrated clearly tures, 50 ml of blood was drawn from a healthy by discontinuous polyacrylamide gel electropho- adult donor. After separation with LSM solution resis (disc-page) (2, 5, 10, 14, 23) and DEAE- (Litton Bionetics), 8 x 107 lymphocytes were cellulose chromatography (20). The expression obtained; one part was frozen immediately, and of different forms of TK is related to the repli- the other two parts were kept growing separately cative state of normal or transformed cultured at 37 C in RPMI 1640 medium with 10% fetal cells rather than the developmental state of the bovine serum, one with 2.5,ug of phytohemagtissue of origin (2). The "fetal" kinase migrates glutinin (PHA) per ml and one without PHA. as a slowly moving species with a relative frac- After 72 h, TK enzymes were extracted from all tion of migration (Rf) value of It is the three groups of cells, according to the method of dominant species in actively growing cells, Munyon et al. (16). Approximately 4 x 107 cells whereas the "adult" kinase, which migrates as a were extracted in a final volume of 1.2 ml. Simfast-moving species with an Rf = 0.50, predomi- ilar enzymes were prepared from P3HR-1 cell nates in nongrowing cells (2). culture, an actively growing EBV-producing Some viruses are capable of inducing virus- lymphoblastoid cell line derived from a Burkitt's specific TK in transformed cell cultures or in lymphoma (8). TK enzymes were extracted from virus-infected cultures, and some others can en- P3HR-1 cells at days 1, 4, 8, and 14 after subculhance cellular TK activities (3-5, 9-11). Within ture in RPMI 1640 with 10% fetal bovine serum the herpesvirus group, there appears to be a without changing the medium during the 14-day wide spectrum. Herpes simplex and pseudora- period. A 150-,ig sample of the protein extracts bies viruses are able to induce virus-specified was electrophoresed on each 100-mm polyacryl- TK; cytomegalovirus stimulates host cell TK, amide gel prepared according to Kit et al. (10). and equine abortion virus (type 1) induces nei- After electrophoresis, each gel was cut into 2.5- ther (5, 9, 14). These differences may be due to mm slices and assayed for TK activities accordthe different modes of natural infection and rep- ing to Estes and Huang (5). lication of these viruses (9, 14). Figure la, b, and c show the electrophoretic Epstein-Barr virus (EBV), a member of the pattern of the TK activities of the three different herpesvirus group that replicates in epithelial treatment schedules of the lymphocytes obcells in vivo (13) but is cultivated in vitro in tained from the same donor. Figure la and c lymphocytes, is quite distinct from the others. represent the nongrowing lymphocytes, which Both Hampar et al. (7) and Glaser et al. (6) have expressed only the adult TK activity with an Rf shown that TK activity increased in Burkitt = 0.6. Figure lb represents the growing lympholymphoblastoid cells and in Burkitt lymphoblas- cytes stimulated by PHA, which expressed toid-somatic cell hybrids, respectively, after in- mainly the fetal TK activity with an Rf = 0.2. duction of EBV and suggested that an EBV gene Figure ld and e depict the TK activity of growmay participate in the increased enzyme activ- ing P3HR-1 cells, in which only the fetal TK ity. Infection of the non-virus-producing EBV activity was detectable. Figure lf and g represent genome-bearing lymphoblastoid cell line called the TK activities of P3HR-1 in the resting state, Raji causes DNA replication and formation of with some adult TK activity detectable; the noninfectious virus particles (19, 22, 24). We majority of the activity migrates as fetal TK. selected this system to determine whether EBV These results are comparable to other findings is able to induce an EBV-specific TK. in tissues, monolayer cultures, and virus-trans- To show that lymphocytes also express the formed cell cultures (2, 5, 10, 20). 203

