Origin of Thymidine Kinase in Adenovirus-Infected Human Cell Lines

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1 JOURNAL OF VIROLOGY, Apr. 1970, p Copyright ( 1970 American Society for Microbiology Vol. 5, No. 4 Printed in U.S.A. Origin of Thymidine Kinase in Adenovirus-Infected Human Cell Lines SAUL KIT, K. NAKAJIMA, AND D. R. DUBBS Division of Bioclhemical Virology, Baylor College of Medicine, Houston, Texas Received for publication 12 January 1970 Human adenovirus type 5 enhances the thymidine kinase activity of KB cells but does not induce the enzyme in kinase-deficient HeLa (BU25) cells. Vaccinia induces thymidine kinase activity in both KB and HeLa (BU25) cells. Human adenovirus types 2, 4, 7, and 12 also fail to induce the enzyme in HeLa (BU25) cells. Vaccinia replicates equally well in the presence or absence of HATG (hypoxanthineaminopterin-thymidine-glycine) in KB and HeLa (BU25) cells. Adenovirus type 5 replicates in KB and in HeLa (BU25) cells in the absence of HATG, and adenovirus type 5 replicates in kinase-positive KB cells in the presence of HATG. However, replication of adenovirus type 5 is grossly inhibited in HeLa (BU25) cells in the presence of HATG. These results suggest that human adenoviruses do not code for a new virus-specific thymidine kinase. Thymidine kinase (dtk) activity is induced in either thymidine kinase-positive (dtk+) or thymidine kinase-deficient (dtk-) cell lines infected with vaccinia or herpes type viruses (3, 5-8, 14, 15). The dtk activity is also enhanced in polyoma virus-infected dtk+ cell lines, but not in dtk- cell lines productively or abortively infected by the virus (1, 22). An enhancement of dtk activity has been observed after productive infection by simian adenoviruses of African green monkey kidney cells (16, 18), and after either productive or abortive infections by human adenoviruses of human, hamster, and monkey cell lines (2, 11, 18, 20, 21, 23-25). All of the cell lines utilized in the adenovirus studies have been dtk+. The present study was undertaken to learn whether dtk activity is induced by human adenovirus(es) during productive infection of dtk- cells. Enzyme activity was therefore measured at various times after infection of kinase-deficient HeLa (BU25) cells (15) by human adenovirus strains. In addition, the replication of adenovirus type 5 in HeLa (BU25) cells grown in media with or without HATG (see below) was measured. Since aminopterin blocks de novo thymidylate synthesis, adenovirus type 5 would be expected to replicate in the presence of HATG only if the virus induces dtk activity in the HeLa (BU25) cells. As controls, enzyme induction by vaccinia virus and replication of vaccinia in the presence and absence of HATG in HeLa (BU25) cells were studied. MATERUILS AND METHODS Cell lines. HeLa (BU25), a subline derived from HeLa S3, is resistant to growth inhibition by bromodeoxyuridine and deficient in dtk activity (15). Cells were grown in Eagles MEM (Auto POW, Flow Laboratories, Rockville, Md.) supplemented with 10% calf serum and subcultured at weekly intervals. KB cells (9) were grown in the same medium but subcultured at 3- to 5-day intervals. Virus strains. Human adenovirus type 5 (strain adenoid 75) was obtained from American Type Culture Collection. Virus stocks were prepared in either primary human embryonic kidney or KB monolayer cultures in MEM with 5% fetal calf serum and 1 mm L-arginine. Cultures were incubated 72 to 96 hr until cell destruction was complete. The infected cell pellets were resuspended in one-tenth the original volume of phosphate-buffered saline (PBS) and sonically treated for 5 min at 10 kc at 4 C in a Raytheon sonic oscillator. Cell debris was removed by low-speed centrifugation. To the supernatant fluid, 0.02% sterile bovine serum albumin was added. Adenovirus type 5 was titrated by plaque assay on 2-day-old KB monolayers. The overlay medium consisted of Eagle Minimal Essential Medium (MEM) with 10% fetal calf serum, 1 mm L-arginine and 1% agar (Difco). A second overlay containing neutral red was added 6 days postinfection (PI), and plaques were counted from 8 to 14 days PI. Vaccinia (IHD) clone 4 (5) was propagated and assayed on CV-1 monolayers. The overlay medium for the plaque assay consisted of MEM containing 2% fetal calf serum and 1% agar. A second overlay containing neutral red was added at 3 days PI, and plaques were counted 4 to 6 days PI. Preparation of enzyme extracts. Confluent monolayer cultures of KB and HeLa (BU25) cells were 446

