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Serological analysis of some enzymes present in pseudorabies virus-infected and noninfected cells
28, 271-281 (1966)
VIROLOGY
Serological
Analysis
of Some
Virus-Infected CHUYA
HAMADA,”
Department
and
TOMOYA
of Microbiology,
Enzymes
Present
Noninfected
IiAMIYA,3
AND
Research Laboratories, Albert Philadelphia, Pennsylvania
in Pseudorabies
Cells’ ALBERT Einstein
S. KAPLAS Medical
Center,
Accepted October 16, 1965 The pattern of activit,y of some of the “DNA-synt,hesiaing” enzymes was examined ilr rabbit kidney cells in t,he st,at,ionary phase of growth before and after infection with pseudorabies virus. It was found that the levels of activity of thymidine kinase, thymidine monophosphate kinase, and I)NA polymerase, which are barely detectable ill noninfected cells, start to increase bet,ween 1 and 2 hours after infection. No sequence in the time of appearance of these enzymes could be detected. The levels of activity of deoxyadenosine monophosphate kinase, deoxycytidine monophosphate kinase, and deoxyguanosine monophosphate kinase remain high in cells which reach the statioiiary phase of growth and are unchanged by infection. The antigenic relationships between thymidine kirrase, DNA polymerase, and deoxyadenosine monophosphat,e kinase present in noninfected cell and in virus-infected cell extracts were determined by testing the effect, of incubation with specific 7.globulins on the enzyme activity. These tests showed that the deoxyadenosine monophosphat.e 1)NA polymerases present in infected and noninfected cells are antigenically related. Thymidine kinase present in infected cells is, however, unrelated ant,igenirally to the protein performing the same function in noninfected cells. INTROJ)IJCTION
RK4 cells in the stationary phase of growth wit’h Pr virus result,s in an increase in the rat,e of synthesis of DNA in these cells (Kaplan and Ben-Porat, 1960). This incarcase is accompanied by a rise in the level of activity of some of the enzymes involved in the synthesis of DXA (Nohara and Kaplan, 1963; Kaplan et al., 1965; Infc&on
of
1 This investigation was supported by grants from the National Institut,es of Health (AI-02132 and AI-03362), and from the Natianal Science Foundation (GB-1386), and by a U.S. Public Health Service Jtesearch Career Program Award (AI-K3-19335) from the Nat,ional Instit,nte of Allergy and Irlfectious Diseases. ? Present, address: Institute for Virus Research, Kyoto University, Japan. 3 Present address: Cancer Research Institute, Kyushu IJniversity, Japan. 4 Abbreviations: RK, rabbit kidney cells; Pr, pseudorabies virus; l>NA, deoxyribonucleic acid; dAMP, deoxyadenosine monophosphate; dCMP,
Kamiya et al., 1965). Similar results have been report’ed for a variety of virus-cell systems (Hanafusa, 1961; Kit et al., 1962; McAuslan and Joklik, 1962; Green and Pifia, 1962; Newton and McWilliam, 1962; Iieir and Gold, 1963; Kit ef al., 1965; Dulbecco et al., 1965). The elevation in the level of enzymat,ic activity in the infected cells may be due to the synthesis in these cells of new enzymes. Whether or not these proteins are different from t,he prot’eins performing the same function in noninfeect’ed celIs is of considerabIe interest,
since
this
information
may
hell,
the
understanding of the respective roles played by the infecting viral genome and the host -..--.deoxycytidine monophosphate; dGMP, deoxyguanosine monophosphate; TMP, thymidine monophosphate; ATP, adenosine triphosphate; Tris, tris(hydroxymethyl)aminomethane; IE, extracts of infected cells; NE, extract,s of noniufected cells. 271
272
HAMADA,
KAMIYA.
cell genome in the control of this aspect of the infective process. There have been a number of reports describing changes in the characteristics of the enzymes induced in cells infected with vaccinia virus (Kit and Dubbs, 1965), herpes simplex virus (Keir, 1965), cowpox virus (McAuslan, 1963), and Pr virus (Nohara and Kaplan, 1963). However, with the exception of Kit and Dubbs (1965), who used partially purified enzyme preparations, these studies were carried out with crude enzyme preparations and their value is therefore limited in attempting to est’ablish unequivocally a structural difference between the enzymes in infected and noninfected cells. On the other hand, instead of being due to the formation of new proteins, the increase in activity of the enzymes may be the result of the creat’ion in the infected cells of conditions in which the enzymes present in the cell at the time of infection are stabilized by substrate (Bojarski and Hiatt, 1960; Wright, 1960; Weissman et al., 1960; Littlefield, 1965), or released from inhibitory control mechanisms (Gerhart and Pardee, 1963 ; Changeux, 1963; Freundlich and Umbarger, 1963). The fact that the increase in the level of activity of these enzymes is prevented by the addition to the infected cells of inhibitors of protein synthesis does not necessarily imply that the increase in enzyme act’ivity is due to the formation of new enzyme protein. Protein synthesis may be required for the creation of the conditions that favor stabilization of the enzymes present in the cells at the t#ime of infection. The experimenm described in this paper are part of an attempt to distinguish between these possibilities. We have found previously t,hat, during t,he early stages of the infective process of Pr virus-infected cells, serologitally distinct’ proteins which bear no precursor relationship to the proteins found in mature virus particles and which cannot be detected in noninfected cells are formed (Hamada and Kaplan, 1965). The possibilit’y was therefore considered that these “early”, nonstructural viral prot’eins include enzymes, the formation of which is induced by infection. Since serological methods provide a sensitive tool by which structural differences of specific proteins may be distinguished,
AND
KAPLAN
even in crude extracts, we tried to determine the antigenic relationship between some of the enzymes present in infected and in noninfected cells. The results of these experiments show the following: (1) the dAMP kinases in cells infected with Pr virus and in noninfected cells are antigenically related; (2) the DNA polymerases present in infected and noninfected cells are also antigenically related; (3) thymidine kinase from infected cells is, on t,he other hand, unrelated antigenitally to the protein performing the same function in noninfected cells. MATERIALS
AND
METHODS
Virus and Cell Culture The properties of Pr virus and the cultivation of primary RK monolayer cultures have been described previously (Kaplan, 1957; Kaplan and Vatter, 1959; Ben-Porat and Kaplan, 1962). Media and Solutions ELS: Earle’s saline, 94.5 %; lactalbumin hydrolyzate (Nutritional Biochemicals), 0.5 %; and inactivated horse serum, 5%. Buffered isotonic sucrose: sucrose, 0.25 M; KCI, 4 X lop3 d//; and Tris, 5 X 10e2 A!, buffered at pH 7.8. Chemicals Thymidine, ATP, and dAlII were purchased from the Mann Research Laboratories. Thymidine-H3 and dCRIP-H3 were purchased from the New England Nuclear Corporation. dGJIP-CY4, dA;\IP-CY4, TMPCl4 and dATP-HS were purchased from Schwarz RioResearch Inc. dCTP-H3 was prepared from dCMP-H3 by the met’hod described by Lehman et al. (19.58). Preparation of Cell Extracts XIonolayer cultures of RK cells in the stationary phase of growth were inoculated with a high enough multiplicity of Pr virus to infect practically all t’he cells. After virus adsorption for 1 hour at 37” t,he inoculum was withdrawn, ELS was added to the cultures and incubation was continued at 37”. At the desired time after infect’ion, the cult,urc medium was removed and the cells were washed twice wit’h 2 ml of buffered
SEROLOGICAL
ANALYSIS