2 204 NOTES J. VIROL. 0.2 fo Vo x e o 0 L 0.4 IL.4 U U V 8 ~~~~~~~~~~~~0.2 x ~~~C TK aciiyatrds-aeofxrcsfo FIG.~ 3 a)authmnimhcts b dlua 8 ~~~~~~~ x Rtf ~~~d Rf FIG. 1. TK activity after disc-page of extracts from (a) adult human lymphocytes, (b) adult human lymphocytes grown in tissue culture medium and stimulated by PHA for 3 days, (c) adult lymphocytes grown in tissue culture medium without PHA, (d) P3HR-1 cells 1 day after subculture, (e) P3HR-1 cells 4 days after subculture, (f) P-HR-1 cells 8 days after subculture, (g) PaIR-1 cells 14 days after subculture. The Rf value is defined as fraction of migration on 5% acrylamide gel as compared with bromophenol blue marker. Infectious EBV was obtained by polyethylene 1640 medium with 10% fetal bovine serum, 50 glycol concentration of the P3HR-1 cell super-,ug of gentamicin, and 60,ug of Tylocine (Grand natant fluid that had been kept at 32 C for 14 Island Biological Co.) per ml. The concentrated days in RPMI 1640 medium with 2% fetal bovine virus contained about 8 x 107 infectious virus serum (1). Five liters of the supernatant fluid particles per ml, as titrated by detection of earlywas concentrated 500-fold in 10 ml of RPMI antigen (EA) induction in superinfected Raji

3 VOL. 26, 1978 NOTES 205 cells (1, 19). Each milliliter of the concentrated cells infected with EBV at various intervals after virus was used to infect 4 x 107 actively growing infection. The 698 control and 698 infected with Raji cells to assure that the multiplicity of infec- EBV after 48 h expressed the same pattern of tion was at least one infectious virus particle per fetal TK activity with an Rf = 0.2 (Fig. 2a and cell. For infection, Raji cells were concentrated b). The Raji control also expressed the fetal TK by centrifugation at 125 x g to 4 x 107 cells per activity only with an Rf = 0.15 (Fig. 2c), whereas ml and inoculated with 1 ml of the polyethylene a distinct form of TK activity was observed in glycol-concentrated EBV at 37 C for 2 h. The the superinfected Raji cells at various times after infected Raji cells were then resuspended at a infection (Fig. 2d through g). This distinct TK concentration of 1 x 106 cells per ml and incu- migrated between the fetal TK and adult TK bated at 37 C for 72 h. At various times after with an Rf= The fetal TK activity of the infection (12, 24, 48, and 72 h), 4 x 107 cells were infected Raji remained unchanged, whereas the removed for TK extraction. Mock infection of newly induced TK activity increased as the in- Raji control cells followed the same procedure. fection proceeded. This unique TK present in The 698 cell line, a B-lymphocyte line derived superinfected Raji appears to be EBV induced. from a lymphosarcoma that contains no detect- To rule out the possibility that this new TK able EBV genome and lacks EBV receptors, was was induced by mycoplasma contamination of inoculated with EBV as above (12, 17, 18). De- the P3HR-1 cells from which we concentrated tection of EA-positive cells in infected and non- EBV, we offer the following results: (i) none of infected Raji and 698 cell cultures was carried the cell cultures used showed any mycoplasma out by fluorescent-antibody technique with contamination as measured by the classic isolahigh-titer, EA-positive, pooled human sera. tion techniques in Hayflick's media; (ii) the 698 Table 1 shows the percentage of EA-positive cells exposed to the same virus preparation did cells in Raji and 698 cells at various times post- not express any new TK activity; (iii) neither infection. Neither the Raji control, the 698 con- the EBV-exposed 698 nor EBV-infected Raji trol, nor 698 cells infected with EBV showed any cells expressed any enhancement of cellular TK EA-positive cells. The infected Raji cells showed activity as usually shown in mycoplasma-in- 30% EA-positive