Archives of Virology 52, 333--339 (1976) © by Springer-Verlag 1976

Thymidine-Kinase in Cytomepalovirus Infected Cells By V. ZXvADA, V. ERBA~,, D. ~EzX6ovX, and V. VODKA Department of Genetics, Microbiology and Biophysics, Facul*y of Sciences, Charles University, Prague, and Depart, ment of Experimental Virology, Institute of Sera and Vaceines, Prague, Czechoslovakia With 4 Figures Accepted August. 31, 1976

Summary In human diploid fibroblast L E P ceils infected with AD 169 strain of human cytomegMovirus (CMV) a sharp increase of cytosol thymidine kinase activity was observed. The properties of the cytosol enzymes from infected and non-infected cells were compared. No significant differences between the enzymes from infected and control cells were observed in substrate specificity, p H dependence, thermostability and relative electrophoretic mobility. H u m a n sera containing high titres of CMV complement-fixing antibodies did not neutralize the enzyme from infected cells. I t is concluded from these results t h a t the increase of cytosol thymidinekinase activity in CMV-infeeted cells was due to an enhancement of cellular thymidine kinase. lntroduetion

Novel virus-coded thymidine kinases have been found in cytosol of cells infected with several members of herpesvirus group. Thus both types of herpes simplex virus and the marmoset herpesvirus (1, 4, 5, 11, 12) specify pyrimidinedeoxynucleoside kinases phosphorylating both thymidine and deoxyeytidine, whereas virus-coded thymidine-specific kinases are synthesized in cells infected with pseudorabies virus, herpesvirus of turkeys, and infectious laryngotracheitis virus (11). On the other hand, virus-specific kinases are lacking in cells infected with equine herpesviruses type 1 and 2 and Herpesvirus aotus (4, 7, 8, 11, 12). No definite conclusions have been reached so far on Epstein-Barr virus (12) and no data are available on cytomegalovirus and varicella-herpes zoster virus. The aim of the present work was to learn whether human cytomegalovirus does induce a novel thymidine kinase in productively infected ceils. 22*