2 VOL. 5, 1970 ORIGIN OF THYMIDINE KINASE 447 used 3 to 4 days and 6 to 7 days, respectively, after subculture. The cultures were rinsed with isotonic saline solution (GKN) and were infected at multiplicities of 13 to 360 plaque-forming units (PFU) per cell for adenovirus and 8 to 13 PFU per cell for vaccinia. After adsorption for 1 hr at 37 C, unadsorbed virus was removed and MEM containing 10% fetal calf serum and 1 mm L-arginine was added. Cell extracts were prepared 6 to 75 hr after infection with adenovirus and 5 to 8 hr after infection with vaccinia (13). The dtk was assayed as previously described (13) with 3H-deoxyuridine as nucleoside acceptor. Virus growth. Monolayer cultures in 2-oz (60 ml) prescription bottles of 3- to 4-day-old KB or 6- to 7-day-old HeLa (BU25) cells were rinsed with GKN and infected with 0.1 ml of adenovirus or vaccinia at a multiplicity of 0.05 PFU per cell. After 1 hr of incubation at 37 C, the unadsorbed virus was removed and the cultures were rinsed three times with GKN. A 5-ml amount of MEM containing 10% fetal calf serum (1 mm L-arginine was added in adenovirus experiments) was added to half of the cultures. To the rest of the cultures, 5 ml of the same medium was added containing HATG (10-5 M aminopterin, 10- M glycine, 10-4 M hypoxanthine, 4 X 10-5M thymidine; 17, 22). The cultures were incubated at 37 C in a CO2 incubator for the indicated times and then frozen (Fig. 2 and 3). The cells and medium were harvested, and cells were disrupted by sonic treatment at 10 kc at 4 C in a Raytheon sonic oscillator for 3 min for adenovirus type 5 and for 2 min for vaccinia-infected cells. Lysates of adenovirusinfected cells were assayed on KB monolayers, and those from vaccinia-infected cells were assayed on CV-1 monolayers. RESULTS dtk activity of virus-infected KB cells. The effects of vaccinia and adenovirus type 5 infections on the dtk activity of kinase-positive KB cells are shown in Fig. 1. It may be seen that vaccinia enhanced the dtk activity of KB cells two- to threefold at 6 hr after infection. The dtk activity of adenovirus type 5-infected KB cells was enhanced two- to fourfold from 16 to 46 hr after infection. These results confirm previous findings (2, 5, 11, 16, 18-21, 23-25) that vaccinia and adenoviruses enhance the dtk activity of dtk+ cell lines. dtk activity of virus-infected HeLa (BU25) cells. HeLa (BU25) cells are deficient in dtk activity (15). The enzyme activities shown in Table 1 for uninfected cells are minimal, and the variations that occur 7 to 72 hr after mock infection are insignificant. Vaccinia virus infection induces approximately 6.6 units of dtk activity in the kinase-deficient HeLa (BU25) cells (Table 1). This level of induction is about the same as that observed at 6 hr after vaccinia infection of KB cells (11 to 4.5 equals 6.5 units; Fig. 1). In contrast, adenovirus type 5 does not Iz 'o Vaccinia - Infected \NfJ \/ Adenovirus Type 5 - Infected Noninfected KB Cells.1 N\ / o N, No HOURS AFTER ADENOVI RUS TYPE 5 OR VACCINIA INFECTION FIG. 1. Thymidine kinase activity of wuiinfected KB and of adenovirus- and vaccinia-infected KB cells. Confluent monolayer cultures (11.4 X 106 cells per culture) of KB cells were infected with adentovirus type 5 and vaccinia virus at input multiplicities of about 100 and 8 PFU per cell, respectively. Cells were harvested at the times after infection indicated on tlle figure and assayed for thymidine kinase activity. induce a significant increase in dtk activity in HeLa (BU25) cells at any time from 7 to 72 hr after infection (Table 1). In similar experiments, human adenovirus types 2, 4, 7, and 12 failed to induce dtk activity in HeLa (BU25) cells from 1 to 50 hr after infection. Vaccinia virus replication in the presence or absence of HATG. One-step growth curves depicting the replication of vaccinia virus in kinasepositive KB and in kinase-deficient HeLa (BU25) cells in the presence or absence of HATG medium are shown in Fig. 2. Aminopterin-containing HATG medium was employed to inhibit dihydrofolate reductase activity, thereby blocking de novo synthesis of thymidylic acid. In the presence of aminopterin, deoxyribonucleic acid (DNA) synthesis can take place only if endogenous thymidine or the thymidine supplied in the HATG medium is utilized. For the utilization of thymidine, thymidine kinase activity is needed. In vaccinia-infected KB cells, both the cellular and the vaccinia-induced thymidine kinases are available. HeLa (BU25) cells are deficient in dtk activity, but the enzyme induced by vaccinia catalyzes the phosphorylation of thymidine. Thus, replication of vaccinia virus proceeds equally well in either KB or HeLa (BU25) cells in the presence or absence of HATG medium (Fig. 2).

3 448 KIT, NAKAJIMA, AND DUBBS J. VIROL. TABLE 1. Thymidine kinase (dtk) activity of adenovirus type 5- and vaccinia-infected HeLa (BU25) cellsa dtk activityb Hr after infection. Noninfected Adenovirus infected Vacciniatype 5-infected 7 0.2b a Input multiplicity was about 13 plaque-forming units (PFU) per cell for vaccinia and about 300 PFU per cell for adenovirus type 5. In further experiments, induction of thymidine kinase was not observed after HeLa (BU25) cells were infected with adenovirus type 5 at input multiplicities of about 10, 50, or 200 PFU per cell. b Expressed as nanomoles of deoxyuridine monophosphate formed per microgram of protein in 10 min at 38 C. Human adenovirus type 5 replication in KB and HeLa (BU25) cells. One-step growth curves depicting the replication of adenovirus type 5 in KB and in HeLa (BU25) cells are shown in Fig. 3. Adenovirus type 5 undergoes a productive infection in both KB and in HeLa (BU25) cells in normal growth medium. In KB cells, where a cellular dtk activity is available, adenovirus type 5 replicates equally well in the presence or absence of HATG. In contrast, replication of adenovirus type 5 is grossly inhibited in the kinase-deficient HeLa (BU25) cells in the presence of aminopterin (Fig. 3). These experiments demonstrate that neither a cellular nor an adenovirus type 5-induced enzyme is present at activity levels needed for utilization of thymidine so as to permit replication of adenovirus type 5 in HATG-treated HeLa (BU25) cells. DISCUSSION Vaccinia and herpes-type viruses induce in various kinase-deficient cell lines dtk enzymes which differ in thermal stability and in kinetic and immunological properties from cellular enzymes (8, 13). The enzyme induced by vaccinia also differs from the enzyme induced by herpes simplex virus in LM(TK-) cells (14). The herpes simplex-induced dtk is immunologically dis- 108 a KB CELLS b HeLa BU 25) CFLLS Without HATG / With HATG 107_/ With HATG q 0.~~~~~~~~ 0 Without HATG HOURS AFTER VACCINIA INFECTION FIG. 2. Replication of vaccinia virus in KB and in HeLa (BU25) cells in the presence or absence of HATG [hypoxanthine (10-4M), aminopterin (10-tM), thymidine (4 X 10-5M), and glycine (10-5M)]. Confluent monolayer cultures were infected at an input multiplicity of 0.05 PFU per cell. Cultures were harvested at the times indicated in the figure, sonically treated, and titrated for virus on indicator CV-1 cells (African green monkey kidney cells). tinct from the enzyme induced by pseudorabies virus (3). These observations and the demonstration that vaccinia and herpes simplex mutants defective in dik-inducing activity can be obtained (5-7) suggest that new virus-specific enzymes are induced in vaccinia- and herpes virus-infected cells. In contrast, polyoma virus enhances the dtk activity of kinase-positive cells but fails to induce the enzyme in kinasedeficient cells (1, 12, 22). The latter findings support the hypothesis that the dtk enhanced by polyoma virus infection is not coded for by a viral gene, but instead, probably reflects the increased synthesis of a cellular enzyme, perhaps modified to some extent by a viral product. The data presented in this study are similar to those obtained with polyoma virus-infected cells. Human adenoviruses enhance the dtk activity of kinase-positive cell lines. The enzyme from adenovirus-infected cells is somewhat changed in thermal stability and kinetic and

4 VOL. 5, 1970 ORIGIN OF THYMIDINE KINASE 449 CL _ L- tl GD L. ( BU 25 HATG HOUR S AFTER ADFNOVI RUS TYPE 5 INFECTION FIG. 3. Replication of adenovirus type 5 in KB and in HeLa (BU25) cells in the presence or absence of HATG. Confluent monolayer cultures were infected at an input multiplicity of 0.05 PFU per cell. Cultures were harvested at the times indicated in the figure, sonically treated, and titrated for virus on indicator KB cells. electrophoretic properties from the enzyme from uninfected cells (11, 18, 23). However, human ICells With HATG adenovirus strains fail to induce dtk activity in kinase-deficient HeLa (BU25) cells. This also suggests that the enzyme enhanced by adenovirus infection is not coded for by a viral gene. A dtk with altered kinetic and immunological properties is induced in kinase-positive cells by simian virus 40 (4, 10, 12, 17). It remains to be seen whether simian virus 40 can induce this Cells Without HATG enzyme in kinase-deficient cell lines. ACKNOWLEDGMENTS This investigation was aided by grants from the American Cancer Society (E-291F), the National Science Foundation (GB 8469), the Robert A. Welch Foundation (Q-163), and by Public Health Service grants (CA , I-K6-Al-2352, and 5-K3-CA 25,797) from the Institute of Allergy and Infectious Diseases and the National Cancer Institute, respectively. We thank Carolyn Smith for able technical assistance. LITERATURE CITED 1. Basilico, C., Y. Matsuya, and H. Green Origin of the thymidine kinase induced by polyoma virus in productively infected cells. J. Virol. 3: Bresnick, E., and F. Rapp Thymidine kinase activity in cells abortively and productively infected with human adenoviruses. Virology 34: Buchan, A., and D. H. Watson The immunological specificity of thymidine kinases in cells infected by viruses of the herpes group. J. Gen. Virol. 4: Carp, R. I Thymidine kinase from normal, simian virus 40-transformed, and simian virus 40-lytically infected cells. J. Virol. 1: Dubbs, D. R., and S. Kit Isolation and properties of vaccinia mutants deficient in thymidine kinase-inducing activity. Virology 22: Dubbs, D. R., and S. Kit Mutant strains of herpes simplex deficient in thymidine kinase-inducing activity. Virology 22: Dubbs, D. R., and S. Kit The effect of temperature on induction of deoxythymidine kinase activity by herpes simplex mutants. Virology 25: Dubbs, D. R., S. Kit, R. A. de Torres, and M. Anken Virogenic properties of bromodeoxyuridine-sensitive and bromodeoxyuridine-resistant simian virus 40-transformed mouse kidney cells. J. Virol. 1: Eagle, H Propagation in a fluid medium of a human epidermoid carcinoma, strain KB. Proc. Soc. Exp. Biol. Med. 89: Hatanaka, M., and R. Dulbecco SV40-specific thymidine kinase. Proc. Nat. Acad. Sci. U.S.A. 58: Hatanaka, M., E. Twiddy, and R. Gilden Electrophoresis of adenovirus-specified thymidine kinase. J. Virol. 4: Kit, S Enzyme induction in cell cultures during productive and abortive infections by papovavirus SV40, p In J. S. Colter and W. Paranchych (ed.), The molecular biology of viruses. Academic Press Inc., New York. 13. Kit, S., and D. R. Dubbs Properties of deoxythymidine kinase partially purified from noninfected and virusinfected mouse fibroblast cells. Virology 26: Kit, S., D. R. Dubbs, and M. Anken Altered properties of thymidine kinase after infection of mouse fibroblast cells with herpes simplex virus. J. Virol. 1: Kit, S., D. R. Dubbs, and P. M. Frearson HeLa cells resistant to bromodeoxyuridine and deficient in thymidine kinase activity. Int. J. Cancer 1:19-30.

5 450 KIT, NAKAJIMA, AND DUBBS J. VIROL. 16. Kit, S., D. R. Dubbs, R. A. de Torres, and J. L. Melnick Enhanced thymidine kinase activity following infection of green monkey kidney cells by simian adenoviruses, simian papovavirus SV40, and an adenovirus SV40 "hybrid." Virology 27: Kit, S., K. Nakajima, T. Kurimura, D. R. Dubbs, and R. Cassingena Monkey-mouse hybrid cell lines containing the SV40 genome in a partially repressed state. Int. J. Cancer 5: Kit, S., L. J. Piekarski, D. R. Dubbs, R. A. de Torres, and M. Anken Enzyme induction in green monkey kidney cultures infected with simiiian adenovirus. J. Virol. 1: Ledinko, N Stimulation of DNA synthesis and thymidine kinase activity in human embryonic kidney cells infected by adenovirus 2 or 12. Cancer Research 27: Ledinko, N Enhanced deoxyribonucleic acid polymiierase activity in human embryoniic kidney cultures infected with adenoviruses 2 or 12. J. Virol. 2: Ledinko, N., and C. K. Y. Fong Kinetics of nucleic acid synthesis in human embryonic kidney cultures infected with adenovirus 2 or 12: Inhibition of cellular deoxyribonucleic acid synthesis. J. Virol. 4: Littlefield, J. W., and C. Basilico Infection of thymidine kinase-deficienit BHK cells with polyomiia virus. Nature (London) 211: Ogino, T., and M. Takahashi Altered pr-operties of thymidine kinase induced in hamster kidney cells by aidenovirus types 5 and 12. Biken J. 12: Pina, M., and M. Green Biochemical studies on adenovirus multiplication. XIV. Macromolecule and enzyme synthesis in cells replicating oncogenic and nononcogenic human adenovirus. Virology 38: Takahashi, M., S. Ueda, and T. Ogino Enihancement of the thymidine kinase activity of hulman embryonic kidney cells and newborn hamster kidney cells by infection with hulmaln adenovirus types 5 and 12. Virology 30: Downloaded from on May 11, 2018 by guest