OF ENZYMES
273
separated by the ethyl alcohol precipitation isotonic sucrose and collected by scraping them into this solution. The cell suspension method. This procedure was repeated twice. was sonicated (90 seconds) and cent>rifuged Inhibition Test of Enzyme Activity by Antiat 18,000 g for 30 minutes; the supernatant r-Globulin fluid was used for the immunization of To an equal volume of enzyme-containing roosters and for the in vitro experiments. cell extract, 0.05 ml of specific r-globulin was As a normal control, noninfected cultures added. The mixture was incubated for of RK cells in the stationary phase of growth various times at 25”, and the enzyme activity were treat’ed exactly in the same manner, except for virus infection. of the mixture then was assayed. Immunization of Roosters and Preparation of Specific r-Globulins Roosters were immunized as follows: 2 ml of enzyme preparation (protein content: 5-6 mg/ml) from infected cells (3.5 hours after inoculation) or from the noninfected RK cells were emulsified with 2 ml of I+eund’s adjuvant (Difco) and injected intramuscularly into young roosters two times per week for 4 weeks. Three to 4 ml of the enzyme preparations without adjuvant was t,hen given intravenously as a booster on the 5th week; 10 days later blood was collected by cardiac puncture. Y-Globulin was fractionated from immune and preimmune sera by an ethyl alcohol precipitation method (Xchol and Deutsch, 1948). After lyophilization, the r-globulins were dissolved in KCI, 1OWM at a protein concentrat,ion of 40 mg/ml. (Sodium ions were avoided because they were inhibitory to the activity of some of the enzymes.) At this concentration the preimmune rooster yglobulin did not show any nonspecific inhibitory effects on the activity of t’he enzymes tested. Preparation of Absorbed Anti-IE -y-Globulin In order to eliminate antibodies against normal RK cell components from anti-IE r-globulin, it was treated as follows: Extracts from normal RK cells in the logarithmic phase of growth were prepared by the method described above. The activities of thymidine kinase and dA1\IP kinase were tested, and only extracts in which t’he activit,ies of these enzymes were high were used. Equal amount’s of anti-IE r-globulin solution and of cell extracts (containing 10 mg protein/ml) were incubated for 1 hour at 37” under constant’ agitation. The samples were centJrifuged and the r-globulins were then
Kinase Assays Thymidine kinase. To 0.1 ml cell extract, 0.65 ml of reaction mixture containing 5 pmoles MgCIZ , 2.5 Fmoles ATP, 25 pmoles Tris, pH 7.8, and 5 mpmoles thymidine-H3 at a specific activity of 1.25 pC/ml*mole was added. TMP kinase. To 0.2 ml cell extract, 0.55 ml of reaction mixture containing 5 pmoles MgClz , 2.5 pmoles ATP, 25 pmoles Tris, pH 7.8, and 14 mpmoles TMP-Cl4 at a specific act,ivity of 7 mpC per mpmole was added. dGMP kinase. To 0.2 ml of cell extract diluted 1: 6 in isotonic sucrose buffer, 0.55 ml of a reaction mixture containing 5 pmoles n,rgc12 ) 2.5 pmoles ATP, 25 pmoles Tris, pH 7.3, and 14 mHmoles dGMP-Cl* at a specific activity of 7 mpC per mpmole was added. clAMP kinase. To 0.1 ml of cell extract diluted 1: 6 in isotonic sucrose buffer, 0.65 ml reaction mixture containing ,5 pmoles NIgCIZ , 2.5 gmoles ATP, 25 pmoles Tris, pH 7.3, and 11.5 mpmoles d AMP-Cl4 at a specific activity of 9 mMC per mpmole was added. dCMP kinase. To 0.1 ml of enzyme extract, 0.65 ml of reaction mixture containing 5 pmoles n’lgciz , 2.,5 pmoles ATP, 25 pmoles Tris, pH 7.3, and 11 mpmoles dCNIPH3 at a specific act.ivity of 0.11 pC/mpmole was added. An aliquot of the cell extract was incubated for 30 minutes at 37” with the appropriate reaction mixture (see above). Under the conditions used, t’he phosphorylation of t’he substrates was linear for at least 45 minutes. The react.ion was terminated by boiling for 2 minutes, cooling rapidly, and diluting the sample. The protein precipitates were removed by centrifugation. The phosphorylated derivatives of the substrates
274
HAMADA,
KAMIYA,
were separated from the latt’er 011columns of Dowex l-HCl (1 X 4 cm) as described below, and the amount of radioactivity associat,ed with each derivative was determined. Thymidine was separat’ed from its phosphorylated derivatives by elution of the columns with 100 ml of water. The phosphorylated derivative was then eluted with 20 ml 0.5 N HCl. TM’ was separat,ed from TDP and TTP by eluting the columns with 50 ml of a mixture of 0.01 N HCl and 0.05 N KCl. TDP and TTP were then eluted with 20 ml 0.5 N HCl. dGlllP was separated from it,s phosphorylated derivatives by elution of the columns with 100 ml of 0.05 N HCl. The phosphorylated derivatives were then eluted with 20 ml of 0.5 N HCl. dARIP was separated from its phosphorylated derivatives by elution of the columns with 100 ml 0.01 N HCl. The phosphorylated derivatives were then eluted wit’h 20 ml 0.5 N HCl. dCMP was separated from its phosphorylated derivatives by elut’ion of the columns with 100 ml 0.01 N HCl. The phosphorylated derivatives were then eluted wit,h 20 ml 0.5 N HCl. DNA
Polymeruse
Assays
Incorporation of dCTP-H3 into DNA. To 0.2 ml of enzyme extract, 0.2 ml of reaction mixture containing 5 pmoles lUgClz, 2.,5 pmoles ATP, 25 pmoles Tris, pH 7.3, 5 m~moles each of dGTP, dATP, and TTP, 5 m~moles dCTP-H3 at a specific activity of 0.2Fi JLC per mpmole, and 125 pg heat-denatured calf t,hymus DnTA was added. Incmpo~atim of dATP-H3 into DNA. To 0.1 ml of sample to be tested for activity, 0.1 ml of reaction mixture containing 2.,5 pmoles ,\lgClz, 1.25 pmoles ATP, 12.5 pmoles Tris, pH 7.3, 2.5 mpmoles each of dGTP, dCTP, TTP, 2.5 mpmoles dATP-H3 at’ a specific act,ivity of 1.25 WC per m~molc and 125 pg heat-denatured calf thymus DNA4 was added. Incorporation
oj” thymidine-H3
into DNA.
To 0.2 ml of cell extract, 0.2 ml of reaction mixture containing 5 pmoles MgC&, 2.5 bmoles ATP, 25 Hmoles Tris, pH 7.3, 5 mNmoles each dGltlP, dCMP, and dAMP, 5 mhmoles t’hymidine-H3 at’ a specific activity of 0.25 PC per m~mole, and 125 pg heat-denatured calf thymus DNA was added.
AND
KAPLAN I
I
0 *
I
TIME
I
I
I
I
I
I
I
I
I
Thymidine - H3 dCTP-H3 (polymerose)
I
I
I
I AFTER
I
2 INFECTION
3 (HOURS)
FIG. 1. The activity in vitro of DNA polymerase (incorporation of dCTP-H’ into DNA) and of the total “DNA-synthesizing” system (incorporation of thymidine-H3 into I>NA). The assays were performed as described in Materials and Methods. Two-tenths milliliter of cell extract, containing approximat,ely 1 mg of protein, was used in each
sample. In each case the samples were incubated at 37” for 2 hours, acidified, and washed to remove acid-soluble material; the amount of radioactivity associated with the acid insoluble mat’erial was determined. Periodically, tests were performed to det,ermine the distribution of the radioactivity, and it was found invariably associated with DNA, as det,ermined by the Schmidt’ and Thamhauser technique (1945). RESCLTS
Changes in the Level of Activity of I’arious Enzymes as a Result of Infection
RK cells in the stationary phase of growth were infected with Pr virus (adsorbed mult,iplicity = 10); at various times after infertion, the cells were harvest,ed, c+ell extracts were prepared, and the activity of various
SEROLOGICAL
I TIME
AFTER
2 INFECTION
ANALYSIS
3 (HOURS)
FIG. 2. The pattern of activity of some of the kinases involved in the synthesis of DNA. The values on the ordinate are for TMP kinase. To obtain the correct values for the other kinases, mult,iply the ordinate by the factor given next to each kinase. For the assay of TMP kinase, approximately 1 mg of cell ext,ract was used per sample, for thymidine kinase 0.5 mg, for dCMP kioase 0.5 mg, for dGMP kinase 150 pg, and for di\MP kinase 75 pg.