334

V. Z~.VADA, V. ERBAN, D. ]~EZX(~OVJ~, a n d V. VONXA :

Materials and Methods Cells H u m a n diploid f i b r o b l a s t s ( L E P cells) d e r i v e d f r o m a 3 m o n t h s old h u m a n e m b r y o were t h e s a m e as i n p r e v i o u s e x p e r i m e n t s (15). T h e y were c u l t i v a t e d a n d m a i n t a i n e d i n EPL medium [modified Parker's medium enriched with the growth-promoting factor of calf s e r u m (14) b u t t a c k i n g c o m p l e t e calf s e r u m ] . Virus T h e A D - 1 6 9 v i r u s of h u m a n CMV was t h e s a m e as in p r e v i o u s t e s t s (16). I t was g r o w n i n L E P cells, tile fluid p h a s e of c u l t u r e s w i t h a d v a n c e d e y t o p a t h i c c h a n g e s b e i n g u s e d for p a s s i n g t h e virus. F r e s h l y h a r v e s t e d fluids f r e q u e n t l y c o n t a i n e d u p to l 0 s TCDs0/ml. V i r u s t i t r a t i o n s were c a r r i e d o u t in L E P - t u b e cultures, u s i n g 5 t u b e s p e r e a c h t e n - f o l d v i r u s dilution. T h e t e s t was r e a d a f t e r 4 weeks. Cell Extracts W h o l e cell e x t r a c t : Cells w a s h e d twice w i t h t r i s - b u f f e r e d saline ( T B S : 0.14 ~ NaC1, 0.01 M tris-I-IC1, p ~ 7.5) a n d s c r a p e d i n t o T B S c o n t a i n i n g 5 r n ~ m e r c a p t o e t h a n o l a n d 5 ~ t h y m i d i n e ( T M T B S ) were p e l l e t e d a t 1000 × g for 15 m i n u t e s , r e s u s p e n d e d i n a s m a l l v o l u m e of T M T B S a n d s t o r e d a t - - 7 0 ° C. Before t h e assay, t h e cells were d i s r u p t e d b y t h r e e cycles of f r e e z e - t h a w i n g a n d t h e m a t e r i a l was c e n t r i f u g e d for 1 h o u r a t 20,000 × g ~ n d 4 ° C. T h e s u p e r n a t a n t fluid r e p r e s e n t i n g t h e w h o l e cell extract, was i m m e d i a t e l y u s e d i n e n z y m e assay. Cytosol f r a c t i o n : S e m i c o n f l u e n t a n d still p r o l i f e r a t i n g n o n d n f e c t e d a n d c o n f l u e n t C M V - i n f e c t e d a n d m o c k - i n f e c t e d L E P cells (72 h o u r s p o s t infection) were h a r v e s t e d for p r e p a r i n g t h e cytosot cell fraction. T h e pellet of w a s h e d cells p r e p a r e d as d e s c r i b e d a b o v e , was w a s h e d w i t h 0.0l ~ tris-HC1 b u f f e r c o n t a i n i n g 5 m ~ m e r c a p t o e t h a n o l a n d 5 ~M t h y m i d i n e , a n d r e s u s p e n d e d in a s m a l l v o l u m e of t h i s buffer. T h e cells were allowed to swell for 10 m i n u t e s a t 4 ° C a n d were d i s r u p t e d in D o u n c e h o m o g e n i z e r . D i s r u p t i o n of cells a n d t h e following processing of t h e cell h o m o g e n a t e were p e r f o r m e d a t 4 ° C. A f t e r a d j u s t i n g t h e NaC1 c o n c e n t r a t i o n to 0.14 M t h e cell h o m o g e n a t e was c e n t r i f u g e d for 10 m i n u t e s a t 1 0 , 0 0 0 × g a n d t h e s u p e r n a t a n t fluid once m o r e for 1 h o u r a t 105,000 × g u n d e r l i q u i d paraffin. T h e s u p e r n a t a n t fluid r e p r e s e n t i n g t h e cytosol cell f r a c t i o n was s t o r e d in s m a l l v o l u m e s a t - - 7 0 ° C u n t i l r e q u i r e d for e n z y m e assays. U n d e r t h e s e c o n d i t i o n s , t h e t h y m i d i n e k i n a s e a c t i v i t y was s t a b l e for several weeks. M i t o c h o n d r i a l f r a c t i o n : F o r s o m e c o n t r o l e x p e r i m e n t s e x t r a c t s rich in m i t o c h o n d r i M t h y m i d i n e k i n a s e were p r e p a r e d f r o m t h e 10,000 × g pellet of ] ) o u n c e - d i s r u p t e d ceils (see a b o v e ) c o n t a i n i n g nuclei a n d m i t o c h o n d r i a . T h e pellet was r e s u s p e n d e d in a s m a l l v o l u m e of T M T B S , d i s r u p t e d b y t h r e e cycles of f r e e z e - t h a w i n g a n d c e n t r i f u g e d for 1 h o u r a t 105,000 × g u n d e r l i q u i d paraffin. T h e s u p e r n a t a n t fluid was s t o r e d a t - - 7 0 ° C. Protein Estimation T h e p r o t e i n c o n c e n t r a t i o n in cell e x t r a c t s was d e t e r m i n e d b y t h e m e t h o d of L o w ~ Y et aL (13). Determination o/ T h y m i d i n e Kinase Activity T h e r o u t i n e a s s a y was p e r f o r m e d a t 36 ° C in 45 × 5 m m t u b e s . T h e r e a c t i o n m i x t u r e c o n t a i n e d 5 0 r a m tris-HCl, p H 8.0; 5 r a m MgC12; 5 r a m A T P ; 18 ~ f i d C - t h y m i d i n e (0.1 tzCi; 53 m C i / m m o l e ; 40 c p m p e r pikomole) plus 25 ~1 cell e x t r a c t (20 to 80 ~g of p r o t e i n ) i n a t o t a l v o l u m e of 125 ~1. T h e r e a c t i o n was c o n d u c t e d for 15 to 60 m i n u t e s a n d t e r m i n a t e d b y i m m e r s i n g t h e t u b e s i n t o boiling w a t e r for 3 m i n u t e s . T h e p r e c i p i t a t e d p r o t e i n was r e m o v e d b y c e n t r i f u g a t i o n a t 5000 × g for 10 m i n u t e s a n d 50 tzl a l i q u o t e s of t h e s u p e r n a t a n t f l u i d were s p o t t e d o n D E A E - c e l l u l o s e p a p e r discs ( W h a t m a n D E 8 1 ) . T h e s e were d r i e d a n d w a s h e d i n 4 r n ~ a m m o n i u m - f o r m a t e (3 × 5 m i n u t e s ) , in distilled w a t e r a n d in e t h a n o l (1 × 10 m i n u t e s ) . T h e r a d i o a c t i v i t y of t h e p h o s p h o r y l a t e d r e a c t i o n p r o d u c t s s t u c k to t h e p a p e r was m e a s u r e d in a t o l u e n e

Thymidine-Kinase in CMV-Infected Cells

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scintillator (SLT-51, Spolana, Czechoslovakia) using a liquid scintillation counter Isoeap 300 model 6870, Nuclear Chicago Div.