cells as early as 12 h after fected cell cultures (21); and (iv) virus was pelinfection, and at 24 h almost every cell showed leted from the concentrated P3HR-1 inoculum EA. The brightness of the fluorescence of the and tested for TK activity on disc-page; al- EA-positive cells increased as time went on. though 300,ug of protein (double the amount of Most of the infected Raji cells remained viable cellular samples) was electrophoresed, there was (as tested by trypan-blue-dye exclusion) during no TK activity greater than background counts the infection period, that is, 95, 85, and 75% across the gel. survival at 12, 24, and 72 h after infection, re- The EBV-associated TK was separable from spectively. This high percentage of superinfected cellular fetal TK by discontinuous glycerol gracells may be due to the fact that the Raji cells dient electrophoresis (disc-gep [5]). Figure 3a were infected at a growing stage and maintained and b show the TK activities of Raji control cells in RPMI 1640 medium with 10% fetal bovine and Raji cells infected with EBV after 72 h as serum at an appropriate concentration (1 x 106 separated by disc-gep. The EBV-induced TK cells per ml). migrated faster than the cellular fetal TK in Figure 2 shows the electrophoretic pattern of both disc-gep and disc-page, which indicates the TK activities of 698 control cells, 698 cells that this protein has a smaller size than the infected with EBV, Raji control cells, and Raji cellular fetal TK and is also more negatively charged. Further purification and characteriza- TABLE 1. EBV EA in Raji and 698 cells tion of this new TK is in process. Raji cells contain endogenous EBV genomes, Percentage of EA-positive cells and 7 to 15% of P3HR-1 cells contain replicating Time Rji in- 698 in- EBV. We did occasionally find a small peak of no. infec- Raji fected 698 fected TK activity in both lines (25 to 30% above backtion (h) control with t ; with ground) that corresponded in position to that EBV EBV found during superinfection (Fig. le). The total I activity of this peak was only 0.5 to 2% of the 24 0 > cellular fetal TK activity and was not always 48 0 > seen > Attempts to induce both Raji and P3HR-1 cells with bromodeoxyuridine have shown poor II 24 0 > EA induction accompanied by small increases in 48 0 > the EBV-associated TK. Induction with iodo-

4 206 NOTES J. VIROL. 0 K 0.2 3E ~~a e X u.is _8_ K 8 8 0Vo b U~~ 0.15 C I Rf 0 I 0.15d 0.37 Rf FIG. 2. TK activity after disc-page of extracts from (a) 698 control, (b) 698 infected with EBVfor 48 h, (c) Raji control, (d) Raji infected with EBV for 12 h, (e) Raji infected with EBV for 24 h, (/) Raji infected with EBV for 48 h, (g) Raji infected with EBV for 72 h.

5 VOL. 26, 1978 NOTES Davis, D. B., W. Munyon, R. Buchsbaum, and R. Chawda Virus type-specific thymidine kinase in cells biochemically transformed by herpes simplex virus O types 1 and 2. J. Virol. 13: OA8 5. Estes, J. E., and E.-S. Huang Stimulation of X cellular thymidine kinases by human cytomegalovirus. X ll aj. Virol. 24: Glaser, R., T. Ogino, J. Zimmerman, Jr., and F. Rapp. u s \ Thymidine kinase activity in Burkitt lymphoblastoid somatic cell hybrids after induction of EB virus. Proc. Soc. Exp. Biol. Med. 142: Hampar, B., J. G. Derge, L M. Martos, and J. L Walker Synthesis of Epstein-Barr virus after activation of the viral genome in a "virus negative" human lymphoblastoid cell (Raji) made resistant to 5- bromo-deoxyuridine. Proc. Natl. Acad. Sci. U.S.A. 69: Hinuma, Y., and J. J. Grace Cloning of immu- 8 noglobulin-producing human leukemic and lymphoma cells in long-term cultures. Proc. Soc. Exp. Biol. Med. 124: O 9. Jamieson, A. T., G. A. Gentry, and J. H. Subak- Sharpe Induction of both thymidine and deoxx ycytidine kinase activity by herpesviruses. J. Gen. Virol. XE b 24: Kit, S., W. C. Leung, and D. Trkula Properties of u 04A8 mitochondrial