enzymes in these extracts was tested as described in Jlaterials and i\Iethods. Figure 1 summarizes the level of activit’y of DNA polymerase in cell extracts at various times after infe&on, as well as the ability of these cell extracts to incorporate thymidinc into DKA in vitro (which requires, in addition t,o the polymerase, the activity of thymidine kinase and t’he four deoxynucleotide kinases). In both cases there was a rapid increase in activity of the enzymes between 1 and 2 hours after infection. Since the level of enzyme activity in the extracts of the noninfect’ed cells was barely detect’able, we tested whether, against this low background, a sequencae of induct,ion in
OF ENZYMES
275
the infected cells of some of the enzymes involved in the synthesis of DKA could be detected. At’ various times after infection the act,ivity of the different, enzymes involved in the synt,hesis of DNA was determined. I’igure 2 shows that t,he activities of dGRlP, dC;\IP, and dARI1’ kinases were relatively high in the noninfected cells and remained unchanged by infection. The levels of artivity of thymidine kinase and of TMP kinase, on t’he other hand, increased aft)er infection. These results are in general agreement with t’hose reported for vaccinia virus-infected cells (Magee, 1962; Green et al., 1964). The first rise in the levels of activity of thymidine kinase and TMP kinase was detect)ed at npproximately the same time as that of DNA polymeraxe (see Fig. 1); i.e., between SO and 100 minutes after infection, and thus no sequence in t’he appearance of increased levels of activity of these enzymes was detectable by the methods used. The increase in t,he level of enzyme activit)y was dependent, upon protein synthesis; the addition of puromycin (20 pg/ml) to the cultures at any t’irne during the infective process prevented completely any further increase in enzyme activity. Bntigenic Relaticmship between the Thy,,licline Kinases Present in Infected and Noninfected Cells In order to t’est whether t,hymidine kinase induced by infection of stationary phase cells is antigenically relat#ed to thymidine kinase found in noninfect’ed, logarithmically growing cells, the following experiment wns performed. Specific r-globulins against NE (noninfected cell ext,racts) and IE (infect,ed cell extracts), as well as preimmune r-globulin, were prepared as described in Materials and Methods; the effect of preincubation of cell extracts from infected (3.5 hours after infection) and noninfect’ed 1ogzLrithmically growing cells with t#hese r-globulins on the act’ivity of thymidine kinase in the extracts was tested. The results of a representative experiment are illustrat’ed in Fig. 3. All three r-globulins remained wit,hout effect on t)he a&vity of thymidine kinase in extracts of noninfect’ed cells; thymidine knasc activity in infected cell ext)racts was, on the other hand, inhibit,ed by anti-IE r-glob-
276
HAMADA, I
I
AND
I
KAPLAN I
I
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I
T
b z !ii 4 5:
I
KAMIYA,
0
x Y NON
INFECTED
INFECTED
6-
-
1.2
s ;: 2
\Pre-immunC
I.0
? 4 & 0
0
3
4
I
2
TIME
OF PRE-INCUBATION
I
0 WITH
2 &-GLOBULIN
3
-
0.8
-
0.6
2 $ E
4
(HOURS)
FIG. 3. Effect of incubation of cell extracts from infected and noninfected cells with various 7.globulins on thymidine kinase activity. Y-Globulin against noninfected cell extract, NE; r-globulin against infected cell extract, IE. 0.05 ml of cell extract was incubated with 0.05 ml of r-globulin at 25”, and at various times thymidine kinase activity was assayed, as described in Materials and Methods. The infected cell extract contained 250 pg protein per 0.05 ml; the noninfected cell extract, 245 pg/O.O5 ml.
ulin only. Since anti-IE r-globulin was inhibitory to thymidine kinase from the homologous preparation only, it seems that this enzyme in infected cells is antigenically unrelated to the enzyme in noninfected cells. The fact that anti-NE r-globulin was inactive against thymidine kinase from both infected and noninfected cells may possibly be due to the relatively low level of this enzyme in extracts of noninfected cells (approximately 41 that of infected cells) and the consequent formation of low levels of ant’ibody against this enzyme. Antigenic Relationship between the dAMP Kinases Present in Infected and Noninfected Cells Figure 4 shows the effect of the various r-globulins on the level of activity of dAMP kinase in cell extracts from infected and noninfected cells. The activity of this enzyme in extracts of infected, as well as of noninfected cells. was reduced to t’he same
extent by incubation with either anti-IE or anti-NE r-globulin. The fact that dAMP kinase from both sources reacted with homologous, as well as with heterologous, -y-globulin indicates that the enzyme found in infected cells is antigenically related to that found in noninfected cells. E$ect of Absorption of Anti-IE T-Globulin with Noninfected Cell Extract on Its Ability to Neutralize the Activity of Enzymes Present in Extracts of Infected Cells In order to confirm further the results obtained in the previous experiments (i.e., that the dAMP kinases in extracts from infected and noninfected cells are antigenically related, whereas the thymidine kinases are not), the following experiment was done: Anti-IE y-globulin was absorbed exhaustively with extracts prepared from noninfected, logarithmically growing cells which contained relatively high levels of all the
SEROLOGICAL
ANALYSIS
277
OF ENZYMES
INFECTED
ePre-immune
IE NE IE NE
I
TIME
1
I
-
I 1
OF PRE-INCUBATION
WITH
&-GLOBULIN
(HOURS)
FIG. 4. Effect of incubation
of cell extracts from infected and noninfected cells with various 7.globulins on dAMP kinase activity. 0.05 ml of cell extract was incubated with 0.05 ml of r-globulin at 25”, and at various times dAMP kinase activity was assayed, as described in Materials and Methods. Both infected and noninfected cell extracts were used at a concentration of 40 pg protein per 0.05 ml.
enzymes concerned with the synthesis of DNA. If the thymidine kinases found in infected and noninfected cells are indeed antigenically different, and the dAMP kinases, on the ot’her hand, are antigenically related, the exhaust#ive absorption of anti-IE r-globulin with extracts of noninfected cells in t,he logarithmic phase of growth should remove the ability of the r-globulin to inactivate dAh4P kinase activity but’ not affect thymidine kinase activity present in infect’ed cell extracts. The effect of preincubation of extracts of infected cells with anti-?;E r-globulin, antiIE r-globulin, and absorbed anti-IE r-globulin on the subsequent activity of dARlP kinase and thymidine kinase in t,hese extracts is given in Fig. Ls. As described above (see Icig. 3), anti-IE r-globulin was effective in reducing the level of thymidine kinase activity, whereas antiNE r-globulin was not. Furt’hermore, absorbed ant,i-IE y-globulin retained its ability to reduce the level of activity of the enzyme (Fig. 5). Thus, the absorption of anti-IE r-globulin with ext,ract’s of noninfected cells
(containing t,hymidine kinase) did not result in a loss of its ability to reduce the activity of thymidine kinase present in infected cells, indicating that thymidine kinase present in the extracts of noninfected cells did not react with -y-globulin specific for thymidine kinase present in the infected cells. This corroborates the results from the previous experiments; i.e., t’hymidine kinase present in infected cells is antigenically unrelated to thymidine kinase found in noninfected cells. The effect of anti-NE, anLIE, and absorbed anti-IE y-globulins on t’he activity of dAn2P kinase showed a different patt,ern (Fig. 5). As expected from previous experiments (Fig. 4), the activity of dAMP kinase present in infected cells was reduced by irlcubation with either anti-II? or ant’i-NE r-globulins. Absorbed anti-IE y-globulin, however, had lost its inhibitory effect, indicating that dA?tIP kinase present in noninfected cells reacted with r-globulin directed specifically against dAMI’ kinasc of infected cells and that the dAMP kinases from both sources are, therefore, serologically related.
278
HAMADA,
KAMIYA,
AND
NE
0
I
2
3
KAPLAN
-
4
TIME OF PRE-INCUBATION
0 WITH
..
I
-
2
#-GLOBULIN
Ak..lhd
3
“I 2
4
(HOURS)
FIG. 5. Effect of incubation
of infected extracts with ZE -,-globulin before and after absorption on the activity of thymidine kinase (left side) and dAMP kinase (right side). 0.05 ml of cell extract was incubated with 0.05 ml of r-globulin. At various times, the activity of theenzymes was assayed as described in Materials and Methods. For the assay of thymidine kinase, cell extract containing 250 pg protein per 0.05 ml was used; and for dAMP kinase, 53 pg/O.O5 ml.
Antigenic Relationship between the DNA Polynzerases Present in Infected Xtationary Phase Cells and Noninfected Logayithmically Growing Cells In t,he preceding section we showed t’hat’ in t,he infected cells, dAMP kinase (the level of act,ivity of which is unchanged by infection) is antigenically related to the enzyme performing t’he same function in noninfected cells, whereas thymidine kinase (the level of activity of which is greatly increased by infection) is not. We tested therefore whether DKL4 polymerase, the activity of which is also increased in the infected cell, would also be serologically distinct from the enzyme performing the same function in noninfected cells. The type of experiment described above for the determination of antigenic relat,ionships between thymidine and dAMP kinases from infected and noninfected cells was therefore performed for DNA polymerase.
Figure 6 shows the effect of preincubation of infected, as well as of noninfected, cell extracts with a&-IE, anti-NE, and absorbed anti-IE r-globulins. Anti-IE r-globulin was effective in reducing DNA polymerase activity present in extract’s of both infected and noninfected cells, indicating that the enzyme induced by infection in stationary phase cells is serologically related to that present in noninfected logarithmically growing cells. AntiNE r-globulins remained, on the other hand, without effect. The fact that immunization of animals with extracts of noninfected cells did not produce specific r-globulins against DNA polymerase may be due to the lability of this enzyme in these extracts (unpublished result,s). Furthermore, after anti-IE r-globulin had been absorbed wit,h extracts of noninfected cells, it lost its ability to reduce DNA polymerase act,ivity in the cell extracts from both sources. Thus, DNA polymerase from noninfected cells react)ed with the y-
SEROLOGICAL
ANALYSIS
279
OF ENZYMES
0.6
2 2 8 2
0.4
5 ‘p
0.2
9 %
0.8
NON INFECTED
INFECTED
2 I 0 TIME
I
I
I
2
3
4
OF PRE-INCUBATION
0 WITH
I
I
I
I
I
2
3
4
Y-GLOBULIN
(HOURS)
FIG. 6. Reaction
of DNA polymerases from infected and noninfected cells with absorbed and Wabsorbed -,-globulins. 0.05 ml of cell extract was incubated with 0.05 ml of r-globulin at 25”. At various times, 0.1 ml of dATP-H3 reaction mixture was added, and the incorporation of dATP-H” into DNA determined, as described in Materials and Methods. Infected cell extract contained 110 rg protein per 0.05 ml; and noninfected cell extract, 655 pg protein/O.05 ml.
globulins directed against DNA polymerase from infected cells that were present in the anti-IE r-globulin. These results indicat’e that DNA polymerases from both sources are ant,igenically related. DISCUSSION
The experiments described in this paper are an attempt, to determine by a serological method whether the enzymes present in Pr virus-infected cells are structurally different from the enzymes performing the same function in noninfected cells. Of the three enzymes tested, dARIP kinase, DNA polymerase, and thymidine kinase, only the latter seems to be antigenically distinct from the same enzyme present in noninfected cells. A difference in the antigenicity between the thymidine kinases present in vaccinia virusinfected and in noninfect,ed cells has also been reported by Kit and Dubbs (1965). The different antigenicity of the thymidine kinases reported here demonstrates that the enzyme present in Pr virus-infected cells is
structurally different from that present in noninfected cells and would, at’ first glance, seem to indicate that infection does indeed induce t’he de nouo synthesis within the cell of a new and different enzyme protein. However, an alteration in the antigenicity of a protein can also occur as a result of a change in its physical st’ate. Thus, certain trcatmerits of crystalline glutamic dehydrogenase result in changes in its molecular form from a polymer to a monomer, t,hereby also changing the antigenicity of the enzyme (Talal et ab., 1964). The increased activity of the t’hymidine kinase present in infected cells, as well as its different antigenicity, could be due, therefore, to the fact, that the infective process creates conditions wit)hin the infect’ed cells that favor a change in the physical state of the enzyme present in the time of infection, thereby changing its stability and antigenicity. Alternatively, the increase in the level of acbivity of t’hymidine kinase may truly reflect the de novo synthesis of an enzyme ident’ical t,o the thymidine kinase pres-
280
HAMADA,
KAMIYA,
ent in noninfected cells. The alteration in the internal cellular milieu caused by virus infection may, however, change some of the charact,eristics of the enzymes (including its antigenicity). A similar situation has been described by Consigli and Ginsberg (1964) in HeLa cells infected with type 5 adenovirus with respect to aspartate t,ranscarbamylase. Further experiments are therefore necessary before unequivocal proof is obtained that t,he increase in thymidine kinase in the Pr virusinfected cells is indeed due to the de novo synthesis in these cells of a different enzyme protein. REFERENCES and KAPLAN, A. S. (1962). The chemical composition of herpes simplex and pseudorabies viruses. Virology 16, 261-266. B~JARSKI, T. B., and HIATT, H. H. (1960). Stabilization of thymidylate kinase activity by thymidylate and by thymidine. Nature 188, 1112-1114. CHANGEUX, J.-P. (1963). Allosteric interactions on biosynthetic I,-threonine deaminase from E. coli K12. Cold Spring Harbor Symp. Qmnt. Biol. 28, 497-504. COSSIGLI, R. A., and GINSBERG, H. S. (1964). ilctivity of aspartate transcarbamylase in uninfected and type 5 adenovirus-infected HeLa cells. J. Bacterial. 8i, 10341043. DI-LBECCO, It., HARTIYELL, L. H., and VOGT, M. (1965). Induction of cellular DNA synthesis by polyoma virus. Proc. Xafl. -Icad. Sci. U.S. 53, 403-410. FREUNIILICH, M., and UMBARGER, H. E. (1903). The efl’ects of analogues of threonine and of isoleucirre on the properties of threonine deaminase. Cold Spring Harbor Symp. Quant. Biol. 28, 505511. GERHART, J. C., and PARDEE, A. B. (1963). The efiect of the feedback inhibitor, CTP, on submlit interactions in aspartate transcarbamylase. Cold Spring Harbor Symp. Q/cant. Biot. 28, 491496. GREEN, M., and P1S.4, M. (19fi2). Stimulation of the l)NA-synthesizing enzymes of cultured hnman cells by vaccinia virus illfection. Virology 17, 603M04. GREEN, M., PITA, M., and CHAOOYA, X7. (1964). Biochemical studies 011 adenovirus multiplication. T’. Enzymes of deoxyribonucleic acid syllthesis in cells infected by adenovirus and vaccirria viruses. J. Biol. Chem. 23Y, 1188-1197. Havana, C., and KAI~LAN, A. 8. (1965). KineGcs of synthesis of various types of antigenic pro-
BEN-P•
RAT,
T.,
AND
KAPLAN
teins in cells infected with pseudorabies virus. J. Bacteriot. 89, 1328-1334. HAXAFCSA, T. (1961). Enzymatic synthesis and breakdown of deoxyribonucleic acid by extracts of L cells infected with vaccinia virus. Biken’s J. 4, 97-110. KAMIY~, T., BEN-P• RAT, T., and KAPLAN, A. S. (19fi5). Control of certain aspects of the irlfective process by progeny viral DNA. Virology 26, 577-589. KAPLAN, A. S. (1957). A study of the herpes simplex virus-rabbit kidney cell system by the plaque technique. Virology 4, 435-457. KAPLAN, A. S., and BEN-P• RAT, T. (1960). The incorporation of C’4-labeled nucleosides into rabbit kidney cells infected with pseudorabies virus. Virology 11, 12-27. KAPLAN, A. S., and VATTER, A. E. (1959). A comparison of herpes simplex and pseudorabies viruses. Virology 7, 394-407. KAPLAN, A. S., BEN-P• RAT, T., and KAMIYA, T. (1965). Incorporation of 5.bromodeoxyuridine and 5.iododeoxyuridine into viral DNA and it,s effect on the infective process. 4nn. N.Y. Acad. Sci. KEIR,
130.
226-239.
H. 141. (1965). Nucleic acid synthesis in mammalian cells infected with deoxyribonucleic acid viruses. Biochem. J. 94, 3-4P. KEIR, H. M., and GOLD, E. (1963). DeoxyriboIlucleic acid nucleotidyltransferase and deoxyribonuclease from cultured cells infected with herpes simplex virus. BioGhim. Biophys. Acta 72, 263-276. KI,I,, S., and DITBBS, I>. It. (1965). Properties of deoxythymidine kinase partially purified from noninfected and virus infected mouse fibroblast cells. Virology 26, 16-27. KIT, S., DIJBBS, D. R., and PIEKARSKI, L. J. (1982). Enhanced thymidine phosphorylating activity of mouse fibroblasts (strain LM) follow ing vaccinia infection. Biochem. Biophys. Res. Commun. 8, 72-75. KIT, S., FREARSON, P. M., and I)UBBS, I>. R. (1965). Enzyme induction iu polyoma-infected mouse embryo cells. Federation Proc. 24, 598. LEHMAX, I. II., BESSMAN, &I. J., SIMMS, E. S., and KORITBERG, A. (1958). Ellzymatic synthesis of deoxyribonucleic acid. I. Preparat,ion of suhstrates and partial purification of an enzyme from Escherichia coli. J. Biol. Chem. 273, l(i3-170. LITTLEFIELD, J. W. (1965). Studies on t.hymidine kinase in cultured mouse fibroblasts. Biochim. Biophys. Beta 95, 14-22. MAGEE, W. (1962). DNA polymerase and deoxyribonucleotide kinase act,ivities in cells iufccted with vaccinia virus. 17iro/ogy 17, 604-607.
SEROLOGICAL
ANALYSIS
MCAUSLAN, B. R. (1963). The induction and repression of thymidine kinase in the poxvirusinfected HeLa cell. Viirolog2/ 21, 3833389. MCAUSLAN, B. R.., and JOKLIK, W. K. (1962). Stimulation of the thymidine phosphorylating system in HeLa cells on infection with poxvirus. BioLhem. Biophys. Res. Commun. 8, 48&491. NEWTON, A. A., and MCWILLIAM, S. (1962). Incorporation of thymidine by L cells infected with herpes virus. Biochem. J. 84, 112-113P. NICHOL, J. C., and DEUTSCH, H. F. (1948). Biophysical studies of blood plasma proteins. VII. Separation of gamma globulin from the sera of various animals. J. Am. Chem. Sot. 70, 80-83. NOHARA, H., and KAPLAN, A. S. (1963). Induction of a new enzyme in rabbit kidney cells by pseudorabies virus. Biochem. Biophys. Res. Commun. 12, 189-193.
OF ENZYMES
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SCHMIDT, G., and THANNHAUSER, S. J. (1945). A method for the determination of desoxyribonucleic acid, ribonucleic acid, and phosphoproteins in animal tissues. J. Biol. Chem. 161, 83-89. TALAL, N., TOMKINS, G. M., MUSHINSKI, J. F., and YIELDING, K. L. (1964). Immunochemical evidence for multiple molecular forms of crystalline glutamic dehydrogenase. J. Mol. Biol. 8, 46-53. WEISSMAN, S. M., SMELLIE, R. M. S., and PAUL, J. (1960). Studies on the biosynthesis of deoxyribonucleic acid by extracts of mammalian cells. IV. The phosphorylation of thymidine. Biochim. Biophys. Acta 45, 101-110. WRIGHT, B. E. (1960). On enzyme-substrate relationships during biochemical differentiation. Proc. Natl. Acad. Sci. U.S. 46, 798-803.
VIROLOGY
Serological
Analysis
of Some
Virus-Infected CHUYA
HAMADA,”
Department
and
TOMOYA
of Microbiology,
Enzymes
Present
Noninfected
IiAMIYA,3
AND
Research Laboratories, Albert Philadelphia, Pennsylvania
in Pseudorabies
Cells’ ALBERT Einstein
S. KAPLAS Medical
Center,
Accepted October 16, 1965 The pattern of activit,y of some of the “DNA-synt,hesiaing” enzymes was examined ilr rabbit kidney cells in t,he st,at,ionary phase of growth before and after infection with pseudorabies virus. It was found that the levels of activity of thymidine kinase, thymidine monophosphate kinase, and I)NA polymerase, which are barely detectable ill noninfected cells, start to increase bet,ween 1 and 2 hours after infection. No sequence in the time of appearance of these enzymes could be detected. The levels of activity of deoxyadenosine monophosphate kinase, deoxycytidine monophosphate kinase, and deoxyguanosine monophosphate kinase remain high in cells which reach the statioiiary phase of growth and are unchanged by infection. The antigenic relationships between thymidine kirrase, DNA polymerase, and deoxyadenosine monophosphat,e kinase present in noninfected cell and in virus-infected cell extracts were determined by testing the effect, of incubation with specific 7.globulins on the enzyme activity. These tests showed that the deoxyadenosine monophosphat.e 1)NA polymerases present in infected and noninfected cells are antigenically related. Thymidine kinase present in infected cells is, however, unrelated ant,igenirally to the protein performing the same function in noninfected cells. INTROJ)IJCTION
RK4 cells in the stationary phase of growth wit’h Pr virus result,s in an increase in the rat,e of synthesis of DNA in these cells (Kaplan and Ben-Porat, 1960). This incarcase is accompanied by a rise in the level of activity of some of the enzymes involved in the synthesis of DXA (Nohara and Kaplan, 1963; Kaplan et al., 1965; Infc&on
of
1 This investigation was supported by grants from the National Institut,es of Health (AI-02132 and AI-03362), and from the Natianal Science Foundation (GB-1386), and by a U.S. Public Health Service Jtesearch Career Program Award (AI-K3-19335) from the Nat,ional Instit,nte of Allergy and Irlfectious Diseases. ? Present, address: Institute for Virus Research, Kyoto University, Japan. 3 Present address: Cancer Research Institute, Kyushu IJniversity, Japan. 4 Abbreviations: RK, rabbit kidney cells; Pr, pseudorabies virus; l>NA, deoxyribonucleic acid; dAMP, deoxyadenosine monophosphate; dCMP,
Kamiya et al., 1965). Similar results have been report’ed for a variety of virus-cell systems (Hanafusa, 1961; Kit et al., 1962; McAuslan and Joklik, 1962; Green and Pifia, 1962; Newton and McWilliam, 1962; Iieir and Gold, 1963; Kit ef al., 1965; Dulbecco et al., 1965). The elevation in the level of enzymat,ic activity in the infected cells may be due to the synthesis in these cells of new enzymes. Whether or not these proteins are different from t,he prot’eins performing the same function in noninfeect’ed celIs is of considerabIe interest,
since
this
information
may
hell,
the
understanding of the respective roles played by the infecting viral genome and the host -..--.deoxycytidine monophosphate; dGMP, deoxyguanosine monophosphate; TMP, thymidine monophosphate; ATP, adenosine triphosphate; Tris, tris(hydroxymethyl)aminomethane; IE, extracts of infected cells; NE, extract,s of noniufected cells. 271
272
HAMADA,
KAMIYA.
cell genome in the control of this aspect of the infective process. There have been a number of reports describing changes in the characteristics of the enzymes induced in cells infected with vaccinia virus (Kit and Dubbs, 1965), herpes simplex virus (Keir, 1965), cowpox virus (McAuslan, 1963), and Pr virus (Nohara and Kaplan, 1963). However, with the exception of Kit and Dubbs (1965), who used partially purified enzyme preparations, these studies were carried out with crude enzyme preparations and their value is therefore limited in attempting to est’ablish unequivocally a structural difference between the enzymes in infected and noninfected cells. On the other hand, instead of being due to the formation of new proteins, the increase in activity of the enzymes may be the result of the creat’ion in the infected cells of conditions in which the enzymes present in the cell at the time of infection are stabilized by substrate (Bojarski and Hiatt, 1960; Wright, 1960; Weissman et al., 1960; Littlefield, 1965), or released from inhibitory control mechanisms (Gerhart and Pardee, 1963 ; Changeux, 1963; Freundlich and Umbarger, 1963). The fact that the increase in the level of activity of these enzymes is prevented by the addition to the infected cells of inhibitors of protein synthesis does not necessarily imply that the increase in enzyme act’ivity is due to the formation of new enzyme protein. Protein synthesis may be required for the creation of the conditions that favor stabilization of the enzymes present in the cells at the t#ime of infection. The experimenm described in this paper are part of an attempt to distinguish between these possibilities. We have found previously t,hat, during t,he early stages of the infective process of Pr virus-infected cells, serologitally distinct’ proteins which bear no precursor relationship to the proteins found in mature virus particles and which cannot be detected in noninfected cells are formed (Hamada and Kaplan, 1965). The possibilit’y was therefore considered that these “early”, nonstructural viral prot’eins include enzymes, the formation of which is induced by infection. Since serological methods provide a sensitive tool by which structural differences of specific proteins may be distinguished,
AND
KAPLAN
even in crude extracts, we tried to determine the antigenic relationship between some of the enzymes present in infected and in noninfected cells. The results of these experiments show the following: (1) the dAMP kinases in cells infected with Pr virus and in noninfected cells are antigenically related; (2) the DNA polymerases present in infected and noninfected cells are also antigenically related; (3) thymidine kinase from infected cells is, on t,he other hand, unrelated antigenitally to the protein performing the same function in noninfected cells. MATERIALS
AND
METHODS
Virus and Cell Culture The properties of Pr virus and the cultivation of primary RK monolayer cultures have been described previously (Kaplan, 1957; Kaplan and Vatter, 1959; Ben-Porat and Kaplan, 1962). Media and Solutions ELS: Earle’s saline, 94.5 %; lactalbumin hydrolyzate (Nutritional Biochemicals), 0.5 %; and inactivated horse serum, 5%. Buffered isotonic sucrose: sucrose, 0.25 M; KCI, 4 X lop3 d//; and Tris, 5 X 10e2 A!, buffered at pH 7.8. Chemicals Thymidine, ATP, and dAlII were purchased from the Mann Research Laboratories. Thymidine-H3 and dCRIP-H3 were purchased from the New England Nuclear Corporation. dGJIP-CY4, dA;\IP-CY4, TMPCl4 and dATP-HS were purchased from Schwarz RioResearch Inc. dCTP-H3 was prepared from dCMP-H3 by the met’hod described by Lehman et al. (19.58). Preparation of Cell Extracts XIonolayer cultures of RK cells in the stationary phase of growth were inoculated with a high enough multiplicity of Pr virus to infect practically all t’he cells. After virus adsorption for 1 hour at 37” t,he inoculum was withdrawn, ELS was added to the cultures and incubation was continued at 37”. At the desired time after infect’ion, the cult,urc medium was removed and the cells were washed twice wit’h 2 ml of buffered
SEROLOGICAL
ANALYSIS
OF ENZYMES
273
separated by the ethyl alcohol precipitation isotonic sucrose and collected by scraping them into this solution. The cell suspension method. This procedure was repeated twice. was sonicated (90 seconds) and cent>rifuged Inhibition Test of Enzyme Activity by Antiat 18,000 g for 30 minutes; the supernatant r-Globulin fluid was used for the immunization of To an equal volume of enzyme-containing roosters and for the in vitro experiments. cell extract, 0.05 ml of specific r-globulin was As a normal control, noninfected cultures added. The mixture was incubated for of RK cells in the stationary phase of growth various times at 25”, and the enzyme activity were treat’ed exactly in the same manner, except for virus infection. of the mixture then was assayed. Immunization of Roosters and Preparation of Specific r-Globulins Roosters were immunized as follows: 2 ml of enzyme preparation (protein content: 5-6 mg/ml) from infected cells (3.5 hours after inoculation) or from the noninfected RK cells were emulsified with 2 ml of I+eund’s adjuvant (Difco) and injected intramuscularly into young roosters two times per week for 4 weeks. Three to 4 ml of the enzyme preparations without adjuvant was t,hen given intravenously as a booster on the 5th week; 10 days later blood was collected by cardiac puncture. Y-Globulin was fractionated from immune and preimmune sera by an ethyl alcohol precipitation method (Xchol and Deutsch, 1948). After lyophilization, the r-globulins were dissolved in KCI, 1OWM at a protein concentrat,ion of 40 mg/ml. (Sodium ions were avoided because they were inhibitory to the activity of some of the enzymes.) At this concentration the preimmune rooster yglobulin did not show any nonspecific inhibitory effects on the activity of t’he enzymes tested. Preparation of Absorbed Anti-IE -y-Globulin In order to eliminate antibodies against normal RK cell components from anti-IE r-globulin, it was treated as follows: Extracts from normal RK cells in the logarithmic phase of growth were prepared by the method described above. The activities of thymidine kinase and dA1\IP kinase were tested, and only extracts in which t’he activit,ies of these enzymes were high were used. Equal amount’s of anti-IE r-globulin solution and of cell extracts (containing 10 mg protein/ml) were incubated for 1 hour at 37” under constant’ agitation. The samples were centJrifuged and the r-globulins were then
Kinase Assays Thymidine kinase. To 0.1 ml cell extract, 0.65 ml of reaction mixture containing 5 pmoles MgCIZ , 2.5 Fmoles ATP, 25 pmoles Tris, pH 7.8, and 5 mpmoles thymidine-H3 at a specific activity of 1.25 pC/ml*mole was added. TMP kinase. To 0.2 ml cell extract, 0.55 ml of reaction mixture containing 5 pmoles MgClz , 2.5 pmoles ATP, 25 pmoles Tris, pH 7.8, and 14 mpmoles TMP-Cl4 at a specific act,ivity of 7 mpC per mpmole was added. dGMP kinase. To 0.2 ml of cell extract diluted 1: 6 in isotonic sucrose buffer, 0.55 ml of a reaction mixture containing 5 pmoles n,rgc12 ) 2.5 pmoles ATP, 25 pmoles Tris, pH 7.3, and 14 mHmoles dGMP-Cl* at a specific activity of 7 mpC per mpmole was added. clAMP kinase. To 0.1 ml of cell extract diluted 1: 6 in isotonic sucrose buffer, 0.65 ml reaction mixture containing ,5 pmoles NIgCIZ , 2.5 gmoles ATP, 25 pmoles Tris, pH 7.3, and 11.5 mpmoles d AMP-Cl4 at a specific activity of 9 mMC per mpmole was added. dCMP kinase. To 0.1 ml of enzyme extract, 0.65 ml of reaction mixture containing 5 pmoles n’lgciz , 2.,5 pmoles ATP, 25 pmoles Tris, pH 7.3, and 11 mpmoles dCNIPH3 at a specific act.ivity of 0.11 pC/mpmole was added. An aliquot of the cell extract was incubated for 30 minutes at 37” with the appropriate reaction mixture (see above). Under the conditions used, t’he phosphorylation of t’he substrates was linear for at least 45 minutes. The react.ion was terminated by boiling for 2 minutes, cooling rapidly, and diluting the sample. The protein precipitates were removed by centrifugation. The phosphorylated derivatives of the substrates
274
HAMADA,
KAMIYA,
were separated from the latt’er 011columns of Dowex l-HCl (1 X 4 cm) as described below, and the amount of radioactivity associat,ed with each derivative was determined. Thymidine was separat’ed from its phosphorylated derivatives by elution of the columns with 100 ml of water. The phosphorylated derivative was then eluted with 20 ml 0.5 N HCl. TM’ was separat,ed from TDP and TTP by eluting the columns with 50 ml of a mixture of 0.01 N HCl and 0.05 N KCl. TDP and TTP were then eluted with 20 ml 0.5 N HCl. dGlllP was separated from it,s phosphorylated derivatives by elution of the columns with 100 ml of 0.05 N HCl. The phosphorylated derivatives were then eluted with 20 ml of 0.5 N HCl. dARIP was separated from its phosphorylated derivatives by elution of the columns with 100 ml 0.01 N HCl. The phosphorylated derivatives were then eluted wit’h 20 ml 0.5 N HCl. dCMP was separated from its phosphorylated derivatives by elut’ion of the columns with 100 ml 0.01 N HCl. The phosphorylated derivatives were then eluted wit,h 20 ml 0.5 N HCl. DNA
Polymeruse
Assays
Incorporation of dCTP-H3 into DNA. To 0.2 ml of enzyme extract, 0.2 ml of reaction mixture containing 5 pmoles lUgClz, 2.,5 pmoles ATP, 25 pmoles Tris, pH 7.3, 5 m~moles each of dGTP, dATP, and TTP, 5 m~moles dCTP-H3 at a specific activity of 0.2Fi JLC per mpmole, and 125 pg heat-denatured calf t,hymus DnTA was added. Incmpo~atim of dATP-H3 into DNA. To 0.1 ml of sample to be tested for activity, 0.1 ml of reaction mixture containing 2.,5 pmoles ,\lgClz, 1.25 pmoles ATP, 12.5 pmoles Tris, pH 7.3, 2.5 mpmoles each of dGTP, dCTP, TTP, 2.5 mpmoles dATP-H3 at’ a specific act,ivity of 1.25 WC per m~molc and 125 pg heat-denatured calf thymus DNA4 was added. Incorporation
oj” thymidine-H3
into DNA.
To 0.2 ml of cell extract, 0.2 ml of reaction mixture containing 5 pmoles MgC&, 2.5 bmoles ATP, 25 Hmoles Tris, pH 7.3, 5 mNmoles each dGltlP, dCMP, and dAMP, 5 mhmoles t’hymidine-H3 at’ a specific activity of 0.25 PC per m~mole, and 125 pg heat-denatured calf thymus DNA was added.
AND
KAPLAN I
I
0 *
I
TIME
I
I
I
I
I
I
I
I
I
Thymidine - H3 dCTP-H3 (polymerose)
I
I
I
I AFTER
I
2 INFECTION
3 (HOURS)
FIG. 1. The activity in vitro of DNA polymerase (incorporation of dCTP-H’ into DNA) and of the total “DNA-synthesizing” system (incorporation of thymidine-H3 into I>NA). The assays were performed as described in Materials and Methods. Two-tenths milliliter of cell extract, containing approximat,ely 1 mg of protein, was used in each
sample. In each case the samples were incubated at 37” for 2 hours, acidified, and washed to remove acid-soluble material; the amount of radioactivity associated with the acid insoluble mat’erial was determined. Periodically, tests were performed to det,ermine the distribution of the radioactivity, and it was found invariably associated with DNA, as det,ermined by the Schmidt’ and Thamhauser technique (1945). RESCLTS
Changes in the Level of Activity of I’arious Enzymes as a Result of Infection
RK cells in the stationary phase of growth were infected with Pr virus (adsorbed mult,iplicity = 10); at various times after infertion, the cells were harvest,ed, c+ell extracts were prepared, and the activity of various
SEROLOGICAL
I TIME
AFTER
2 INFECTION
ANALYSIS
3 (HOURS)
FIG. 2. The pattern of activity of some of the kinases involved in the synthesis of DNA. The values on the ordinate are for TMP kinase. To obtain the correct values for the other kinases, mult,iply the ordinate by the factor given next to each kinase. For the assay of TMP kinase, approximately 1 mg of cell ext,ract was used per sample, for thymidine kinase 0.5 mg, for dCMP kioase 0.5 mg, for dGMP kinase 150 pg, and for di\MP kinase 75 pg.
enzymes in these extracts was tested as described in Jlaterials and i\Iethods. Figure 1 summarizes the level of activit’y of DNA polymerase in cell extracts at various times after infe&on, as well as the ability of these cell extracts to incorporate thymidinc into DKA in vitro (which requires, in addition t,o the polymerase, the activity of thymidine kinase and t’he four deoxynucleotide kinases). In both cases there was a rapid increase in activity of the enzymes between 1 and 2 hours after infection. Since the level of enzyme activity in the extracts of the noninfect’ed cells was barely detect’able, we tested whether, against this low background, a sequencae of induct,ion in
OF ENZYMES
275
the infected cells of some of the enzymes involved in the synthesis of DKA could be detected. At’ various times after infection the act,ivity of the different, enzymes involved in the synt,hesis of DNA was determined. I’igure 2 shows that t,he activities of dGRlP, dC;\IP, and dARI1’ kinases were relatively high in the noninfected cells and remained unchanged by infection. The levels of artivity of thymidine kinase and of TMP kinase, on t’he other hand, increased aft)er infection. These results are in general agreement with t’hose reported for vaccinia virus-infected cells (Magee, 1962; Green et al., 1964). The first rise in the levels of activity of thymidine kinase and TMP kinase was detect)ed at npproximately the same time as that of DNA polymeraxe (see Fig. 1); i.e., between SO and 100 minutes after infection, and thus no sequence in t’he appearance of increased levels of activity of these enzymes was detectable by the methods used. The increase in t,he level of enzyme activit)y was dependent, upon protein synthesis; the addition of puromycin (20 pg/ml) to the cultures at any t’irne during the infective process prevented completely any further increase in enzyme activity. Bntigenic Relaticmship between the Thy,,licline Kinases Present in Infected and Noninfected Cells In order to t’est whether t,hymidine kinase induced by infection of stationary phase cells is antigenically relat#ed to thymidine kinase found in noninfect’ed, logarithmically growing cells, the following experiment wns performed. Specific r-globulins against NE (noninfected cell ext,racts) and IE (infect,ed cell extracts), as well as preimmune r-globulin, were prepared as described in Materials and Methods; the effect of preincubation of cell extracts from infected (3.5 hours after infection) and noninfect’ed 1ogzLrithmically growing cells with t#hese r-globulins on the act’ivity of thymidine kinase in the extracts was tested. The results of a representative experiment are illustrat’ed in Fig. 3. All three r-globulins remained wit,hout effect on t)he a&vity of thymidine kinase in extracts of noninfect’ed cells; thymidine knasc activity in infected cell ext)racts was, on the other hand, inhibit,ed by anti-IE r-glob-
276
HAMADA, I
I
AND
I
KAPLAN I
I
I
I
T
b z !ii 4 5:
I
KAMIYA,
0
x Y NON
INFECTED
INFECTED
6-
-
1.2
s ;: 2
\Pre-immunC
I.0
? 4 & 0
0
3
4
I
2
TIME
OF PRE-INCUBATION
I
0 WITH
2 &-GLOBULIN
3
-
0.8
-
0.6
2 $ E
4
(HOURS)
FIG. 3. Effect of incubation of cell extracts from infected and noninfected cells with various 7.globulins on thymidine kinase activity. Y-Globulin against noninfected cell extract, NE; r-globulin against infected cell extract, IE. 0.05 ml of cell extract was incubated with 0.05 ml of r-globulin at 25”, and at various times thymidine kinase activity was assayed, as described in Materials and Methods. The infected cell extract contained 250 pg protein per 0.05 ml; the noninfected cell extract, 245 pg/O.O5 ml.
ulin only. Since anti-IE r-globulin was inhibitory to thymidine kinase from the homologous preparation only, it seems that this enzyme in infected cells is antigenically unrelated to the enzyme in noninfected cells. The fact that anti-NE r-globulin was inactive against thymidine kinase from both infected and noninfected cells may possibly be due to the relatively low level of this enzyme in extracts of noninfected cells (approximately 41 that of infected cells) and the consequent formation of low levels of ant’ibody against this enzyme. Antigenic Relationship between the dAMP Kinases Present in Infected and Noninfected Cells Figure 4 shows the effect of the various r-globulins on the level of activity of dAMP kinase in cell extracts from infected and noninfected cells. The activity of this enzyme in extracts of infected, as well as of noninfected cells. was reduced to t’he same
extent by incubation with either anti-IE or anti-NE r-globulin. The fact that dAMP kinase from both sources reacted with homologous, as well as with heterologous, -y-globulin indicates that the enzyme found in infected cells is antigenically related to that found in noninfected cells. E$ect of Absorption of Anti-IE T-Globulin with Noninfected Cell Extract on Its Ability to Neutralize the Activity of Enzymes Present in Extracts of Infected Cells In order to confirm further the results obtained in the previous experiments (i.e., that the dAMP kinases in extracts from infected and noninfected cells are antigenically related, whereas the thymidine kinases are not), the following experiment was done: Anti-IE y-globulin was absorbed exhaustively with extracts prepared from noninfected, logarithmically growing cells which contained relatively high levels of all the
SEROLOGICAL
ANALYSIS
277
OF ENZYMES
INFECTED
ePre-immune
IE NE IE NE
I
TIME
1
I
-
I 1
OF PRE-INCUBATION
WITH
&-GLOBULIN
(HOURS)
FIG. 4. Effect of incubation
of cell extracts from infected and noninfected cells with various 7.globulins on dAMP kinase activity. 0.05 ml of cell extract was incubated with 0.05 ml of r-globulin at 25”, and at various times dAMP kinase activity was assayed, as described in Materials and Methods. Both infected and noninfected cell extracts were used at a concentration of 40 pg protein per 0.05 ml.
enzymes concerned with the synthesis of DNA. If the thymidine kinases found in infected and noninfected cells are indeed antigenically different, and the dAMP kinases, on the ot’her hand, are antigenically related, the exhaust#ive absorption of anti-IE r-globulin with extracts of noninfected cells in t,he logarithmic phase of growth should remove the ability of the r-globulin to inactivate dAh4P kinase activity but’ not affect thymidine kinase activity present in infect’ed cell extracts. The effect of preincubation of extracts of infected cells with anti-?;E r-globulin, antiIE r-globulin, and absorbed anti-IE r-globulin on the subsequent activity of dARlP kinase and thymidine kinase in t,hese extracts is given in Fig. Ls. As described above (see Icig. 3), anti-IE r-globulin was effective in reducing the level of thymidine kinase activity, whereas antiNE r-globulin was not. Furt’hermore, absorbed ant,i-IE y-globulin retained its ability to reduce the level of activity of the enzyme (Fig. 5). Thus, the absorption of anti-IE r-globulin with ext,ract’s of noninfected cells
(containing t,hymidine kinase) did not result in a loss of its ability to reduce the activity of thymidine kinase present in infected cells, indicating that thymidine kinase present in the extracts of noninfected cells did not react with -y-globulin specific for thymidine kinase present in the infected cells. This corroborates the results from the previous experiments; i.e., t’hymidine kinase present in infected cells is antigenically unrelated to thymidine kinase found in noninfected cells. The effect of anti-NE, anLIE, and absorbed anti-IE y-globulins on t’he activity of dAn2P kinase showed a different patt,ern (Fig. 5). As expected from previous experiments (Fig. 4), the activity of dAMP kinase present in infected cells was reduced by irlcubation with either anti-II? or ant’i-NE r-globulins. Absorbed anti-IE y-globulin, however, had lost its inhibitory effect, indicating that dA?tIP kinase present in noninfected cells reacted with r-globulin directed specifically against dAMI’ kinasc of infected cells and that the dAMP kinases from both sources are, therefore, serologically related.
278
HAMADA,
KAMIYA,
AND
NE
0
I
2
3
KAPLAN
-
4
TIME OF PRE-INCUBATION
0 WITH
..
I
-
2
#-GLOBULIN
Ak..lhd
3
“I 2
4
(HOURS)
FIG. 5. Effect of incubation
of infected extracts with ZE -,-globulin before and after absorption on the activity of thymidine kinase (left side) and dAMP kinase (right side). 0.05 ml of cell extract was incubated with 0.05 ml of r-globulin. At various times, the activity of theenzymes was assayed as described in Materials and Methods. For the assay of thymidine kinase, cell extract containing 250 pg protein per 0.05 ml was used; and for dAMP kinase, 53 pg/O.O5 ml.
Antigenic Relationship between the DNA Polynzerases Present in Infected Xtationary Phase Cells and Noninfected Logayithmically Growing Cells In t,he preceding section we showed t’hat’ in t,he infected cells, dAMP kinase (the level of act,ivity of which is unchanged by infection) is antigenically related to the enzyme performing t’he same function in noninfected cells, whereas thymidine kinase (the level of activity of which is greatly increased by infection) is not. We tested therefore whether DKL4 polymerase, the activity of which is also increased in the infected cell, would also be serologically distinct from the enzyme performing the same function in noninfected cells. The type of experiment described above for the determination of antigenic relat,ionships between thymidine and dAMP kinases from infected and noninfected cells was therefore performed for DNA polymerase.
Figure 6 shows the effect of preincubation of infected, as well as of noninfected, cell extracts with a&-IE, anti-NE, and absorbed anti-IE r-globulins. Anti-IE r-globulin was effective in reducing DNA polymerase activity present in extract’s of both infected and noninfected cells, indicating that the enzyme induced by infection in stationary phase cells is serologically related to that present in noninfected logarithmically growing cells. AntiNE r-globulins remained, on the other hand, without effect. The fact that immunization of animals with extracts of noninfected cells did not produce specific r-globulins against DNA polymerase may be due to the lability of this enzyme in these extracts (unpublished result,s). Furthermore, after anti-IE r-globulin had been absorbed wit,h extracts of noninfected cells, it lost its ability to reduce DNA polymerase act,ivity in the cell extracts from both sources. Thus, DNA polymerase from noninfected cells react)ed with the y-
SEROLOGICAL
ANALYSIS
279
OF ENZYMES
0.6
2 2 8 2
0.4
5 ‘p
0.2
9 %
0.8
NON INFECTED
INFECTED
2 I 0 TIME
I
I
I
2
3
4
OF PRE-INCUBATION
0 WITH
I
I
I
I
I
2
3
4
Y-GLOBULIN
(HOURS)
FIG. 6. Reaction
of DNA polymerases from infected and noninfected cells with absorbed and Wabsorbed -,-globulins. 0.05 ml of cell extract was incubated with 0.05 ml of r-globulin at 25”. At various times, 0.1 ml of dATP-H3 reaction mixture was added, and the incorporation of dATP-H” into DNA determined, as described in Materials and Methods. Infected cell extract contained 110 rg protein per 0.05 ml; and noninfected cell extract, 655 pg protein/O.05 ml.
globulins directed against DNA polymerase from infected cells that were present in the anti-IE r-globulin. These results indicat’e that DNA polymerases from both sources are ant,igenically related. DISCUSSION
The experiments described in this paper are an attempt, to determine by a serological method whether the enzymes present in Pr virus-infected cells are structurally different from the enzymes performing the same function in noninfected cells. Of the three enzymes tested, dARIP kinase, DNA polymerase, and thymidine kinase, only the latter seems to be antigenically distinct from the same enzyme present in noninfected cells. A difference in the antigenicity between the thymidine kinases present in vaccinia virusinfected and in noninfect,ed cells has also been reported by Kit and Dubbs (1965). The different antigenicity of the thymidine kinases reported here demonstrates that the enzyme present in Pr virus-infected cells is
structurally different from that present in noninfected cells and would, at’ first glance, seem to indicate that infection does indeed induce t’he de nouo synthesis within the cell of a new and different enzyme protein. However, an alteration in the antigenicity of a protein can also occur as a result of a change in its physical st’ate. Thus, certain trcatmerits of crystalline glutamic dehydrogenase result in changes in its molecular form from a polymer to a monomer, t,hereby also changing the antigenicity of the enzyme (Talal et ab., 1964). The increased activity of the t’hymidine kinase present in infected cells, as well as its different antigenicity, could be due, therefore, to the fact, that the infective process creates conditions wit)hin the infect’ed cells that favor a change in the physical state of the enzyme present in the time of infection, thereby changing its stability and antigenicity. Alternatively, the increase in the level of acbivity of t’hymidine kinase may truly reflect the de novo synthesis of an enzyme ident’ical t,o the thymidine kinase pres-
280
HAMADA,
KAMIYA,
ent in noninfected cells. The alteration in the internal cellular milieu caused by virus infection may, however, change some of the charact,eristics of the enzymes (including its antigenicity). A similar situation has been described by Consigli and Ginsberg (1964) in HeLa cells infected with type 5 adenovirus with respect to aspartate t,ranscarbamylase. Further experiments are therefore necessary before unequivocal proof is obtained that t,he increase in thymidine kinase in the Pr virusinfected cells is indeed due to the de novo synthesis in these cells of a different enzyme protein. REFERENCES and KAPLAN, A. S. (1962). The chemical composition of herpes simplex and pseudorabies viruses. Virology 16, 261-266. B~JARSKI, T. B., and HIATT, H. H. (1960). Stabilization of thymidylate kinase activity by thymidylate and by thymidine. Nature 188, 1112-1114. CHANGEUX, J.-P. (1963). Allosteric interactions on biosynthetic I,-threonine deaminase from E. coli K12. Cold Spring Harbor Symp. Qmnt. Biol. 28, 497-504. COSSIGLI, R. A., and GINSBERG, H. S. (1964). ilctivity of aspartate transcarbamylase in uninfected and type 5 adenovirus-infected HeLa cells. J. Bacterial. 8i, 10341043. DI-LBECCO, It., HARTIYELL, L. H., and VOGT, M. (1965). Induction of cellular DNA synthesis by polyoma virus. Proc. Xafl. -Icad. Sci. U.S. 53, 403-410. FREUNIILICH, M., and UMBARGER, H. E. (1903). The efl’ects of analogues of threonine and of isoleucirre on the properties of threonine deaminase. Cold Spring Harbor Symp. Quant. Biol. 28, 505511. GERHART, J. C., and PARDEE, A. B. (1963). The efiect of the feedback inhibitor, CTP, on submlit interactions in aspartate transcarbamylase. Cold Spring Harbor Symp. Q/cant. Biot. 28, 491496. GREEN, M., and P1S.4, M. (19fi2). Stimulation of the l)NA-synthesizing enzymes of cultured hnman cells by vaccinia virus illfection. Virology 17, 603M04. GREEN, M., PITA, M., and CHAOOYA, X7. (1964). Biochemical studies 011 adenovirus multiplication. T’. Enzymes of deoxyribonucleic acid syllthesis in cells infected by adenovirus and vaccirria viruses. J. Biol. Chem. 23Y, 1188-1197. Havana, C., and KAI~LAN, A. 8. (1965). KineGcs of synthesis of various types of antigenic pro-
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SEROLOGICAL
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MCAUSLAN, B. R. (1963). The induction and repression of thymidine kinase in the poxvirusinfected HeLa cell. Viirolog2/ 21, 3833389. MCAUSLAN, B. R.., and JOKLIK, W. K. (1962). Stimulation of the thymidine phosphorylating system in HeLa cells on infection with poxvirus. BioLhem. Biophys. Res. Commun. 8, 48&491. NEWTON, A. A., and MCWILLIAM, S. (1962). Incorporation of thymidine by L cells infected with herpes virus. Biochem. J. 84, 112-113P. NICHOL, J. C., and DEUTSCH, H. F. (1948). Biophysical studies of blood plasma proteins. VII. Separation of gamma globulin from the sera of various animals. J. Am. Chem. Sot. 70, 80-83. NOHARA, H., and KAPLAN, A. S. (1963). Induction of a new enzyme in rabbit kidney cells by pseudorabies virus. Biochem. Biophys. Res. Commun. 12, 189-193.
OF ENZYMES
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SCHMIDT, G., and THANNHAUSER, S. J. (1945). A method for the determination of desoxyribonucleic acid, ribonucleic acid, and phosphoproteins in animal tissues. J. Biol. Chem. 161, 83-89. TALAL, N., TOMKINS, G. M., MUSHINSKI, J. F., and YIELDING, K. L. (1964). Immunochemical evidence for multiple molecular forms of crystalline glutamic dehydrogenase. J. Mol. Biol. 8, 46-53. WEISSMAN, S. M., SMELLIE, R. M. S., and PAUL, J. (1960). Studies on the biosynthesis of deoxyribonucleic acid by extracts of mammalian cells. IV. The phosphorylation of thymidine. Biochim. Biophys. Acta 45, 101-110. WRIGHT, B. E. (1960). On enzyme-substrate relationships during biochemical differentiation. Proc. Natl. Acad. Sci. U.S. 46, 798-803.