Deter'ruination o/Deoxycytidine Kinase Acti~;ity Deoxycytidine kinase was assayed following the protocol for thymidine kinase except t h a t 14C-deoxycytidine, 0.05 fzCi (25mCi/mmole) was substituted for 14Cthymidine in the reaction mixture.

Disc Polyacrylamide Gel Electrophoresis Disc P A G E analyses were performed in 5 per cent acrylamide gels according to the procedure described by KIT et al. (6, 7, 8) except t h a t the concentrations of thymidine and A T P in gel polymerization mixtures and buffer solutious were 2 ,aM and 2 m~, respectively. The temperature was maintained at 4 ° C during eleetrophoresis which was performed at 2.5--3 mA/gel for 150 minutes. The gels were sliced to 2 m m sections which were transferred to vials containing 100 tzl of buffer solution consisting of 50 ms~ tris-HC1, p H 8.0; 5 mM MgC12; 1 InM mereaptoethanol; 1 fzM thymidine (unlabeled), and stored for several hours at --30° C until all gels were sliced. Then 100 ~zl of the buffer solution (50 m3~ tris-HC1; 5 m~f MgC12) supplemented with ATP (10 m~) and 14C-thymidine or deoxyeytidine (0.05 ~Ci) were added to each vial. After incubation at 38 ° C for 9 hours, t00 ~1 Miquotes from each vial were spotted on DE-81 paper discs and these were processed as in the routine thymidine kinase assay. With some of the disc P A G E runs the 2 m m thick gel slices were cut across and thymidine kinase and deoxyeytidine kinase activities were determined separately. Results

Time Course o/ Thymidine Kina.se and Deoxycytidine Kinase Activitie~ after C M V In/ection As o b s e r v e d in several e x p e r i m e n t s p e r f o r m e d a t i n p u t multiplicities ranging f r o m 3.5 to 25 TCDs0 p e r cell, there was a s h a r p increase of t h y m i d i n e kiuase a c t i v i t y in CMV infected confluent cells. The m a x i m u m level of the e n z y m e a c t i v i t y p r e s e n t in t h e whole cell e x t r a c t was r e a c h e d 24 to 72 hours p o s t infection, preceding t h e a p p e a r a n c e of infectious virus progenies. The e n z y m e levels in infected cells e x c e e d e d five- to eight-times t h e a c t i v i t y p r e s e n t in mock-infected cells. As follows from t h e e x p e r i m e n t p r e s e n t e d in F i g u r e 1, the post-infection increase of t h y m i d i n e kinase a c t i v i t y was followed b y a slower increase of d e o x y cytidine kinase a c t i v i t y as well. S u b s e q u e n t t e s t i n d i c a t e d t h a t the post-infection increase of the t h y m i d i n e kinase a c t i v i t y was m M n i y localized in t h e cytosol cell fraction. As it will be shown l a t e r (see below), the disc P A G E eleetrophoresis r e v e a l e d t h a t these p r e p a r a t i o n s were free of m i t o c h o n d r i a l t h y m i d i n e - k i n a s e . The l a t t e r enzyme, w i t h an Rrn value of 0.62, r e p r e s e n t e d the m a j o r electrophorctic p e a k in t h e mitochondrial extracts. W h e r e a s t h e levels of t h y m i d i n e - k i n a s e a c t i v i t y in cytosol from CMV-infected cells were in t h e range of 2 5 0 - - 5 0 0 c p m / ~ g p r o t e i n / p e r hour, t h e c o r r e s p o n d i n g values in mock-infected confluent cells d i d n o t exceed 20 cpm. On the other h a n d , t h e t h y m i d i n e - k i n a s e a c t i v i t y in the cytosol from semiconfluent a n d still proliferating cell cultures was in the range of 150--300 cpm/Ixg p r o t e i n / h o u r , t h u s ~ p p r o x i m a t i n g t h e vMues d e t e r m i n e d in t h e infected cells. F o r this r e a s o n t,he semiconfluent cultures were used as controls in the s u b s e q u e n t s t u d y a i m e d a t elucidating w h e t h e r t h e postinfection increase in t h e enzyme a c t i v i t y reflected

336

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Fig. 1. Time course of thymidine kinase (full line) and deoxyeytidine kinase (dotted line) activities in CMV-infeeted (closed circles) and mockdnfected (open circles) cells. Activities of enzymes were measured in whole cell extracts and expressed as nucleoside phosphorylated (epm × 10 2) per ~g protein and 1 hour incubation. The curve in the upper part of the figure indicates the C2~IVvirus titres as determined in parallel samples. I n p u t multiplicity of infection was 25 TCDa0 per cell

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Fig. 2. Disc PAGE analyses of thymidine kinase activities present in eytosol fractions from CMV infected cells (closed circles; 53 ~g protein per gel) and noninfeeted semiconfluent ceils (open circles; 42 ~g protein per gel). Numbers above the peaks signify eleetrophoretie mobilities relative to the tracking dye (Rm)

Thymidine-Kinase in CMV-Infected Cells

337

the synthesis of a novel enzyme or was due to an enhancement of the hostspecific enzyme.

Properties o/Cytvsol Thymidine Kinaae Activities/tom CM V-In/ected and Non-In/ected Cells To compare the relative eleetrophoretic mobilities of both cytosol thymidine kinase activities, several runs of disc polyacrylamide gel eleetrophoresis were performed in 5 per cent gels. These were sliced Co 2 mm sections and assayed for thymidine kinase activity. As illustrated in Figure 2 there was no difference between the relative eleetrophoretic mobilities (gm) of both activities, the mean value calculated from several measurements being 0.175 for both. I t can also be seen that the cytosol cell fractions were virtually free of mitochondrial thymidine-kinase. Similarly, the investigation of substrate specificity did not reveal any differenee between cytesol thymidine kinase from CMV-infected and control cells. Both activities were monospecific for thymidine as indicated by the absence of deoxyeytidine-phosphorylating activity in electrophoretie peaks of eytosoI thymidine kinase activities. Accordingly, deoxyeytidine (unlabeled) added in fifty-fold excess did not compete with thymidine for thymidine-phosphorylating activity in cytosol from either CMV-infected or control cells. Both activities utilized only ATP as an efficient phosphate-group donor, the efficiency of GTP, CTP and U T P being less than 10 per cent of that of ATP. The pH dependence (Fig. 3) and the heat inactivation curves (Fig. 4) were essentially identical for the enzymes from both the infected and non-infected cells.

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semiconfluent cells (open circles; 21 ~g protein per 125 ~1 of the reaction mixture). 50 ms~ tris-maleinate buffer was substituted for the standard tris-HC1 buffer in the reaction mixtures. Incubation time was 20 minutes. The epm vMues are given per 50 ~zl of the reaction mixture

338

V. ZXVA:DA,V. ERBAN, D. ~]~zXSovX, and V. VONKA:

Three h u m a n sera with high CMV complement-fixing antibody levels (1:8 to 1:160) were tested for their ability to neutralize the thymidine kinase a.ctivity present in the eytosol of CMV-infected eells. No inhibition effect was observed.

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:Fig. 4. Hea$ inactivation of eytosol thymidine kinase activities from C2¢IVdnfeeted eells (dosed circles) and non-infected semieonfluent (open eireles) cells. Cytosol fractions diluted with TMTBS to 20 ~g protein/25 ~1 were held at 54 ° C and samples were withdra~m at intervals for thymidine kinase activity assays

Discussion The infection with the AD-169 strain of human CMV resulted in an early increase in thymidine-kinase activity in the eytosol of confluent stationary phase human diploid cells. The subsequent tests aimed at elucidating the nature and origin of this enzyme did not reveal any significant differences between the eytosol enzymes from infected and control cells in their substrate specificity, p i t optima, thermostability and, most important, in their relative eleetrophoretie mobility. I t should be recalled t h a t all herpesvirus-speeifie thymidine-kinases possess •m vMues in the range of 0.6 to 0.7 resembling thus more the mitoehondrial than the respective cellular eytosol enzyme (11). I n addition, none of the human sera possessing high levels of CMV complement-fixing antibodies, exhibited an inhibitory effect on the enzyme from the infected cells. On the basis of all these results we conclude t h a t - - a t variance with herpes simplex virus and several herpesviruses of lower a n i m a l s - - t h e AD-169 strain of h n m a n CM-V does not induce a novel thymidine-kinase in productively infected eells. The postinfeetion increase of eytosol thymidine-kinase activity was obviously due to the enhancement of the host-specific eytosol enzyme, as observed with several papova- and adenoviruses (for references see 9, 10). Thus the novel DNA polymerase observed in CM-V infected cells (2, 3) seems to be at present the only candidate for virus distinct enzyme in CMV-infeeted cells. Before making any generalization concerning the incapability of h u m a n C3~V, to induce a novel thymidine-kinase, the present results should be confirmed with other CMV strains. Thymidine-kinase deficient mutants of I-ISV, marmoset herpesvirus and pseudorabies virus exist, at least as laboratory strains (1, 11). The possibility that AD-169 virus represents such a mutant, though unlikely, cannot be ruled out at this moment.

Thymidine-Kinase in CMV-Infeeted Cells

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Aeknowledflments The excellent technical assistance of Mrs. D. Po~i~ovX is appreciated.

References t. DUBBS, D. R., KIT, S. : Mutant strains of herpes simplex deficient in vhymidine kinase inducing activity. Virology 22, 493 502 (1964). 2. I-Ii~hr, K., FURWgAWA,T., PLOWKIN,S. A. : Induction of DNA polymerase in WI-38 and guinea pig cells infected with h u m a n eytomegalovirus (HCMV). Virology 70, 251---255 (1976). 3. HwAs"~, E. S. : H u m a n eytomegMovirus. III. Virus-induced DNA polymerase. J. Virol. 16, 298--310 (1975). 4. JANIESO~, A. T., G~XT~Y, G. A., S~RA~-S~ARPE, J. I-[.: Induction of both thymidine and deoxyeytidine kinase activity by herpes viruses. J. gen. Virol. 24, 465--480 (1974). 5. JA~IESON, A. T., SUBAK-S~ARPE,J. H. : Biochemical studies on the herpes simplex virus.specified deoxypyrimidine kinase activity. J. gen, Virol. 24, 481--492 (1974). 6. KI% S., LIoN,G, W.-C., TR~:ULA,D. : Properties of mitoehondrial thymidine kinase of parental and enzyme-deficient HeLa cells. Arch. Bichem. Biophys. 158, 503 to 513 (1973). 7. KI~, S., LEU~G, W.-C., JO~GE~SEN, G. N., DTJ~BS, D. R.: Distinctive properties of thymidine kinase isozymes induced by h u m a n and avian herpesviruses. Int. J. Cancer 14, 598--610 (1974). 8. KIT, S., LEUSrC, W.-C., TRKULA, D., JOUGENSE~, G.-N.: Gel eleetrophoresis and isoeleetrie focusing of mitoehondriM and viral induced thymidine kinases. Int. J. Cancer 13, 203--218 (1974). 9. KIT, S., LEUNG, W.-C., TRI4ULA, D., DUBB8, D. R., JOt~GENSEN, G.-~.: Gel electrophoresis, isoelectrie focusing and localization of thymidine kinase in normal and simian virus 40.infected cells. Intervirology 2, 137--151 (1974). 10. K~T, S., LEV~rc, W.-C., JORGE~rSEN, G.-N., T~:ULA, D., DVBBS, D. R. : Subeellular localization and properties of thymidine kinase from adeno-virus-infeeted cells. J. ten. Virol. 24, 28t---292 (t974). 11. KIT, S., LEUNG, W.-C., JORG]~NSEN, G. N , TI~:ULA, D., DUBBS, D. R.: Viralinduced thymidine kinase isozymes. I n : J. L. MELmC~: (ed.), Progr. med. Virol., Vol. 21, t3--24. Basel: Karger 1975. 12. Lgu~rG, W.-C., DUBBS, D. R., T~KVLA, D., KI~, S. : Mitoehondrial and herpesvirusspecific deoxypyrimidine kinases. J. Virol. 16, 486--497 (1975). 13. LowRY, O. H., ROS~BROUGH, N. J., :FA~, A. L., I~ASrDALL, R. J. : Protein measurement. with the Folin phenol reagent. J. biol. Chem. 193, 265--275 (1951). t4. MICt~L, J., t~I.EZXSOVX,D. : Cultivation of mammalian cells in a medium with growth promoting proteins from calf serum. Acta virol. 10, 254--259 (1966). 15. tlEziSovX, D., BAR:~OVX, Z. : Experience in a tong-term cultivation of own h u m a n diploid cell strains in synthetic medium with growth promoting proteins. Progr. irnmunobiol. Stand, 3, 111--112 (1969). 16. VONKA, V., BENYESII-MELNICK, M. : Interactions of human eytomegalovirus with human fibroblasts. J. Bacteriol. 91, 213--220 (1965). Authors' address : Dr, V. ZIVaDA, Department Vinifn~ 5, CS-128 44 Prague 2, Czechoslovakia.

of Microbiology,

Received July 12, 1976

Charles University,

Thymidine-kinase in cytomegalovirus infected cells.

Archives of Virology 52, 333--339 (1976) © by Springer-Verlag 1976 Thymidine-Kinase in Cytomepalovirus Infected Cells By V. ZXvADA, V. ERBA~,, D. ~Ez...
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