thymidine kinase in parental and enzyme-deficient HeLa cells. Arch. Biochem. Biophys : Kit, S., W. C. Leung, D. Trkula, and G. Jorgensen Gel electrophoresis and isoelectric focusing of ^_ mitochondrial and viral-induced thymidine kinases. Int. J. Cancer 13: Klein, G., T. Lindahl, M. Jondal, W. Leibold, J. Me- Rnezes, K. Nilsson, and C. Sundstrom Contin- Rf~uous lymphoid cell lines with characteristics of B cells FIG. 3. TK activity after disc-gep (10 to 50%) of (bone-marrow-derived) lacking the Epstein-Barr-virus extracts from (a) Raji control and (b) Raji infected genome and derived from three human lymphomas. with EBVfor 72 h. Proc. Natl. Acad. Sci. U.S.A. 71: Lemon, S. M., L. M. Hutt, J. E. Shaw, Jui-Lien H. Li, and J. S. Pagano Replication of EBV in epithedeoxyuridine is in progress and will be reported lial cells during infectious mononucleosis. Nature (Lonlater, together with P3HR-1 EBV superinfection don) 268: of BJA-B (human B cell, EBV negative and 14. Leung, W.-C., D. R. Dubbs, D. Trkula, and S. Kit. infectable infectab*.ler with EBV[15]) v cord1975. Mitochondria and 15]) and human cord- herpesvirus-specific deoxypyr- idine kinases. J. Virol. 16: blood cells (not infectable by P3HR-1 EBV). 15. Menezes, J., W. Leibold, G. Klein, and G. Clements. These results have been reproduced several Establishment and characterization of an Epsteintimes over a 1-year period and indicate that Barr virus (EBV)-negative lymphoblastoid B cell line EBV induces a virus-specific TK (BJA-B) from an during super- exceptional EBV-genome negative African Burkitt's lymphoma. Biomedicine 22: infection. 16. Munyon, W., R. Buchsbaum, E. Paoletti, J. Mann, E. Kraiselburd, and D. Davis This work was supported by Public Health Service grant thymidine Electrophoresis of kinase activity synthesized by cells trans- AI12717 from the National Institute of Allergy and Infectious formed by herpes simplex virus. Virology 49: Diseases, CA P30-CA16086 from the National Cancer 1 on, K. and C. vinds stblshen Institute, and by training grant T32 CA09156 and fellowship 17. N dlsson, K., and C. Sundstrim Establfshment 7 F22 CA and characteristics of two unique cel lines from patients with lymphosarcoma. Int. J. Cancer 13: LITERATURE CITED 18. Nonoyama, M., and J. S. Pagano Complementary RNA specific to the DNA of the Epstein-Barr 1. Adams, A Preparation of Epstein-Barr virus from virus: detection of EB viral genome in nonproductive P3HR-I cells and isolation of the virus DNA, p cells. Nature (London) New Biol. 233: In D. V. Ablashi, H. G. Haslestad, and G. de-the' (ed.), 19. Nonoyama, M., and J. S. Pagano Replication of Epstein-Barr virus, production, concentration, and pu- viral deoxyribonucleic acid in Epstein-Barr virus infecrification. Internal technical report International tion. J. Virol. 9: Agency for Research on Cancer, Lyons, France. 20. Okuda, H., T. Arima, T. Hashimato, and S. Fujui. 2. Alder, R., and B. R. McAuslan Expression of Multiple forms of deoxythymidine kinase in varthymidine kinase variants is a function of the replicative ious tissues. Cancer Res. 32: state of cells. Cell 2: Russell, W. C Alterations in the nucleic acid me- 3. Carp, R. L Thymidine kinase from normal, simian tabolism of tissue culture cells infected by mycoplasma. virus 40-transformed and simian virus 40 lytically in- Nature (London) 212: fected cells. J. Virol. 1: Shaw, J. E., T. Seebeck, Jui-Lien H. Li, and J. S.

6 208 NOTES J. VIROL. Pagano Epstein-Barr virus DNA synthesized in superinfected Raji 247: cells. Virology 77: Taylor, 24. A. T., M. Yajima, A. Y., and M. Stafford, and 0. W. Nonoyama. Jones Mechanisms of Properties of infection with thymidine kinase Esptein-Barr partially virus. I. purified Viral from DNA replihuman fetal cation and and adult formation of tissue. J. Biol. non-infectious Chem. virus particles in super-infected Raji cells. J. Virol. 19: