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Changes in metabolic and enzymatic activities of monkey kidney cells after infection with adenovirus 2
VIROLOGY
28, 679-692 (1966)
Changes
in Metabolic
and
Ceils after
Enzymatic Infection
NADA The Salk Institute
Activities
with
of Monkey
Adenovirus
Kidney
2
LEDINKO’
for Biological
Studies,
San Diego,
California
Accepted December 16, 1965 Primary cultures of monkey kidney were infected with type 2 adenovirus. The formation of infective virus was first detected at approximately 21 hours after infection. Final yields of l&50 plaque forming units per cell were obtained at approximately 50 hours. The rate of DNA synthesis, as measured by the rate of incorporation of H3-thymidine into DNA, did not change after infection. The rates of incorporation of H3-uridine into RNA, and of Ha-leucine into protein, however, were markedly inhibited in infected cells before infective virus appeared. A striking decrease in the activity of the DNA-dependent RNA polymerase also occurred before virus maturation began. The effects of type 2 adenovirus infection of monkey kidney cells on the activities of deoxythymidine kinase, DNA polymerase, deoxycytidylate deaminase, and aspartate transcarbamylase were investigated. The deoxythymidine kinase activity began to increase at approximately 20 hours after infection, and at 50 hours the activity was four- to sixfold higher than that of the uninfected cells. At 4@60 hours, the deoxycytidylate deaminase and aspartate transcarbamylase activities were slightly but consistently greater than those of control cells by a factor of 1.6-2. The DNA polymerase activity was not changed. The increase in the deoxythymidine kinase activity induced after infection depended on de novo protein synthesis. The induced deoxythymidine kinase activity of the infected cells was more sensitive to feedback inhibition by dTDP, and dTTP, than the enzyme activity of the control cells. INTRODUCTION
Adenovirus is a DNAZ-containing virus which multiplies in the nucleus of the cell r Present address: Putnam Memorial Hospital Institute for Medical Research, Bennington, Vermont 05201. z Abbreviations: DNA, deoxyribonucleic acid; RNA, ribonucleic acid; dCMP, deoxycytidine monophosphate; dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosdTDP, deoxythymidine diphosphate; phate; dTTP, deoxythymidine triphosphate; dATP, deoxyadenosine triphosphate; dGTP, deoxyguanosine triphosphate; dCTP, deoxycytidine triphosphate; ATP, adenosine triphosphate; GTP, guanosine triphosphate; UTP, uridine triphosphate; CTP, cytidine triphosphate; RNase, ribonuclease; DNase, deoxyribonuclease; S.A., specific activity; PFU, plaque-forming unit; cpm, counts per minute; CGM, cell growth medium. 679
(Brandon and McLean, 1962). Observations on the metabolism of adenovirus-infected KB and HeLa cells have indicated that the synthesis of DNA, RNA, and protein continues after infection (Ginsberg and Dixon, 1959; Green, 1962; Wilcox and Ginsberg, 1963; Polasa and Green, 1965). However, the synthesis of these macromolecules dimishes late in the infectious process when replication is complete or almost complete (Green, 1959; Green and Daesch, 1961; Green et nl., 1964). The possibility that the replication of adenovirus DNA is mediated by virusinduced changes in the DNA synthesizing enzymes has been investigated. No appreciable change in the activities of the enzymes involved in DNA synthesis has been found in adenovirus-infected cells (Green et al., 1564). In contrast, increases in some of the
680
LEDINKO
DNA synthesizing enzymes have been induced after infection with a number of other DNA-containing animal viruses. For example, there is a marked increase in the activity of deoxythymidine kinase in cells infected with vaccinia (Kit et al., 1962, 1963; McAuslan, 1963a), herpes simplex (Kit and Dubbs, 1963), pseudorabies (Nohara and Kaplan, 1963), and polyoma (Dulbecco et al., 1965) viruses. In the present study, we have observed the induction of the deoxythymidine kinase activity of monkey kidney cells after adenovirus infection. Using this host cell, we have found that the rate of synthesis of host cell RNA and protein was inhibited soon after infection, but the rate of DNA synthesis was unaffected. The inhibition of RNA and protein synthesis was accompanied by a striking decrease in the activity of the DNAdependent RNA polymerase. MATERIALS
Solutions
AND
METHODS
and Media
Cell growth medium (CGM): Cultures were grown in reinforced Eagle’s medium (Vogt and Dulbecco, 1963) containing 5% fetal calf serum. Virus growth medium (VGM): Virus growth medium consisted of reinforced Eagle’s medium (Vogt and Dulbecco, 1963) containing 1% fetal calf serum which had been heated at 56’ for 30 minutes. In addition, the 0.1 mM concentration of arginine in Eagle’s original basic medium was increased to a final concentration of 0.8 mM because of the requirement of type 2 adenovirus growth for arginine (Rouse et al., 1963). Tris-buffered saline (TBS): The Trisbuffered saline was prepared according to the procedure described by Winocour (1963). TD: The procedure for making TBS was used, except that CaC12, MgCL, and dextrose were omitted. EDTA: Disodium ethylenediamine tetraacetate was prepared as a 0.02 % solution in TD. Cells. Primary rhesus monkey kidney monolayer cultures were used. Cell suspensions containing approximately 1 X lo6 viable monkey kidney cells per milliliter in
medium containing SVs antiserum were obtained from Microbiological Associates, Bethesda. Approximately 4 to 6 X lo5 cells were placed in a 60-mm plastic petri dish containing 4 ml of CGM. The cultures were incubated at 37” in a humidified 7% COzair mixture for 5 days. After 1 or 2 days of incubation, the CGM was replaced. At 5 days a confluent or almost confluent (about 90% of the surface of the dish was covered) monolayer of cells had formed. ‘c/‘irus. Type 2 adenovirus stock was prepared in KB cells (Microbiological Associates). Approximately 2 X lo6 KB cells were placed in a 60-mm plastic petri dish containing 4 ml of CGM. The dishes were incubated at 37” in a humidified 7% COz-air mixture for 2-3 days, at which time an almost confluent (about 90% of the surface of the dish was covered) monolayer of cells had formed. The monolayers were washed twice with TBS and 0.2 ml of virus suspension at an input multiplicity of approximately 10 was added onto the cell layer. After an adsorption period of 1 hour at 37”, the cells were covered with 2 ml of VGM. After incubation at 37” for 2 days, the infected cells were scraped into TBS, and then the cells were frozen and thawed 3 or 4 times. Cell debris was removed by centrifugation at 800 g for 15 minutes, and the virus-containing supernatant was frozen at -20”. Virus preparations which were purified by density gradient centrifugation in cesium chloride (Green and P&a, 1964) were used in most experiments. Infection of cells. Five-day-old monkey kidney monolayer cultures were washed twice with TBS and 0.2 ml of virus suspension was added onto the cell layer. After adsorption for 60 minutes at 37”, the cells were washed twice with TBS, covered with 2 ml of VGRI, and incubated at 37” in a humidified 7% Con-air mixture for stated times. Control cultures were treated under the same conditions but no virus was added. Multiplicity of infection (PF U/cell). The multiplicity given refers to adsorbed multiplicity as determined by measuring the difference in plaque titer before and after exposure to cells. The number of cells was determined by detaching monolayers with
ADENOVIRUS
AND HOST METABOLIC
EDTA and counting the cells in a hemacytometer. Plaque assay. The plaque assay was carried out with KB cells using a modification of the procedure of Rouse et aZ. (1963). Confluent monolayer cultures of KB cells (obtained 3-4 days after plating 2.5 X lo6 cells in 4 ml of CGM in a 60-mm petri dish) were washed twice with TBS and 0.2 ml of virus suspension was added onto the cell layer. After an adsorption period of 1 hour at 37”, the cells were covered with 5 ml of CGM containing 0.9% agar. The pH was maintained at approximately 7.2. The plates were incubated at 37” in a humidified 7% COZair mixture. After 5 days, each plate received an additional 4 ml of the same medium. After 10 days the plates were overlaid with 4 ml of the same medium, which contained, in addition, 0.0025 % neutral red obtained from J. T. Baker Chemical Co., Phillipsburg, New Jersey). Plaques were counted on the third or fourth day after the neutral red was added. The assay for infective centers. Cultures were infected as described above, washed once with TBS, and then an amount of specific adenovirus type 2 antiserum in excess of the amount necessary to neutralize over 99% of virus was added. The cultures were incubated for 20 minutes at room temperature, and then were washed twice with TD. Single-cell suspensions were prepared by detaching cells from the dish with a mixture containing 0.025% trypsin and 0.01% EDTA (Howes, 1959). Cells were counted with a hemacytometer, and appropriate dilutions were made in TBS containing 0.1% gelatin. Then 0.2 ml of the cell suspension was placed on confluent KB monolayers and allowed to adsorb for 30 minutes at 37”. The monolayers were then gently overlaid with 5 ml of CGM containing 0.9% agar without removing the inoculum. The remaining procedure followed is described in detail under the plaque assay. Protein determination. This was done by the method of Lowry et al. (1951). DNA determination. This was done by the method of Burton (1956). Determination of the rates of synthesis of DNA, RNA, and protein. Aliquots of cell
CHANGES
681
cultures received the following radioactive precursors added to the following final concentrations in a total volume of 2 ml: either thymidine-H3-methyl (5 PC/ml, 1.9 C/ mmole); or uridine-H3 (2 &/ml, 2.9 C/ mmole); or L-leucine-H3 (2 pC/ml, 0.69 C/mmole). The cultures which received the tritiated precursors were incubated for 1 hour. The rate of DNA synthesis was determined from the incorporation of thymidine-H3 into DNA. The cells were washed twice with cold TD, scraped into TD with a rubber policeman, and centrifuged. The cell pellet was extracted three times with cold 0.3 M trichloroacetic acid. The acidinsoluble material was filtered onto Millipore membranes (type HA), placed in 5 ml toluene-scintillator mixture, and counted in a Nuclear Chicago liquid scintillation spectrometer. The rate of RNA synthesis was determined from the incorporation of uridine-H3 into RNA using the method of Reich et al. (1962). Aliquots of an alkaline hydrolyzate (0.5 N KOH; 18 hours, 37”) which had been neutralized with perchloric acid were tested. The rate of prot’ein synthesis was determined from the incorporation of L-leucine-H3 into protein using the technique of McAuslan (196313). DNase activity was determined using the method of Baltimore and Franklin (1962). RNase activity was determined using the method of Baltimore and Franklin (1962). Deoxythymidine kinase activity. Cell extracts were prepared by the method of Dulbecco et al. (1965), and the supernatants were assayed by the procedure of McAuslan (1963a) using H3-thymidine as the substrate. The substrate and the phosphorylated derivatives were isolated by paper chromatography in an isopropanol-NH3 solvent (Chargaff and Davidson, 1955). DNA polymeruse activity. The activity of the DNA polymerase was assayed by measuring the conversion of acid-soluble dATPH3 into an acid-jnsoluble product using essentially the procedure of Keir (1962). The reaction mixture contained, in a total volume of 0.15 ml: 10 pmoles of Tris-HCl buffer, pH 7.5, 1 pmole of MgClz, 0.3 pmole of mercaptoethanol, 0.08 pmole of EDTA, 9 pmoles of KCl, 100 pg of t’hermally de-
682
LEDINKO
natured (100’ for 10 minutes) salmon sperm DNA (obtained from Worthington Biochemical Co.), 50 pmoles each of dCTP, dGTP, dTTP, and dATP-H3 (1.25 C/ mmole), and 0.05 ml of the cell extract (prepared for the deoxythymidine kinase assay) containing 50-100 pg of protein. After incubation at 37” for 30 minutes, the acid-insoluble product was isolated as was described previously (Keir, 1962), and the radioactivity was measured in the Nuclear Chicago liquid scintillation spectrometer. When 100 pg of heat-denatured monkey kidney DNA prepared by the sodium dodecyl sulfate-phenol extraction method of Dulbecco et al. (1965) was used in place of the salmon sperm DNA, identical results were obtained. dCMP deaminase activity. Cell extracts were prepared by the method of Dulbecco et al. (1965). The assay system of Maley and Maley (1962) was used with H3-dCMP as the substrate. dUMP was separated from dCMP by paper chromatography in a solvent consisting of 85 % isopropanol, 15 % 0.3 N HCl. Aspartate transcarbamylase activity. Cell extracts prepared for the thymidine kinase assay were assayed by the method of Bresnick (1962) using DL-(~C’~) aspartic acid as the substrate. The reaction was carried out at pH 8.0, and 0.001 &! mercaptoethanol and 1.1 X 1t4 M EDTA were present in the reaction mixture in addition to the substrates. The reaction was terminated after incubation at 37” for 30 minutes, by boiling for 3 minutes. Ureidosuccinate was separated from aspartate by paper chromatography in a solvent consisting of 60% n-Butanol, 15 % glacial acetic acid, and 25 % water. Aspartate was located by ninhydrin and ureidosuccinate by Ehrlich’s reagent (Fink et al., 1954). DNA-dependent RNA polymerase activity. The activity of the DNA-dependent RNA polymerase was assayed in nuclear fractions using the method of Baltimore and Franklin (1962). The enzyme activity was assayed by measuring the conversion of acid-soluble CTP-H3 into an acid-insoluble product. The reaction mixture, in 0.5 ml, contained 50 pmoles of Tris-HCl buffer, pH 7.9, 1.5
pmoles of MnClz, 10 pmoles of NaF, 2.5 pmoles of mercaptoethanol, 0.05 ml of saturated (room temperature) (NHJzS04, 60 kg each of GTP, UTP, and ATP, 0.05 PC CTP-H3-(NH4)4 (2.5 C/mmole), and 0.2 ml of a nuclear suspension containing 200-600 pg of protein. The assay mixture was incubated for 60 minutes at 37”) and then the acid-insoluble product was isolated, as described previously (Baltimore and Franklin, 1962)) and assayed for radioactivity in a Nuclear Chicago liquid scintillation spectrometer. The product was completely hydrolyzed to trichloroacetic acid-soluble material by treatment with 0.3 N NaOH for 16 hours at 37”) indicating that the product was RNA. For assays in cytoplasmic extracts the procedure of Holland and Peterson (1964) was used. RESULTS
Deoxythymidine Kinase Activity Cultured Cells
of Various
Previous results have indicated that adenovirus infection of KB cells did not result in detectable changes in the activity of deoxythymidine kinase (Green et al., 1964). The possibility existed that virus-mediated changes in deoxythymidine kinase activity might be more readily observed with another host cell which had a lower background level of the enzyme activity. In the following experiment the activity of the deoxythymidine kinase of a number of different kinds of uninfected cells was measured. The activity of deoxythymidine kinase was measured in cell-free extracts prepared from primary cultures of monkey kidney and from the continuous cell lines of FL amnion, KB, and HEp-2. Five-day-old cultures containing confluent monolayers of each cell type were used. It may be seen in Table 1 that the deoxythymidine kinase activity of the extracts of the monkey kidney cultures was approximately one-third to one-fifth of the activity observed in the extracts prepared from the permanent cultures. This finding prompt’ed an investigation of the effects of adenovirus infection on the deoxythymidine kinase activity of monkey kidney cells.
ADENOVIRUS
AND
HOST TABLE
DEOXYTHYMIDINE Expt. no.
KINASE
Cell tested
ACTIVITY
METABOLIC
683
CHANGES
1 OF VARIOUS CULTURED CELLP
Source and type of culture
Age of culture (days)
S.A. of deoxythymidine kinase
1
Monkey kidney FL amnion KB HEp-2
Simian, Human, Human, Human,
primary continuous continuous continuous
5 5 5 5
0.20 1.03 0.60 0.67
2
Monkey kidney FL amnion KB
Simian, primary Human, continuous Human, continuous
5 5 5
0.16 0.89 0.52
Cultures of FL, KB, and HEp-2 (Microbiological Associates) were prepared according to the following procedure. Approximately 2 X lo6 cells were placed in a 60-mm plastic petri dish containing 4 ml of CGM. The cultures were incubated at 37” in a humidified 7% Cog-air mixture. The medium was replaced on the third day. By 5 days the cultures contained 12 to 18 X lo6 cells. Cultures of monkey kidney were prepared by seeding culture dishes with approximately 1 X lo6 cells using the procedure described in Materials and Methods. By 5 days the cultures contained 3 to 5 X lo6 cells. At 5 days each culture had formed a monolayer of cells which covered more than 95y0 of the surface of the dish. At this time 4 cultures of each cell were collected, and extracts of the cells were prepared as described in Materials and Methods. The cell extracts were tested for deoxythymidine kinase activity using the assay described in Materials and Methods. Specific activity @.A.) of deoxythymidine kinase was expressed as counts per minute dTMP per microgram protein. The radioactivity recovered in dTMP was calculated as the percentage of the total radioactivity on the chromatogram found in dTMP and thymidine.
Colony Formation and Infective Center Assays
9 #’
0’
. IO
’ 20
. 30
40
’ 50
. 60
CONTROL
70’
HOURS
FIG. 1. Growth of adenovirus-infected and uninfected monkey kidney cells. Monolayer cultures of monkey kidney were infected as described in Materials and Methods. The multiplicity was 23. Control cells were treated in the same way, but no virus was added. After the final washing, the cells were covered with 4 ml of VGM. At the indicated times, 2-4 cultures were washed with TD. The cells were then detached from the dish with a mixture containing 0.025y0 trypsin and 0.01% EDTA, as recommended by Howes (1959), and counted with a hemacytometer. Approximately 90% of the infected cells were viable at 64 hours as measured by the failure to stain with trypan blue.
Before attempting to study the effects of adenovirus infection on the enzyme activity of monkey kidney cells, it was essential to show that the majority of cells became infected at the time of virus addition. The proportion of cells infected was estimated in two ways. The first method was based on the finding that adenovirus-infected cells did not divide. As shown in Fig. 1, the number of cells in an infected monkey kidney culture remained approximately the same for at least 64 hours, while by this time, the uninfected cell number had increased by a factor of approximately 3. The number of cells infected could, therefore, be estimated from the proportion of cells which were no longer able to form colonies after infection. The second method utilized the assay for infective centers. Both methods have been applied previously to adenovirus-infected KB cells (Green and Daesch, 1961; Green et al., 1964). The results of a representative experiment designed to test the ability of the adenovirusinfected and control monkey kidney cells to
684
LEDINKO TABLE COLONY
FORMATION
OF UNINFECTED
Number of cells plated per dish
Cells
1. Uninfected 2. Infected 3. Uninfected
100 100 100 each
plus infected
2
AND ADENOVIRUS-INFECTED
MONKEY
KIDNEY
Total number of Number of colonies cells plated found per dish 300 300 300 each
40, 43, 47 0, 1, 1 39, 41, 44
CELLS”
Per cent of cells plated which formed colonies 43 0.7 41
0 Monolayer cultures of monkey kidney were infected as described in Materials and Methods. The multiplicity was 16. Control cells were treated in the same way, but no virus was added. At 1 hour after virus was added, several infected and control cultures were washed twice with TBS, and then an amount of specific adenovirus type 2 antiserum in excess of the amount necessary to neutralize over 99% of free virus was added. Cultures were incubated for 20 minutes at room temperature, and then were washed twice with TD. Single-cell suspensions were prepared by detaching cells from the dish with a mixture containing O.O25oj, trypsin and 0.01% EDTA as recommended by Howes (1959). The cells were counted with a hemacytometer. The uninfected cells, the infected cells and a mixture of both, were suspended in 4 ml of Eagle’s medium supplemented with 10% pooled human serum and 3% calf serum (Green et al., 1964), and placed in 60-mm plastic petri dishes. Macroscopic colonies were counted 10-12 days later; 37-46y0 of the uninfected cells formed colonies in different experiments. TABLE GROWTH
3
OF ADENOVIRUS IN CELLSa
MONKEY
KIDNEY
Hours after virus addition
Total PFU produced per cell
l-20 21 22 24 26 28 30 40 50 60 70
<0.003* 0.007 0.009 0.02 0.06 0.14 0.31 7.1 18.1 20.4 20.0
a Monolayer cultures of monkey kidney were infected as described in Materials and Methods. The multiplicity was 12. After the final washing, the cells were covered with 2 ml of VGM. At the indicated times, 3 cultures were collected. The supernatant was removed, and the cells were washed twice with TBS. The cells from each dish were then scraped into 1 ml of TBS with a rubber policeman. The cells were frozen and thawed 4 times, and the supernatant was assayed for intracellular virus. The figure given above for the total PFU found per cell represents the sum of extracellular and intracellular PFU found. b The virus titer found was approximately the same as that of the residual virus remaining after repeated washing of the cells at 1 hour after virus addition.
form colonies are shown in Table 2. At 1 hour after the addition of virus the cells were plated in Eagle’s medium containing 10% pooled human serum which neutralized type 2 adenovirus (Green et al., 1964). It can be seen that 43 % of the uninfected cells formed colonies. In contrast, only 0.7% of the cells taken from cultures 1 hour after virus was added were able to grow. Infection was therefore 98 % complete. When a mixture of uninfected and infected cells was plated, the infected cells did not prevent the growth of the uninfected cells. This finding indicates that possible infection of the originally uninfected cells by virus released from the infected cells was prevented under the above plating conditions. The assay for infective centers was carried out according to the procedure described in the text. When the adsorbed multiplicity was 4 PFU’s per cell, 52-60 % of the cells formed plaques, but at adsorbed multiplicities of 25-40 PFU per cell, only 37-46 % of the cells formed infective centers. The reason for this reduction in the number of plaque formers at higher multiplicities of infection is not known. In all the experiments reported below, infection was at least 90% complete as measured by the failure of the infected cells to form colonies.
ADENOVIRUS
AND
HOST
METABOLIC
685
CHANGES
after infection, and less hours. In other experiments, between 10 and 50 PFU cell at approximately 50 tion.
than 20% at 50 a final yield of was produced per hours after infec-
Deoxythymidine Kinase Activity of Uninfected and Adenovirus-Infected Monkey Kidney Cells
CONTROL
HOURS
FIG. 2. The specific activity of deoxythymidine kinase of adenovirus-infected and uninfected monkey kidney cells at different times after infection. Monolayer cultures of monkey kidney were infected as described in Materials and Methods. The multiplicity was 21. A parallel series of cultures were treated in the same way, but no virus was added. After the final washing, the cells were covered with 2 ml of VGM. At the indicated times cell-free extracts of the uninfected and infected cells were prepared and the deoxythymidine kinase activity was measured. The S. A. of deoxythymidine kinase was expressed as counts per minute dTMP per microgram protein. The radioactivity recovered in dTMP was calculated as the percentage of the total radioactivity found on the chromatogram in dTMP and thymidine. At 50 hours, a total of 28 PFU were produced per cell.
Growth of Adenovirus in Monkey Kidney
Cells
The growth of adenovirus in monkey kidney cells has been measured. The results of a representative experiment are shown in Table 3. The first increase in virus titer was found at approximately 21 hours. At this time 0.607 PFU was produced per cell. The amount of virus produced then increased progressively to reach a yield of 18.1 PFU per cell at 50 hours after infection. At this time virus maturation was complete or virtually complete. At 60 and 70 hours after infection 20 PFU were found per cell. Extracellular virus was found to be less than 5 % of the intracellular virus at 40 hours
The deoxythymidine kinase activity of extracts prepared from the control and adenovirus-infected monkey kidney cells was assayed at different times after infection. It can be seen in Fig. 2 that the activity of deoxythymidine kinase began to increase at approximately 20 hours after infection very shortly before infective virus was first produced. The activity then increased to reach a maximum at 50 hours. At this time the enzyme activity of extracts prepared from infected cells was greater than that of control cell extracts by a factor of approximately 6. A sharp drop in the activity of thymidine kinase occurred after 50 hours. However, even at 70 hours after infection, the activity of the infected cell extracts was approximately threefold higher than that of the uninfected cell extracts. Properties of the Adenovirus-Induced thymidine Kinase
Deoxy-
The possibility existed that the increase in the activity of the deoxythymidine kinase found in adenovirus-infected cells was due to the synthesis of a new enzyme coded by the viral nucleic acid. The virus-induced enzyme might, therefore, have properties which differed from the normal cell enzyme. The thermostability of the deoxythymidine kinase in crude extracts of control cells and cells infected 48 hours was tested by the method of McAuslan (1963b). No difference in thermostability was found. Deoxythymidine kinase obtained from a variety of sources is inhibited by dTT (Bresnick et al., 1964). The possibility existed that the deoxythymidine kinase activity of the uninfected and infected cell extracts was inhibited by different levels of dTTP. The effect of dTTP, as well as of dTDP, on the
686
LEDINKO TABLE
4
INHIBITION OF DEOXYTHYMIDINE KINASE ACTIVITY BY dTTP AND dTDPa
Substance added (mpmoles)
Enzyme source
Ratio of activity of extract in pr;r,;~e sence of thymine derivatives
Uninfected tract
cell
ex-
Infected (48 hours) cell extract
dTTP,
0.1 0.2 0.4 l.Ob
0.78 0.71 0.68 0.65
dTDP,
0.2 1.0 2.0b
0.76 0.68 0.64
dTTP,
0.1 0.2 0.4 l.O*
0.32 0.24 0.18 0.14
dTDP,
0.2 1.0 2.Ob
0.31 0.16 0.13
a Monolayer cultures of monkey kidney were infected as described in Materials and Methods. The multiplicity was 19. A parallel series of cultures were treated in the same way but no virus was added. At 48 hours after infection, cell-free extracts of the uninfected and infected cells were prepared. The extracts were chromatographed on columns of Sephadex G-25 to remove low molecular weight substances. The activity of the deoxythymidine kinase was then measured. The S.A. (cpm dTMP/pg protein) of the infected cell extracts was 0.92, and that of the uninfected cell extracts was 0.18. The radioactivity recovered in dTMP was calculated as the percentage of the total radioactivity found on the chromatogram in dTMP and thymidine. b Concentrations of inhibitor up to 100 mpmoles did not cause any more inhibition of enzyme activity.
activity of the deoxythymidine kinase was tested. Extracts of the infected and uninfected cells were first passed through columns of Sephadex G-25 to remove low molecular weight substances. The results obtained are shown in Table 4. Concen-
trations of dTTP which inhibited the activity of the enzyme of control cell extracts by approximately 30-40% inhibited the enzyme activity of infected cells by approximately SO-90%. When dTDP was tested a similar difference was found in the extent of inhibition of the activity of the enzyme obtained from control and infected cells. E$ects of Puromycin on the Activity of Deoxythymidine Kinase The next experiment was carried out to determine whether the continuous increase in the deoxythymidine kinase activity found after adenovirus infection depended on de novo protein synthesis. The results are shown in Table 5. At 19 hours after infection the ratio of the deoxythymidine kinase activity of the infected cells to that of the control cells was 1.4, and at 24 hours 2.8. At these times puromycin (2 X 1w4 M) was added to aliquots of the infected and uninfected cells. In the absence of puromycin the ratio of the enzyme activity of infected cells to that control cells increased to 4.2 at 42 hours after infection, and to 5.1 at 47 hours. In contrast, when puromycin was added at 19, or at 24 hours after infection, the ratio found at these times remained approximately the same for at least 23 hours. The S. A. of the uninfected cell extracts was approximately the same in the untreated and puromycin-treated cultures. These findings indicate that the continuous increase in deoxythymidine kinase activity in infected cells depended on de novo protein synthesis. Activities of DNA Polymerase, dCMP Deaminase, and Aspartate Transcarbamylase after Adenovirus Infection of Monkey Kidney Cells A considerable increase in the deoxythymidine kinase activity (four to sixfold) always occurred after adenovirus infection of monkey kidney cells. The possibility existed that adenovirus replication was associated with alterations in the activities of other enzymes involved in the processes leading to DNA synthesis. For example, a stimulation of the deoxythymidine kinase, DNA polymerase, and dCMP deaminase activities has been
ADENOVIRUS
AND
HOST
METABOLIC
TABLE
5
THE EFFECT OF PUROMYCIN ON THE DEOXYTHYMIDINE KINASE ACTIVITY ADENOVIRUS-INFECTED MONKEY KIDNEY CELLW No puromycin Hours after infection
19 24 42 47
added
Puromycin
0.28b 0.64 1.09 0.97
Control
0.20 0.23 0.26 0.19
OF UNINFECTED AND
(2 X 1OW M) added
Added at 19 hours Infected
687
CHANGES
Added at 24 hours
‘~~~~~’
1.4 2.8 4.2 5.1
Infected
Control
l~~n~~o~’
Infected
Control
Infected: control
0.28 0.29 0.49 NDC
0.20 0.19 0.22 ND
1.4 1.5 1.8 ND
0.64 0.58 0.60
0.23 0.18 0.20
2.8 3.2 3.0
(1Monolayer cultures of monkey kidney were infected as described in Materials and Methods. The multiplicity was 20. Control cells were treated in the same way, but no virus was added. After the final washing the cells were covered with 2 ml of VGM. At the indicated times, extracts of the infected and uninfectecl cells were prepared. At 19 hours after infection, 0.1 ml of a solution of puromycin hydrochloride (4 X lOwa M) was added to 1.9 ml of the culture medium of aliquots of the infected and uninfected cultures; and to a parallel series of cultures at 24 hours. Extracts of the uninfected and infected added puromycin-treated cultures were prepared at the indicated times. The amount of puromycin was sufficient to inhibit 92% of the protein synthesis, as determined by measuring the uptake of L-leutine-Ha according to the technique described in Materials and Methods. The activity of deoxythymidine kinase was measured and the S.A. of deoxythymidine kinase was expressed as counts per minute dTMP per microgram protein. The radioactivity recovered in dTMP was calculated as the percentage of the total radioactivity on the chromatogram found in dTMP and deoxythymidine. b Values indicate specific activity of deoxythymidine kinase of cell extracts. c ND, not done.
observed in polyoma-infected mouse kidney cells (Dulbecco et al., 1965). Moreover, an increa.se in the activity of aspartate transcarbamylase has been reported in type 5 adenovirus-infected HeLa cells (Consigli and Ginsberg, 1964). In the following experiment, the activities of deoxythymidine kinase, DNA polymerase, dCMP deaminase, and aspartate transcarbamylase were measured in adenovirusinfected monkey kidney cells at different times after infection. The results of a representative experiment are given in Table 6. The deoxythymidine kinase activity of the infected cells was approximately fourfold higher than that of the uninfected cells at 47 hours after infection, and approximately threefold higher at 41 and 64 hours. The activities of dCMP deaminase and aspartate transcarbamylase of the infected cell extracts did not differ more than twofold from those of the control cells. However, at 40-60 hours after infection, the dCMP deaminase and aspartate transcarbamylase activities were consistently greater than those of the control cells by a factor of 1.6-2.
In contrast, the DNA polymerase activity wasnot significantly increased at 20-70 hours after infection. No difference in the DNA polymerase activity between adenovirus type 2-infected Kl3 cells and uninfected cells has been found by Green et al. (1964). The Rates of DNA, RNA, and Protein Synthesis in Adenovirus-Injected Monkey Kidney Cells The results of the preceding experiment indicated that the activity of deoxythymidine kinase was considerably increased after adenovirus infection of monkey kidney cells, but that the activity of DNA polymerase was not demonstrably changed. These findings were then related to measurements of the rates of incorporation of H3-thymidine into the DNA of infected cells. At the same time, the rates of incorporation of H3uridine into RNA and of H3-leucine into protein were also measured. As may be seen in Fig. 3, the rates of incorporation per cell of precursors into the DNA, RNA, and protein of the uninfected cells decreased slowly but steadily during a
688
LEDINKO TABLE
6
ACTIVITIES OF DEOXYTHYMIDINE KINASE, DNA POLYMERASE, dCMP DEAMINASE, TRANSCARBAMYLASE AFTER ADENOVIRUS INFECTION OF MONKEY KIDNEY
THE
DNA polymerase dCMP deaminase Aspartate Deoxythymidine kinase Hours after Con- Infected: yF$- ‘~~~t~o~’ Infected ‘On- Infected’ Infected a~id~~on Infected trol control Infected trol control 2 17 24 41 47 64 71
0.33” 0.27 0.46 0.81 1.20 0.75 0.57
0.30 0.30 0.29 0.29 0.28 0.26 0.26
1.1 0.9 1.6 2.8 4.3 2.9 2.2
12.6 8.5 14.1 10.0 14.4 10.3 7.3
14.0 12.2 12.8 11.1 11.1 10.3 10.4
0.9 0.7 1.1 0.9 1.3 1.0 0.7
0.40 0.30 0.35 0.24 0.24 0.13 0.09
0.40 0.38 0.23 0.14 0.12 0.08 0.07
1.0 0.8 1.5 1.7 2.0 1.6 1.3
AND ASPARTATE CELLS”
transcarbamylase Control
Infected. control’
0.008 0.008 0.017 0.020 0.020 0.019 0.018
1.1 1.1 1.3 1.9 1.8 1.6 1.5
0.009 0.01 0.022 0.038 0.036 0.030 0.027
a Monolayer cultures of monkey kidney were infected as described in Materials and Methods. The multiplicity was 10. A parallel series of cultures were treated in the same way, but no virus was added. At the indicated times, extracts of the infected and uninfected cells were prepared, and the enzyme activities were measured. The S.A. of deoxythymidine kinase was expressed as cpm dTMP/pg protein. The radioactivity recovered in dTMP was calculated as the percentage of the total radioactivity found on the chromatogram in dTMP and deoxythymidine. The S.A. of DNA polymerase was expressed as cpm acid-insoluble product/pg protein. The S.A. of dCMP deaminase was expressed as cpm dUMP/pg protein. The radioactivity recovered in dUMP was calculated as the percentage of the total radioactivity found on the chromatogram in dUMP and dCMP. The S.A. of aspartate transcarbamylase was expressed as cpm ureidosuccinate/pg protein. The radioactivity found in ureidosuccinate was calculated as the percentage of the total radioactivity found in ureidosuccinate and aspartate. b Values indicate specific activity of cell extracts. H3 Uridine
H3 Leucine
H3 Thymidine
50
h \ A;,,
0
0
IO
20
0 .
30
40
50
60
lb
20
30 40 HOURS
50
$0
lb
2.0
30
40
50
$0
FIG. 3. The effect of adenovirus infection on the rates of synthesis of DNA, RNA, and protein in monkey kidney cells. Monolayer cultures of monkey kidney were infected as described in Materials and Methods. The multiplicity was 22. A parallel series of cultures were treated in the same way, but no virus was added. At the indicated times aliquots of the infected and uninfected cell cultures were subjected to l-hour pulses of the radioactive precursors and the rates of DNA, RNA, and protein synthesis were determined as described in Materials and Methods. Abscissa: period of incubation after infection (hours). Ordinate: rate of protein synthesis: cpm L-leucine-H3 incorporated/l04 cells/hour. Rate of RNA synthesis: cpm uridine-H3 incorporated/l03 cells/hour. Rate of DNA synthesis: cpm thymidine-H3 incorporated/l03 cells/hour.
ADE~~~~IR~JS TABLE
AND HOST METABOLIC
7
TWE INCOWORATION ok THPMIDINE-Ha FOR 60 MINUTES INTO TI-KE DNA AND ACID-S• LUXUZ FRACTIONS OF AD~NOYIR~~-I~F~C~~~ AND UNINFECTED
%fOMKEY
Hours after virus addition 2 16 19 22 40 50 60
KIDNEY
CELLS”
Ratio, cpm infected cell fraction: cpm uninfected ceil -fraction --.-_I Nucleic acid Acid-soluble pool 1.12 0.94 0.92 1.35 0.82 0.68 0.60
ND ND 0.98 1.13 X.24 0.90 0.85
CHANGES
689
cursors into RKA and protein was approximately 5 7%or less of the original rate. The effect of adenovirus infection on the rate of thyn~idi~e incorporation into the acid-solubIo fraction of monkey kidney cells IOC
SC
BC
70
I+----.1 I tntracellular
a Monolayer cultures of monkey kidney cells were infected as described in Materials and Methods. The multiplicity was 25. Control cells were t,reated in the same way, but no virus was added. At the indicated times the medium of replicate cultures of the infected and uninfected cells was decanted a& 2 ml of VGM containing thymi-
dine-J.%3 (5 MC/ml, 1.9 C/mmole) was added to each culture. At 30, and again at 60, minutes of incubation, cells were washed twice with cold TD, scraped from the culture dish into TD with a rubber policeman, and centrifuged. The cell pellet was extracted three times with cold 0.3 M trichloroaeetic a.cid. The pooled superuatants from these extract,ions represented the acid-soluble pool. The pellets were further extracted with 0.3 M trichloroacetic acid at 90” for 15 mil~utes to give the m&eic acid fraction. The radioactivity of the acid-soluble fraction was similar at 30 and 60 minutes, indicating that the pool was rapidly saturated with th~~idil~e. The results are given as the ratios of the counts per minute incorporated in the infected cells to that incorporated in the uninfected cells.
period of 65 hours. At 65 hours the incorporation of precursors was approximately 40-50% of t,he original rate. The rate of incorporation per cell of ~3-thy~~ne into the DNA of infected cdls declined to approxi~tely the same extent as that of the uninfected cells. In striking contrast, the incorporations of II”-uridine into the RNA and of IWeucine into protein were much more severely ~hibit~ in the infected cells than in the controls. At 65 hours after aection the rate of incorporation of the pre-
Virus
formed
of Infected
Nuclei
1
20
IO
f
_..,,... 4 20
40
60
30
HOURS
FIG. 4. Adenovir~~s formation and the time course of the decrease in the DNA-dep~udent RNA polymerase activity in infected monkey kidney cells. Monolayer cultures of monkey kidney were infected as described in materials and Methods. The m~lltiplicit~ was 18. Control cultures were treated in the same way, but no virus was added. After the final washing, the cultures were covered with 2 ml of VGM. At the indicated times, S-10 replicate cultures of the infected and uninfected cells were removed, and the cell nuclei were isoIated. The activity of the ~NA-dependent RNA pol~erase was assayed in the nuclear fractions. The results are expressed as the percentage of the S.A. of the enzyme found at the time of virus addition. At this time t,he S.A. of the RNA polymerase, expressed as counts per minute CTP-Ha incorporated into acid-insoluble material per microgram of protein, was 6.7, Intracellular virus growth was determined using the method described in Table 3. The results are expressed as the percentage of the PFU produced per cell at 70 hours. At this t,ime 36 PFXJ per eelI were found.
690
LEDINKO
was also investigated. It may be seen in Table 7 that, as with the incorporation of thymidine into the DNA, the rate of incorporation of thymidine into the acid-soluble fraction of infected cells was similar to that of the uninfected cells. The results of preliminary experiments have indicated that the rates of incorporation of uridine and leucine into the acid-soluble pool of infected cells were also similar to those of the control cells. Activity of DNA-Primed RNA after Adenovirus Infection Kidney Cells
Polymerase of Monkey
The possibility existed that the marked inhibition of RNA and protein synthesis observed in infectNed monkey kidney cells was accompanied by a decline in the activity of the DNA-directed RNA polymerase. This enzyme is believed to play a key role in protein synthesis. The RNA polymerase activity of nuclei isolated from adenovirusinfected and control monkey kidney cells at different times after infection is shown in Fig. 4. A decrease in the activity of RNA polymerase of the infected cells began at approximately 15 hours after infection. Less than half of bhe initial activity was found at 20 hours after infection, and only 25 % of the activity remained at 24 hours. This level was maintained up to 60-70 hours after infection. The activity of the RNA polymerase of infected cells was related to the time course of intracellular virus formation (Fig. 4). At 24 hours when only 25% of the original activity of RNA polymerase remained, less than 0.1% of the progeny infective virus had been formed. DISCUSSION
One effect of type 2 adenovirus infection uncovered in this study is the inhibition of the activity of the host cell DNA-dependent RNA polymerase. This inhibition occurred before virus maturation began. At the time when maturation began, the level of the RNA polymerase activity was only between 20 and 30% of that of the uninfected cell. A loss of the RNA polymerase activity has also been found to occur after infection of Escherichia coli with the DNA-containing phage T4 (Skold and Buchanan, 1964), and after infection of L cells with the RNA-
containing mengovirus (Baltimore and Franklin, 1962). The mechanism of the inhibition of RNA polymerase activity is not known. An attempt was made to examine the possibility that the inhibition of the activity of the RNA polymerase in the nuclei isolated from adenovirus-infected cells was not related to virus synthesis. The inhibition might have been due to degradation of the DNA template or of the RNA product. The activities of the DNase and RNase of the nuclei prepared from control cells and cells infected 48 hours were, therefore, measured. No DNase activity was found, and the small amount of RNase activity observed was approximately the same in the control and infected cell nuclear fractions. Other findings also suggested that degradation of the primer DNA in infected cells did not occur. The ratios of DNA to protein in the nuclei of control cells and cells infected 48 hours were similar. In addition, the addition of 100 to 200 pugof highly polymerized salmon sperm DNA to the assay system did not increase the activity of the RNA polymerase in the control and infected cell nuclear fractions. The possibility also existed that the nuclei prepared from the infected cells were very fragile and that the RNA polymerase had leaked out. However, no activity was found in the cytoplasmic fraction. Moreover, the DNA polymerase activity of the nuclei isolated from the infected cells was of the same order of magnitude as that of the nuclei prepared from the control cells. The question arises of the relevance of the marked inhibition of RNA and protein synthesis to the biosynthesis of adenovirus occurring in the monkey kidney cells. The total yield of adenovirus found was between 10 to 50 PFU per cell. This yield is considerably lower than was found using the permanent KB cells where approximately 10,000 PFU were produced per cell (Green, 1962). Furthermore, the first detectable increase in intracellular virus in monkey kidney cells was found at approximately 21 hours after infection, whereas maturation in the KB cells started at 13-14 hours after infection (Green, 1962). Adenovirus replication in the KB cell does not inhibit macromolecular synthesis until late
ADENOVIRUS
AND
HOST
in the infectious process. The possibility existed that the early inhibition of the RNA and protein synthesis observed in the infected monkey kidney cells was unrelated to virus synthesis. It was possible, for example, that the protein factor present in adenovirus lysates which causes the early cytopathic effect (Pereira, 1958) was also responsible for the inhibition of the RNA and protein synthesis. This possibility was eliminated by the use of virus purified by density gradient centrifugation in cesium chloride. No inhibition was observed when cells were treated with extracts prepared from uninfected cells. It appears therefore very likely that the inhibition of the cell RNA and protein synthesis is a function of the adenovirus. Several possibilities exist to account for the differences noted between the previous results by others and the findings reported here. Aside from differences in virus types and media, possibly the outstanding difference is in the kind of cell type tested. We chose to work with primary cultures derived from normal tissue, whereas previous studies were carried out with permanent cell lines. The choice of monkey kidney cells was initially dictated by the finding that these cells in confluent monolayers have consistently low background levels of deoxythymidine kinase activity. Cells of permanent lines, on the contrary, tend to have a high level of deoxythymidine kinase activity at high cell density (data given, and unpublished observations). It is conceivable that in the established cell lines a loss in metabolic control systems has occurred which permits high enzymatic and metabolic activity at high cell density. What relationship, if any, exists between this possible loss in regulatory activity and the failure of adenovirus infection to produce any marked early effects on the machinery of the permanent cell is not known. It is of considerable interest that the activity of the deoxythymidine kinase was induced after adenovirus infection. The activity of this enzyme began to increase at approximately the time when virus maturation started. When maturation was complete, or almost complete, the deoxythymidine kinase activity was four- to sixfold
METABOLIC
CHANGES
691
higher than that of the control cells. The increase in the enzyme activity depended on de novo protein synthesis. It is possible that the observed increase of the deoxythymidine kinase activity resulted from the synthesis of a new enzyme induced by adenovirus, the synthesis of a latent host enzyme, or simply from an increase in the rate of synthesis of the regular host enzyme. The significance of the enhanced deoxythymidine kinase activity in adenovirus infected monkey kidney cells is not known. Note added in proof. After this manuscript was submitted for publication, it was reported that the activity of deoxythymidine kinase was enhanced after infection of green monkey kidney cells by simian adenoviruses, simian papovavirus SV40, and an adenovirus-SV40 “hybrid”. (Kit, S., Dubbs, D. R., deTorres, R. A., and Melnick, J. L., Virology 27,453-457 (1965).) ACKNOWLEDGMENTS The author is indebted to Dr. R. Dulbecco for valuable advice and to Dr. J. Buchanan for his interest and helpful suggestions. This investigation was supported by a Public Health Service Special Research Fellowship from the National Institute of General Medical Sciences to the author, and by a Research Grant from the Public Health Service, National Institutes of Health, No. CA-07592. REFERENCES BALTIMORE, D., and FR.~NKLIN, R. M. (1962). The effect of mengovirus infection on the activity of the DNA dependent RNA polymerase of n-cells. Proc. Natl. Acad. Sci. U.S. 48, 13831390. BRANDON, F. B., and MCLEBN, I. W. (1962). Adenovirus. Advan. Virus Res. 9, 157-193. BRESNICK, E. (1962). Feedback inhibition by deoxyribonucleotides of aspartate transcarbamylase activity in liver preparations. Biochim. Biophys. Acta 61, 598605. BRESNICK, E., THOMPSON, U.B., MORRIS, H.P., and LIEBELT, A. G. (1964). Inhibition of thymidine kinase activity in liver and hepatomas by TTP and d-CTP. Biochem. Biophys. Res. Commun. 16, 278-284. BURTON, K. (1956). A study of the conditions and mechanisms of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-323. CHARG~FF, E., and DAVIDSON, J. N. (1955). In
692
LEDINKO
“the Nucleic Acids” (E. Chargaff and J. N. Davidson, eds.), Vol. 1, p. 252. Academic Press, New York. CONSIGLI, R. A., and GINSBERG, H. S. (1964). Activity of aspartate transcarbamylase in uninfected and type 5 adenovirus-infected HeLa cells. J. Bacterial. 87, 1034-1043. DULBECCO, R., H‘IRT\~ELL, L. H., and VOGT, M. (1965). Induction of cellular DNA synthesis by polyoma virus. Proc. Natl. Acad. Sci. U.S. 53, 403410. FINK, R. M., CLINE, R. E., and KOCH, H. M. G. (1954). Chromatographic detection of pyrimidine reduction products: microbiological application. Federation Proc. 13, 207-208. GINSBERG, H. S., and DIXON, M. K. (1959). Deoxyribonucleic acid (DNA) and protein alterations in HeLa cells infected with type 4 adenovirus. J. Esptl. Med. 109, 407422. GREEN, M. (1959). Biochemical studies on adenovirus multiplication. I. Stimulation of phosincorporation into deoxyribonucleic phorus acid and ribonucleic acid. Virology 9, 343-358. GREEN, M. (1962). Studies on the biosynthesis of viral DNA. Cold Spring Harbor Symp. Quant. Biol. 27, 219-235. GREEN, M., and DAESCH, G. E. (1961). Biochemical studies on adenovirus multiplication. II. Kinetics of nucleic acid and protein synthesis in suspension cultures. Virology 13, 169-176. GREEN, M., and P&A, M. (1964). Biochemical multiplication. VI. studies on adenovirus Properties of highly purified tumorigenic huand their DNA’S, Proc. man adenoviruses, Natl. Acad. Sci. U.S. 51, 1251-1259. GREEN, M., PII~A, M., and CHAGOYA, V. (1964). Biochemical studies on adenovirus multiplication. V. Enzymes of deoxyribonucleic acid synthesis in cells infected by adenovirus and vaccinia virus. J. Biol. Chem. 239, 1188-1197. HOLLAND, J. J., and PETERSON, J. A. (1964). Nucleic acid and protein synthesis during poliovirus infection of human cells. J. Mol. Biol. 8, 556-573. HOWES, D. W. (1959). The growth cycle of poliovirus in cultured cells. II. Maturation and release of virus in suspended cell populations. Virology 9, 96-109. KEIR, H. M. (1962). Stimulation and inhibition of deoxyribonucleic acid nucleotidyltransferase by oligodeoxyribonucleotides. Biochem. d, 85, 265-276. KIT, S., and DOBBS, D. R. (1963). Acquisition of thymidine kinase activity by herpes simplex infected mouse fibroblast cells. B&c&m. Biophys. Res. Commun. 11, 55-53.
KIT, S., DUBBS, D. R., and PIEKARSKI, L. J. (1962). Enhanced thymidine phosphorylating activity of mouse fibroblasts (strain L-M) following vaccinia infection. Biochem. Biophys. Res. Commun. 8, 72-75. KIT, S., PIEKARSKI, L. J., and DUBBS, D. It. (1963). Induction of thymidine kinase by vaccinia-infected mouse fibroblasts. J. Mol. Biol. 6, 22-33. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275. MCAUSLAN, B. R. (1963a). Control of induced thymidine kinase activity in the poxvirusinfected cell. Virology 20, 162-168. MCAUSLAN, B. R. (1963b). The induction and repression of thymidine kinase in the poxvirusinfected HeLa cell. Virology 21, 383-389. MALEY, G. F., and MALEY, F. (1962). Nucleotide interconversions. J. Biol. Chem. 237, PC3311PC3314. NOHARA, H., and KAPLAN, A. S. (1963). DNAsynthesizing enzymes in pseudorabies virusinfected rabbit kidney cells. Federation Proc. 22, 615. PEREIRA, H. G. (1958). A protein factor responsible for the early cytopathic effect of adenoviruses. Virology 6, 601-611. POLASA, H., and GREEN, M. (1965). Biochemical studies on adenovirus multiplication. VIII. Analysis of protein synthesis. Virology 25, 68-79. REICH, E., FRANKLIN, R. M., SHATKIN, A. J., and TATUM, E. L. (1962). Action of actinomycin D on animal cells and viruses. Proc. N&Z. Acad. Sci. U.S. 48, 1238-1245. ROUSE, H. C., BONIFAS, V. H., and SCHLESINGER, R. W. (1963). Dependence of adenovirus replication on arginine and inhibition of plaque formation by pleuropneumonia-like organisms. Virology 20, 357-365. SKOLD, O., and BUCHANAN, J. M. (1964). Inhibition of deoxyribonucleic acid-directed ribonucleic acid polymerase in Escherichia coli after infection with bacteriophage T4. Proc. Natl. Acad. Sci. U.S. 51, 553360. VOGT, M., and DULBECCO, R. (1963). Steps in the neoplastic transformation of hamster embryo cells by polyoma virus. Proc. Natl. Acad. Sci. U.S. 49, 171-179. WILCOX, W. C., and GINSBERG, H. S. (1963). Protein synthesis in type 5 adenovirus infected cells. Effect of p-fluorophenylalanine on synthesis of protein, nucleic acids, and infectious virus. Virology 20, 269-280. WINOCOUR, E. (1963). Purification of polyoma virus. Virology 19, 158-168.
28, 679-692 (1966)
Changes
in Metabolic
and
Ceils after
Enzymatic Infection
NADA The Salk Institute
Activities
with
of Monkey
Adenovirus
Kidney
2
LEDINKO’
for Biological
Studies,
San Diego,
California
Accepted December 16, 1965 Primary cultures of monkey kidney were infected with type 2 adenovirus. The formation of infective virus was first detected at approximately 21 hours after infection. Final yields of l&50 plaque forming units per cell were obtained at approximately 50 hours. The rate of DNA synthesis, as measured by the rate of incorporation of H3-thymidine into DNA, did not change after infection. The rates of incorporation of H3-uridine into RNA, and of Ha-leucine into protein, however, were markedly inhibited in infected cells before infective virus appeared. A striking decrease in the activity of the DNA-dependent RNA polymerase also occurred before virus maturation began. The effects of type 2 adenovirus infection of monkey kidney cells on the activities of deoxythymidine kinase, DNA polymerase, deoxycytidylate deaminase, and aspartate transcarbamylase were investigated. The deoxythymidine kinase activity began to increase at approximately 20 hours after infection, and at 50 hours the activity was four- to sixfold higher than that of the uninfected cells. At 4@60 hours, the deoxycytidylate deaminase and aspartate transcarbamylase activities were slightly but consistently greater than those of control cells by a factor of 1.6-2. The DNA polymerase activity was not changed. The increase in the deoxythymidine kinase activity induced after infection depended on de novo protein synthesis. The induced deoxythymidine kinase activity of the infected cells was more sensitive to feedback inhibition by dTDP, and dTTP, than the enzyme activity of the control cells. INTRODUCTION
Adenovirus is a DNAZ-containing virus which multiplies in the nucleus of the cell r Present address: Putnam Memorial Hospital Institute for Medical Research, Bennington, Vermont 05201. z Abbreviations: DNA, deoxyribonucleic acid; RNA, ribonucleic acid; dCMP, deoxycytidine monophosphate; dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosdTDP, deoxythymidine diphosphate; phate; dTTP, deoxythymidine triphosphate; dATP, deoxyadenosine triphosphate; dGTP, deoxyguanosine triphosphate; dCTP, deoxycytidine triphosphate; ATP, adenosine triphosphate; GTP, guanosine triphosphate; UTP, uridine triphosphate; CTP, cytidine triphosphate; RNase, ribonuclease; DNase, deoxyribonuclease; S.A., specific activity; PFU, plaque-forming unit; cpm, counts per minute; CGM, cell growth medium. 679
(Brandon and McLean, 1962). Observations on the metabolism of adenovirus-infected KB and HeLa cells have indicated that the synthesis of DNA, RNA, and protein continues after infection (Ginsberg and Dixon, 1959; Green, 1962; Wilcox and Ginsberg, 1963; Polasa and Green, 1965). However, the synthesis of these macromolecules dimishes late in the infectious process when replication is complete or almost complete (Green, 1959; Green and Daesch, 1961; Green et nl., 1964). The possibility that the replication of adenovirus DNA is mediated by virusinduced changes in the DNA synthesizing enzymes has been investigated. No appreciable change in the activities of the enzymes involved in DNA synthesis has been found in adenovirus-infected cells (Green et al., 1564). In contrast, increases in some of the
680
LEDINKO
DNA synthesizing enzymes have been induced after infection with a number of other DNA-containing animal viruses. For example, there is a marked increase in the activity of deoxythymidine kinase in cells infected with vaccinia (Kit et al., 1962, 1963; McAuslan, 1963a), herpes simplex (Kit and Dubbs, 1963), pseudorabies (Nohara and Kaplan, 1963), and polyoma (Dulbecco et al., 1965) viruses. In the present study, we have observed the induction of the deoxythymidine kinase activity of monkey kidney cells after adenovirus infection. Using this host cell, we have found that the rate of synthesis of host cell RNA and protein was inhibited soon after infection, but the rate of DNA synthesis was unaffected. The inhibition of RNA and protein synthesis was accompanied by a striking decrease in the activity of the DNAdependent RNA polymerase. MATERIALS
Solutions
AND
METHODS
and Media
Cell growth medium (CGM): Cultures were grown in reinforced Eagle’s medium (Vogt and Dulbecco, 1963) containing 5% fetal calf serum. Virus growth medium (VGM): Virus growth medium consisted of reinforced Eagle’s medium (Vogt and Dulbecco, 1963) containing 1% fetal calf serum which had been heated at 56’ for 30 minutes. In addition, the 0.1 mM concentration of arginine in Eagle’s original basic medium was increased to a final concentration of 0.8 mM because of the requirement of type 2 adenovirus growth for arginine (Rouse et al., 1963). Tris-buffered saline (TBS): The Trisbuffered saline was prepared according to the procedure described by Winocour (1963). TD: The procedure for making TBS was used, except that CaC12, MgCL, and dextrose were omitted. EDTA: Disodium ethylenediamine tetraacetate was prepared as a 0.02 % solution in TD. Cells. Primary rhesus monkey kidney monolayer cultures were used. Cell suspensions containing approximately 1 X lo6 viable monkey kidney cells per milliliter in
medium containing SVs antiserum were obtained from Microbiological Associates, Bethesda. Approximately 4 to 6 X lo5 cells were placed in a 60-mm plastic petri dish containing 4 ml of CGM. The cultures were incubated at 37” in a humidified 7% COzair mixture for 5 days. After 1 or 2 days of incubation, the CGM was replaced. At 5 days a confluent or almost confluent (about 90% of the surface of the dish was covered) monolayer of cells had formed. ‘c/‘irus. Type 2 adenovirus stock was prepared in KB cells (Microbiological Associates). Approximately 2 X lo6 KB cells were placed in a 60-mm plastic petri dish containing 4 ml of CGM. The dishes were incubated at 37” in a humidified 7% COz-air mixture for 2-3 days, at which time an almost confluent (about 90% of the surface of the dish was covered) monolayer of cells had formed. The monolayers were washed twice with TBS and 0.2 ml of virus suspension at an input multiplicity of approximately 10 was added onto the cell layer. After an adsorption period of 1 hour at 37”, the cells were covered with 2 ml of VGM. After incubation at 37” for 2 days, the infected cells were scraped into TBS, and then the cells were frozen and thawed 3 or 4 times. Cell debris was removed by centrifugation at 800 g for 15 minutes, and the virus-containing supernatant was frozen at -20”. Virus preparations which were purified by density gradient centrifugation in cesium chloride (Green and P&a, 1964) were used in most experiments. Infection of cells. Five-day-old monkey kidney monolayer cultures were washed twice with TBS and 0.2 ml of virus suspension was added onto the cell layer. After adsorption for 60 minutes at 37”, the cells were washed twice with TBS, covered with 2 ml of VGRI, and incubated at 37” in a humidified 7% Con-air mixture for stated times. Control cultures were treated under the same conditions but no virus was added. Multiplicity of infection (PF U/cell). The multiplicity given refers to adsorbed multiplicity as determined by measuring the difference in plaque titer before and after exposure to cells. The number of cells was determined by detaching monolayers with
ADENOVIRUS
AND HOST METABOLIC
EDTA and counting the cells in a hemacytometer. Plaque assay. The plaque assay was carried out with KB cells using a modification of the procedure of Rouse et aZ. (1963). Confluent monolayer cultures of KB cells (obtained 3-4 days after plating 2.5 X lo6 cells in 4 ml of CGM in a 60-mm petri dish) were washed twice with TBS and 0.2 ml of virus suspension was added onto the cell layer. After an adsorption period of 1 hour at 37”, the cells were covered with 5 ml of CGM containing 0.9% agar. The pH was maintained at approximately 7.2. The plates were incubated at 37” in a humidified 7% COZair mixture. After 5 days, each plate received an additional 4 ml of the same medium. After 10 days the plates were overlaid with 4 ml of the same medium, which contained, in addition, 0.0025 % neutral red obtained from J. T. Baker Chemical Co., Phillipsburg, New Jersey). Plaques were counted on the third or fourth day after the neutral red was added. The assay for infective centers. Cultures were infected as described above, washed once with TBS, and then an amount of specific adenovirus type 2 antiserum in excess of the amount necessary to neutralize over 99% of virus was added. The cultures were incubated for 20 minutes at room temperature, and then were washed twice with TD. Single-cell suspensions were prepared by detaching cells from the dish with a mixture containing 0.025% trypsin and 0.01% EDTA (Howes, 1959). Cells were counted with a hemacytometer, and appropriate dilutions were made in TBS containing 0.1% gelatin. Then 0.2 ml of the cell suspension was placed on confluent KB monolayers and allowed to adsorb for 30 minutes at 37”. The monolayers were then gently overlaid with 5 ml of CGM containing 0.9% agar without removing the inoculum. The remaining procedure followed is described in detail under the plaque assay. Protein determination. This was done by the method of Lowry et al. (1951). DNA determination. This was done by the method of Burton (1956). Determination of the rates of synthesis of DNA, RNA, and protein. Aliquots of cell
CHANGES
681
cultures received the following radioactive precursors added to the following final concentrations in a total volume of 2 ml: either thymidine-H3-methyl (5 PC/ml, 1.9 C/ mmole); or uridine-H3 (2 &/ml, 2.9 C/ mmole); or L-leucine-H3 (2 pC/ml, 0.69 C/mmole). The cultures which received the tritiated precursors were incubated for 1 hour. The rate of DNA synthesis was determined from the incorporation of thymidine-H3 into DNA. The cells were washed twice with cold TD, scraped into TD with a rubber policeman, and centrifuged. The cell pellet was extracted three times with cold 0.3 M trichloroacetic acid. The acidinsoluble material was filtered onto Millipore membranes (type HA), placed in 5 ml toluene-scintillator mixture, and counted in a Nuclear Chicago liquid scintillation spectrometer. The rate of RNA synthesis was determined from the incorporation of uridine-H3 into RNA using the method of Reich et al. (1962). Aliquots of an alkaline hydrolyzate (0.5 N KOH; 18 hours, 37”) which had been neutralized with perchloric acid were tested. The rate of prot’ein synthesis was determined from the incorporation of L-leucine-H3 into protein using the technique of McAuslan (196313). DNase activity was determined using the method of Baltimore and Franklin (1962). RNase activity was determined using the method of Baltimore and Franklin (1962). Deoxythymidine kinase activity. Cell extracts were prepared by the method of Dulbecco et al. (1965), and the supernatants were assayed by the procedure of McAuslan (1963a) using H3-thymidine as the substrate. The substrate and the phosphorylated derivatives were isolated by paper chromatography in an isopropanol-NH3 solvent (Chargaff and Davidson, 1955). DNA polymeruse activity. The activity of the DNA polymerase was assayed by measuring the conversion of acid-soluble dATPH3 into an acid-jnsoluble product using essentially the procedure of Keir (1962). The reaction mixture contained, in a total volume of 0.15 ml: 10 pmoles of Tris-HCl buffer, pH 7.5, 1 pmole of MgClz, 0.3 pmole of mercaptoethanol, 0.08 pmole of EDTA, 9 pmoles of KCl, 100 pg of t’hermally de-
682
LEDINKO
natured (100’ for 10 minutes) salmon sperm DNA (obtained from Worthington Biochemical Co.), 50 pmoles each of dCTP, dGTP, dTTP, and dATP-H3 (1.25 C/ mmole), and 0.05 ml of the cell extract (prepared for the deoxythymidine kinase assay) containing 50-100 pg of protein. After incubation at 37” for 30 minutes, the acid-insoluble product was isolated as was described previously (Keir, 1962), and the radioactivity was measured in the Nuclear Chicago liquid scintillation spectrometer. When 100 pg of heat-denatured monkey kidney DNA prepared by the sodium dodecyl sulfate-phenol extraction method of Dulbecco et al. (1965) was used in place of the salmon sperm DNA, identical results were obtained. dCMP deaminase activity. Cell extracts were prepared by the method of Dulbecco et al. (1965). The assay system of Maley and Maley (1962) was used with H3-dCMP as the substrate. dUMP was separated from dCMP by paper chromatography in a solvent consisting of 85 % isopropanol, 15 % 0.3 N HCl. Aspartate transcarbamylase activity. Cell extracts prepared for the thymidine kinase assay were assayed by the method of Bresnick (1962) using DL-(~C’~) aspartic acid as the substrate. The reaction was carried out at pH 8.0, and 0.001 &! mercaptoethanol and 1.1 X 1t4 M EDTA were present in the reaction mixture in addition to the substrates. The reaction was terminated after incubation at 37” for 30 minutes, by boiling for 3 minutes. Ureidosuccinate was separated from aspartate by paper chromatography in a solvent consisting of 60% n-Butanol, 15 % glacial acetic acid, and 25 % water. Aspartate was located by ninhydrin and ureidosuccinate by Ehrlich’s reagent (Fink et al., 1954). DNA-dependent RNA polymerase activity. The activity of the DNA-dependent RNA polymerase was assayed in nuclear fractions using the method of Baltimore and Franklin (1962). The enzyme activity was assayed by measuring the conversion of acid-soluble CTP-H3 into an acid-insoluble product. The reaction mixture, in 0.5 ml, contained 50 pmoles of Tris-HCl buffer, pH 7.9, 1.5
pmoles of MnClz, 10 pmoles of NaF, 2.5 pmoles of mercaptoethanol, 0.05 ml of saturated (room temperature) (NHJzS04, 60 kg each of GTP, UTP, and ATP, 0.05 PC CTP-H3-(NH4)4 (2.5 C/mmole), and 0.2 ml of a nuclear suspension containing 200-600 pg of protein. The assay mixture was incubated for 60 minutes at 37”) and then the acid-insoluble product was isolated, as described previously (Baltimore and Franklin, 1962)) and assayed for radioactivity in a Nuclear Chicago liquid scintillation spectrometer. The product was completely hydrolyzed to trichloroacetic acid-soluble material by treatment with 0.3 N NaOH for 16 hours at 37”) indicating that the product was RNA. For assays in cytoplasmic extracts the procedure of Holland and Peterson (1964) was used. RESULTS
Deoxythymidine Kinase Activity Cultured Cells
of Various
Previous results have indicated that adenovirus infection of KB cells did not result in detectable changes in the activity of deoxythymidine kinase (Green et al., 1964). The possibility existed that virus-mediated changes in deoxythymidine kinase activity might be more readily observed with another host cell which had a lower background level of the enzyme activity. In the following experiment the activity of the deoxythymidine kinase of a number of different kinds of uninfected cells was measured. The activity of deoxythymidine kinase was measured in cell-free extracts prepared from primary cultures of monkey kidney and from the continuous cell lines of FL amnion, KB, and HEp-2. Five-day-old cultures containing confluent monolayers of each cell type were used. It may be seen in Table 1 that the deoxythymidine kinase activity of the extracts of the monkey kidney cultures was approximately one-third to one-fifth of the activity observed in the extracts prepared from the permanent cultures. This finding prompt’ed an investigation of the effects of adenovirus infection on the deoxythymidine kinase activity of monkey kidney cells.
ADENOVIRUS
AND
HOST TABLE
DEOXYTHYMIDINE Expt. no.
KINASE
Cell tested
ACTIVITY
METABOLIC
683
CHANGES
1 OF VARIOUS CULTURED CELLP
Source and type of culture
Age of culture (days)
S.A. of deoxythymidine kinase
1
Monkey kidney FL amnion KB HEp-2
Simian, Human, Human, Human,
primary continuous continuous continuous
5 5 5 5
0.20 1.03 0.60 0.67
2
Monkey kidney FL amnion KB
Simian, primary Human, continuous Human, continuous
5 5 5
0.16 0.89 0.52
Cultures of FL, KB, and HEp-2 (Microbiological Associates) were prepared according to the following procedure. Approximately 2 X lo6 cells were placed in a 60-mm plastic petri dish containing 4 ml of CGM. The cultures were incubated at 37” in a humidified 7% Cog-air mixture. The medium was replaced on the third day. By 5 days the cultures contained 12 to 18 X lo6 cells. Cultures of monkey kidney were prepared by seeding culture dishes with approximately 1 X lo6 cells using the procedure described in Materials and Methods. By 5 days the cultures contained 3 to 5 X lo6 cells. At 5 days each culture had formed a monolayer of cells which covered more than 95y0 of the surface of the dish. At this time 4 cultures of each cell were collected, and extracts of the cells were prepared as described in Materials and Methods. The cell extracts were tested for deoxythymidine kinase activity using the assay described in Materials and Methods. Specific activity @.A.) of deoxythymidine kinase was expressed as counts per minute dTMP per microgram protein. The radioactivity recovered in dTMP was calculated as the percentage of the total radioactivity on the chromatogram found in dTMP and thymidine.
Colony Formation and Infective Center Assays
9 #’
0’
. IO
’ 20
. 30
40
’ 50
. 60
CONTROL
70’
HOURS
FIG. 1. Growth of adenovirus-infected and uninfected monkey kidney cells. Monolayer cultures of monkey kidney were infected as described in Materials and Methods. The multiplicity was 23. Control cells were treated in the same way, but no virus was added. After the final washing, the cells were covered with 4 ml of VGM. At the indicated times, 2-4 cultures were washed with TD. The cells were then detached from the dish with a mixture containing 0.025y0 trypsin and 0.01% EDTA, as recommended by Howes (1959), and counted with a hemacytometer. Approximately 90% of the infected cells were viable at 64 hours as measured by the failure to stain with trypan blue.
Before attempting to study the effects of adenovirus infection on the enzyme activity of monkey kidney cells, it was essential to show that the majority of cells became infected at the time of virus addition. The proportion of cells infected was estimated in two ways. The first method was based on the finding that adenovirus-infected cells did not divide. As shown in Fig. 1, the number of cells in an infected monkey kidney culture remained approximately the same for at least 64 hours, while by this time, the uninfected cell number had increased by a factor of approximately 3. The number of cells infected could, therefore, be estimated from the proportion of cells which were no longer able to form colonies after infection. The second method utilized the assay for infective centers. Both methods have been applied previously to adenovirus-infected KB cells (Green and Daesch, 1961; Green et al., 1964). The results of a representative experiment designed to test the ability of the adenovirusinfected and control monkey kidney cells to
684
LEDINKO TABLE COLONY
FORMATION
OF UNINFECTED
Number of cells plated per dish
Cells
1. Uninfected 2. Infected 3. Uninfected
100 100 100 each
plus infected
2
AND ADENOVIRUS-INFECTED
MONKEY
KIDNEY
Total number of Number of colonies cells plated found per dish 300 300 300 each
40, 43, 47 0, 1, 1 39, 41, 44
CELLS”
Per cent of cells plated which formed colonies 43 0.7 41
0 Monolayer cultures of monkey kidney were infected as described in Materials and Methods. The multiplicity was 16. Control cells were treated in the same way, but no virus was added. At 1 hour after virus was added, several infected and control cultures were washed twice with TBS, and then an amount of specific adenovirus type 2 antiserum in excess of the amount necessary to neutralize over 99% of free virus was added. Cultures were incubated for 20 minutes at room temperature, and then were washed twice with TD. Single-cell suspensions were prepared by detaching cells from the dish with a mixture containing O.O25oj, trypsin and 0.01% EDTA as recommended by Howes (1959). The cells were counted with a hemacytometer. The uninfected cells, the infected cells and a mixture of both, were suspended in 4 ml of Eagle’s medium supplemented with 10% pooled human serum and 3% calf serum (Green et al., 1964), and placed in 60-mm plastic petri dishes. Macroscopic colonies were counted 10-12 days later; 37-46y0 of the uninfected cells formed colonies in different experiments. TABLE GROWTH
3
OF ADENOVIRUS IN CELLSa
MONKEY
KIDNEY
Hours after virus addition
Total PFU produced per cell
l-20 21 22 24 26 28 30 40 50 60 70
<0.003* 0.007 0.009 0.02 0.06 0.14 0.31 7.1 18.1 20.4 20.0
a Monolayer cultures of monkey kidney were infected as described in Materials and Methods. The multiplicity was 12. After the final washing, the cells were covered with 2 ml of VGM. At the indicated times, 3 cultures were collected. The supernatant was removed, and the cells were washed twice with TBS. The cells from each dish were then scraped into 1 ml of TBS with a rubber policeman. The cells were frozen and thawed 4 times, and the supernatant was assayed for intracellular virus. The figure given above for the total PFU found per cell represents the sum of extracellular and intracellular PFU found. b The virus titer found was approximately the same as that of the residual virus remaining after repeated washing of the cells at 1 hour after virus addition.
form colonies are shown in Table 2. At 1 hour after the addition of virus the cells were plated in Eagle’s medium containing 10% pooled human serum which neutralized type 2 adenovirus (Green et al., 1964). It can be seen that 43 % of the uninfected cells formed colonies. In contrast, only 0.7% of the cells taken from cultures 1 hour after virus was added were able to grow. Infection was therefore 98 % complete. When a mixture of uninfected and infected cells was plated, the infected cells did not prevent the growth of the uninfected cells. This finding indicates that possible infection of the originally uninfected cells by virus released from the infected cells was prevented under the above plating conditions. The assay for infective centers was carried out according to the procedure described in the text. When the adsorbed multiplicity was 4 PFU’s per cell, 52-60 % of the cells formed plaques, but at adsorbed multiplicities of 25-40 PFU per cell, only 37-46 % of the cells formed infective centers. The reason for this reduction in the number of plaque formers at higher multiplicities of infection is not known. In all the experiments reported below, infection was at least 90% complete as measured by the failure of the infected cells to form colonies.
ADENOVIRUS
AND
HOST
METABOLIC
685
CHANGES
after infection, and less hours. In other experiments, between 10 and 50 PFU cell at approximately 50 tion.
than 20% at 50 a final yield of was produced per hours after infec-
Deoxythymidine Kinase Activity of Uninfected and Adenovirus-Infected Monkey Kidney Cells
CONTROL
HOURS
FIG. 2. The specific activity of deoxythymidine kinase of adenovirus-infected and uninfected monkey kidney cells at different times after infection. Monolayer cultures of monkey kidney were infected as described in Materials and Methods. The multiplicity was 21. A parallel series of cultures were treated in the same way, but no virus was added. After the final washing, the cells were covered with 2 ml of VGM. At the indicated times cell-free extracts of the uninfected and infected cells were prepared and the deoxythymidine kinase activity was measured. The S. A. of deoxythymidine kinase was expressed as counts per minute dTMP per microgram protein. The radioactivity recovered in dTMP was calculated as the percentage of the total radioactivity found on the chromatogram in dTMP and thymidine. At 50 hours, a total of 28 PFU were produced per cell.
Growth of Adenovirus in Monkey Kidney
Cells
The growth of adenovirus in monkey kidney cells has been measured. The results of a representative experiment are shown in Table 3. The first increase in virus titer was found at approximately 21 hours. At this time 0.607 PFU was produced per cell. The amount of virus produced then increased progressively to reach a yield of 18.1 PFU per cell at 50 hours after infection. At this time virus maturation was complete or virtually complete. At 60 and 70 hours after infection 20 PFU were found per cell. Extracellular virus was found to be less than 5 % of the intracellular virus at 40 hours
The deoxythymidine kinase activity of extracts prepared from the control and adenovirus-infected monkey kidney cells was assayed at different times after infection. It can be seen in Fig. 2 that the activity of deoxythymidine kinase began to increase at approximately 20 hours after infection very shortly before infective virus was first produced. The activity then increased to reach a maximum at 50 hours. At this time the enzyme activity of extracts prepared from infected cells was greater than that of control cell extracts by a factor of approximately 6. A sharp drop in the activity of thymidine kinase occurred after 50 hours. However, even at 70 hours after infection, the activity of the infected cell extracts was approximately threefold higher than that of the uninfected cell extracts. Properties of the Adenovirus-Induced thymidine Kinase
Deoxy-
The possibility existed that the increase in the activity of the deoxythymidine kinase found in adenovirus-infected cells was due to the synthesis of a new enzyme coded by the viral nucleic acid. The virus-induced enzyme might, therefore, have properties which differed from the normal cell enzyme. The thermostability of the deoxythymidine kinase in crude extracts of control cells and cells infected 48 hours was tested by the method of McAuslan (1963b). No difference in thermostability was found. Deoxythymidine kinase obtained from a variety of sources is inhibited by dTT (Bresnick et al., 1964). The possibility existed that the deoxythymidine kinase activity of the uninfected and infected cell extracts was inhibited by different levels of dTTP. The effect of dTTP, as well as of dTDP, on the
686
LEDINKO TABLE
4
INHIBITION OF DEOXYTHYMIDINE KINASE ACTIVITY BY dTTP AND dTDPa
Substance added (mpmoles)
Enzyme source
Ratio of activity of extract in pr;r,;~e sence of thymine derivatives
Uninfected tract
cell
ex-
Infected (48 hours) cell extract
dTTP,
0.1 0.2 0.4 l.Ob
0.78 0.71 0.68 0.65
dTDP,
0.2 1.0 2.0b
0.76 0.68 0.64
dTTP,
0.1 0.2 0.4 l.O*
0.32 0.24 0.18 0.14
dTDP,
0.2 1.0 2.Ob
0.31 0.16 0.13
a Monolayer cultures of monkey kidney were infected as described in Materials and Methods. The multiplicity was 19. A parallel series of cultures were treated in the same way but no virus was added. At 48 hours after infection, cell-free extracts of the uninfected and infected cells were prepared. The extracts were chromatographed on columns of Sephadex G-25 to remove low molecular weight substances. The activity of the deoxythymidine kinase was then measured. The S.A. (cpm dTMP/pg protein) of the infected cell extracts was 0.92, and that of the uninfected cell extracts was 0.18. The radioactivity recovered in dTMP was calculated as the percentage of the total radioactivity found on the chromatogram in dTMP and thymidine. b Concentrations of inhibitor up to 100 mpmoles did not cause any more inhibition of enzyme activity.
activity of the deoxythymidine kinase was tested. Extracts of the infected and uninfected cells were first passed through columns of Sephadex G-25 to remove low molecular weight substances. The results obtained are shown in Table 4. Concen-
trations of dTTP which inhibited the activity of the enzyme of control cell extracts by approximately 30-40% inhibited the enzyme activity of infected cells by approximately SO-90%. When dTDP was tested a similar difference was found in the extent of inhibition of the activity of the enzyme obtained from control and infected cells. E$ects of Puromycin on the Activity of Deoxythymidine Kinase The next experiment was carried out to determine whether the continuous increase in the deoxythymidine kinase activity found after adenovirus infection depended on de novo protein synthesis. The results are shown in Table 5. At 19 hours after infection the ratio of the deoxythymidine kinase activity of the infected cells to that of the control cells was 1.4, and at 24 hours 2.8. At these times puromycin (2 X 1w4 M) was added to aliquots of the infected and uninfected cells. In the absence of puromycin the ratio of the enzyme activity of infected cells to that control cells increased to 4.2 at 42 hours after infection, and to 5.1 at 47 hours. In contrast, when puromycin was added at 19, or at 24 hours after infection, the ratio found at these times remained approximately the same for at least 23 hours. The S. A. of the uninfected cell extracts was approximately the same in the untreated and puromycin-treated cultures. These findings indicate that the continuous increase in deoxythymidine kinase activity in infected cells depended on de novo protein synthesis. Activities of DNA Polymerase, dCMP Deaminase, and Aspartate Transcarbamylase after Adenovirus Infection of Monkey Kidney Cells A considerable increase in the deoxythymidine kinase activity (four to sixfold) always occurred after adenovirus infection of monkey kidney cells. The possibility existed that adenovirus replication was associated with alterations in the activities of other enzymes involved in the processes leading to DNA synthesis. For example, a stimulation of the deoxythymidine kinase, DNA polymerase, and dCMP deaminase activities has been
ADENOVIRUS
AND
HOST
METABOLIC
TABLE
5
THE EFFECT OF PUROMYCIN ON THE DEOXYTHYMIDINE KINASE ACTIVITY ADENOVIRUS-INFECTED MONKEY KIDNEY CELLW No puromycin Hours after infection
19 24 42 47
added
Puromycin
0.28b 0.64 1.09 0.97
Control
0.20 0.23 0.26 0.19
OF UNINFECTED AND
(2 X 1OW M) added
Added at 19 hours Infected
687
CHANGES
Added at 24 hours
‘~~~~~’
1.4 2.8 4.2 5.1
Infected
Control
l~~n~~o~’
Infected
Control
Infected: control
0.28 0.29 0.49 NDC
0.20 0.19 0.22 ND
1.4 1.5 1.8 ND
0.64 0.58 0.60
0.23 0.18 0.20
2.8 3.2 3.0
(1Monolayer cultures of monkey kidney were infected as described in Materials and Methods. The multiplicity was 20. Control cells were treated in the same way, but no virus was added. After the final washing the cells were covered with 2 ml of VGM. At the indicated times, extracts of the infected and uninfectecl cells were prepared. At 19 hours after infection, 0.1 ml of a solution of puromycin hydrochloride (4 X lOwa M) was added to 1.9 ml of the culture medium of aliquots of the infected and uninfected cultures; and to a parallel series of cultures at 24 hours. Extracts of the uninfected and infected added puromycin-treated cultures were prepared at the indicated times. The amount of puromycin was sufficient to inhibit 92% of the protein synthesis, as determined by measuring the uptake of L-leutine-Ha according to the technique described in Materials and Methods. The activity of deoxythymidine kinase was measured and the S.A. of deoxythymidine kinase was expressed as counts per minute dTMP per microgram protein. The radioactivity recovered in dTMP was calculated as the percentage of the total radioactivity on the chromatogram found in dTMP and deoxythymidine. b Values indicate specific activity of deoxythymidine kinase of cell extracts. c ND, not done.
observed in polyoma-infected mouse kidney cells (Dulbecco et al., 1965). Moreover, an increa.se in the activity of aspartate transcarbamylase has been reported in type 5 adenovirus-infected HeLa cells (Consigli and Ginsberg, 1964). In the following experiment, the activities of deoxythymidine kinase, DNA polymerase, dCMP deaminase, and aspartate transcarbamylase were measured in adenovirusinfected monkey kidney cells at different times after infection. The results of a representative experiment are given in Table 6. The deoxythymidine kinase activity of the infected cells was approximately fourfold higher than that of the uninfected cells at 47 hours after infection, and approximately threefold higher at 41 and 64 hours. The activities of dCMP deaminase and aspartate transcarbamylase of the infected cell extracts did not differ more than twofold from those of the control cells. However, at 40-60 hours after infection, the dCMP deaminase and aspartate transcarbamylase activities were consistently greater than those of the control cells by a factor of 1.6-2.
In contrast, the DNA polymerase activity wasnot significantly increased at 20-70 hours after infection. No difference in the DNA polymerase activity between adenovirus type 2-infected Kl3 cells and uninfected cells has been found by Green et al. (1964). The Rates of DNA, RNA, and Protein Synthesis in Adenovirus-Injected Monkey Kidney Cells The results of the preceding experiment indicated that the activity of deoxythymidine kinase was considerably increased after adenovirus infection of monkey kidney cells, but that the activity of DNA polymerase was not demonstrably changed. These findings were then related to measurements of the rates of incorporation of H3-thymidine into the DNA of infected cells. At the same time, the rates of incorporation of H3uridine into RNA and of H3-leucine into protein were also measured. As may be seen in Fig. 3, the rates of incorporation per cell of precursors into the DNA, RNA, and protein of the uninfected cells decreased slowly but steadily during a
688
LEDINKO TABLE
6
ACTIVITIES OF DEOXYTHYMIDINE KINASE, DNA POLYMERASE, dCMP DEAMINASE, TRANSCARBAMYLASE AFTER ADENOVIRUS INFECTION OF MONKEY KIDNEY
THE
DNA polymerase dCMP deaminase Aspartate Deoxythymidine kinase Hours after Con- Infected: yF$- ‘~~~t~o~’ Infected ‘On- Infected’ Infected a~id~~on Infected trol control Infected trol control 2 17 24 41 47 64 71
0.33” 0.27 0.46 0.81 1.20 0.75 0.57
0.30 0.30 0.29 0.29 0.28 0.26 0.26
1.1 0.9 1.6 2.8 4.3 2.9 2.2
12.6 8.5 14.1 10.0 14.4 10.3 7.3
14.0 12.2 12.8 11.1 11.1 10.3 10.4
0.9 0.7 1.1 0.9 1.3 1.0 0.7
0.40 0.30 0.35 0.24 0.24 0.13 0.09
0.40 0.38 0.23 0.14 0.12 0.08 0.07
1.0 0.8 1.5 1.7 2.0 1.6 1.3
AND ASPARTATE CELLS”
transcarbamylase Control
Infected. control’
0.008 0.008 0.017 0.020 0.020 0.019 0.018
1.1 1.1 1.3 1.9 1.8 1.6 1.5
0.009 0.01 0.022 0.038 0.036 0.030 0.027
a Monolayer cultures of monkey kidney were infected as described in Materials and Methods. The multiplicity was 10. A parallel series of cultures were treated in the same way, but no virus was added. At the indicated times, extracts of the infected and uninfected cells were prepared, and the enzyme activities were measured. The S.A. of deoxythymidine kinase was expressed as cpm dTMP/pg protein. The radioactivity recovered in dTMP was calculated as the percentage of the total radioactivity found on the chromatogram in dTMP and deoxythymidine. The S.A. of DNA polymerase was expressed as cpm acid-insoluble product/pg protein. The S.A. of dCMP deaminase was expressed as cpm dUMP/pg protein. The radioactivity recovered in dUMP was calculated as the percentage of the total radioactivity found on the chromatogram in dUMP and dCMP. The S.A. of aspartate transcarbamylase was expressed as cpm ureidosuccinate/pg protein. The radioactivity found in ureidosuccinate was calculated as the percentage of the total radioactivity found in ureidosuccinate and aspartate. b Values indicate specific activity of cell extracts. H3 Uridine
H3 Leucine
H3 Thymidine
50
h \ A;,,
0
0
IO
20
0 .
30
40
50
60
lb
20
30 40 HOURS
50
$0
lb
2.0
30
40
50
$0
FIG. 3. The effect of adenovirus infection on the rates of synthesis of DNA, RNA, and protein in monkey kidney cells. Monolayer cultures of monkey kidney were infected as described in Materials and Methods. The multiplicity was 22. A parallel series of cultures were treated in the same way, but no virus was added. At the indicated times aliquots of the infected and uninfected cell cultures were subjected to l-hour pulses of the radioactive precursors and the rates of DNA, RNA, and protein synthesis were determined as described in Materials and Methods. Abscissa: period of incubation after infection (hours). Ordinate: rate of protein synthesis: cpm L-leucine-H3 incorporated/l04 cells/hour. Rate of RNA synthesis: cpm uridine-H3 incorporated/l03 cells/hour. Rate of DNA synthesis: cpm thymidine-H3 incorporated/l03 cells/hour.
ADE~~~~IR~JS TABLE
AND HOST METABOLIC
7
TWE INCOWORATION ok THPMIDINE-Ha FOR 60 MINUTES INTO TI-KE DNA AND ACID-S• LUXUZ FRACTIONS OF AD~NOYIR~~-I~F~C~~~ AND UNINFECTED
%fOMKEY
Hours after virus addition 2 16 19 22 40 50 60
KIDNEY
CELLS”
Ratio, cpm infected cell fraction: cpm uninfected ceil -fraction --.-_I Nucleic acid Acid-soluble pool 1.12 0.94 0.92 1.35 0.82 0.68 0.60
ND ND 0.98 1.13 X.24 0.90 0.85
CHANGES
689
cursors into RKA and protein was approximately 5 7%or less of the original rate. The effect of adenovirus infection on the rate of thyn~idi~e incorporation into the acid-solubIo fraction of monkey kidney cells IOC
SC
BC
70
I+----.1 I tntracellular
a Monolayer cultures of monkey kidney cells were infected as described in Materials and Methods. The multiplicity was 25. Control cells were t,reated in the same way, but no virus was added. At the indicated times the medium of replicate cultures of the infected and uninfected cells was decanted a& 2 ml of VGM containing thymi-
dine-J.%3 (5 MC/ml, 1.9 C/mmole) was added to each culture. At 30, and again at 60, minutes of incubation, cells were washed twice with cold TD, scraped from the culture dish into TD with a rubber policeman, and centrifuged. The cell pellet was extracted three times with cold 0.3 M trichloroaeetic a.cid. The pooled superuatants from these extract,ions represented the acid-soluble pool. The pellets were further extracted with 0.3 M trichloroacetic acid at 90” for 15 mil~utes to give the m&eic acid fraction. The radioactivity of the acid-soluble fraction was similar at 30 and 60 minutes, indicating that the pool was rapidly saturated with th~~idil~e. The results are given as the ratios of the counts per minute incorporated in the infected cells to that incorporated in the uninfected cells.
period of 65 hours. At 65 hours the incorporation of precursors was approximately 40-50% of t,he original rate. The rate of incorporation per cell of ~3-thy~~ne into the DNA of infected cdls declined to approxi~tely the same extent as that of the uninfected cells. In striking contrast, the incorporations of II”-uridine into the RNA and of IWeucine into protein were much more severely ~hibit~ in the infected cells than in the controls. At 65 hours after aection the rate of incorporation of the pre-
Virus
formed
of Infected
Nuclei
1
20
IO
f
_..,,... 4 20
40
60
30
HOURS
FIG. 4. Adenovir~~s formation and the time course of the decrease in the DNA-dep~udent RNA polymerase activity in infected monkey kidney cells. Monolayer cultures of monkey kidney were infected as described in materials and Methods. The m~lltiplicit~ was 18. Control cultures were treated in the same way, but no virus was added. After the final washing, the cultures were covered with 2 ml of VGM. At the indicated times, S-10 replicate cultures of the infected and uninfected cells were removed, and the cell nuclei were isoIated. The activity of the ~NA-dependent RNA pol~erase was assayed in the nuclear fractions. The results are expressed as the percentage of the S.A. of the enzyme found at the time of virus addition. At this time t,he S.A. of the RNA polymerase, expressed as counts per minute CTP-Ha incorporated into acid-insoluble material per microgram of protein, was 6.7, Intracellular virus growth was determined using the method described in Table 3. The results are expressed as the percentage of the PFU produced per cell at 70 hours. At this t,ime 36 PFXJ per eelI were found.
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was also investigated. It may be seen in Table 7 that, as with the incorporation of thymidine into the DNA, the rate of incorporation of thymidine into the acid-soluble fraction of infected cells was similar to that of the uninfected cells. The results of preliminary experiments have indicated that the rates of incorporation of uridine and leucine into the acid-soluble pool of infected cells were also similar to those of the control cells. Activity of DNA-Primed RNA after Adenovirus Infection Kidney Cells
Polymerase of Monkey
The possibility existed that the marked inhibition of RNA and protein synthesis observed in infectNed monkey kidney cells was accompanied by a decline in the activity of the DNA-directed RNA polymerase. This enzyme is believed to play a key role in protein synthesis. The RNA polymerase activity of nuclei isolated from adenovirusinfected and control monkey kidney cells at different times after infection is shown in Fig. 4. A decrease in the activity of RNA polymerase of the infected cells began at approximately 15 hours after infection. Less than half of bhe initial activity was found at 20 hours after infection, and only 25 % of the activity remained at 24 hours. This level was maintained up to 60-70 hours after infection. The activity of the RNA polymerase of infected cells was related to the time course of intracellular virus formation (Fig. 4). At 24 hours when only 25% of the original activity of RNA polymerase remained, less than 0.1% of the progeny infective virus had been formed. DISCUSSION
One effect of type 2 adenovirus infection uncovered in this study is the inhibition of the activity of the host cell DNA-dependent RNA polymerase. This inhibition occurred before virus maturation began. At the time when maturation began, the level of the RNA polymerase activity was only between 20 and 30% of that of the uninfected cell. A loss of the RNA polymerase activity has also been found to occur after infection of Escherichia coli with the DNA-containing phage T4 (Skold and Buchanan, 1964), and after infection of L cells with the RNA-
containing mengovirus (Baltimore and Franklin, 1962). The mechanism of the inhibition of RNA polymerase activity is not known. An attempt was made to examine the possibility that the inhibition of the activity of the RNA polymerase in the nuclei isolated from adenovirus-infected cells was not related to virus synthesis. The inhibition might have been due to degradation of the DNA template or of the RNA product. The activities of the DNase and RNase of the nuclei prepared from control cells and cells infected 48 hours were, therefore, measured. No DNase activity was found, and the small amount of RNase activity observed was approximately the same in the control and infected cell nuclear fractions. Other findings also suggested that degradation of the primer DNA in infected cells did not occur. The ratios of DNA to protein in the nuclei of control cells and cells infected 48 hours were similar. In addition, the addition of 100 to 200 pugof highly polymerized salmon sperm DNA to the assay system did not increase the activity of the RNA polymerase in the control and infected cell nuclear fractions. The possibility also existed that the nuclei prepared from the infected cells were very fragile and that the RNA polymerase had leaked out. However, no activity was found in the cytoplasmic fraction. Moreover, the DNA polymerase activity of the nuclei isolated from the infected cells was of the same order of magnitude as that of the nuclei prepared from the control cells. The question arises of the relevance of the marked inhibition of RNA and protein synthesis to the biosynthesis of adenovirus occurring in the monkey kidney cells. The total yield of adenovirus found was between 10 to 50 PFU per cell. This yield is considerably lower than was found using the permanent KB cells where approximately 10,000 PFU were produced per cell (Green, 1962). Furthermore, the first detectable increase in intracellular virus in monkey kidney cells was found at approximately 21 hours after infection, whereas maturation in the KB cells started at 13-14 hours after infection (Green, 1962). Adenovirus replication in the KB cell does not inhibit macromolecular synthesis until late
ADENOVIRUS
AND
HOST
in the infectious process. The possibility existed that the early inhibition of the RNA and protein synthesis observed in the infected monkey kidney cells was unrelated to virus synthesis. It was possible, for example, that the protein factor present in adenovirus lysates which causes the early cytopathic effect (Pereira, 1958) was also responsible for the inhibition of the RNA and protein synthesis. This possibility was eliminated by the use of virus purified by density gradient centrifugation in cesium chloride. No inhibition was observed when cells were treated with extracts prepared from uninfected cells. It appears therefore very likely that the inhibition of the cell RNA and protein synthesis is a function of the adenovirus. Several possibilities exist to account for the differences noted between the previous results by others and the findings reported here. Aside from differences in virus types and media, possibly the outstanding difference is in the kind of cell type tested. We chose to work with primary cultures derived from normal tissue, whereas previous studies were carried out with permanent cell lines. The choice of monkey kidney cells was initially dictated by the finding that these cells in confluent monolayers have consistently low background levels of deoxythymidine kinase activity. Cells of permanent lines, on the contrary, tend to have a high level of deoxythymidine kinase activity at high cell density (data given, and unpublished observations). It is conceivable that in the established cell lines a loss in metabolic control systems has occurred which permits high enzymatic and metabolic activity at high cell density. What relationship, if any, exists between this possible loss in regulatory activity and the failure of adenovirus infection to produce any marked early effects on the machinery of the permanent cell is not known. It is of considerable interest that the activity of the deoxythymidine kinase was induced after adenovirus infection. The activity of this enzyme began to increase at approximately the time when virus maturation started. When maturation was complete, or almost complete, the deoxythymidine kinase activity was four- to sixfold
METABOLIC
CHANGES
691
higher than that of the control cells. The increase in the enzyme activity depended on de novo protein synthesis. It is possible that the observed increase of the deoxythymidine kinase activity resulted from the synthesis of a new enzyme induced by adenovirus, the synthesis of a latent host enzyme, or simply from an increase in the rate of synthesis of the regular host enzyme. The significance of the enhanced deoxythymidine kinase activity in adenovirus infected monkey kidney cells is not known. Note added in proof. After this manuscript was submitted for publication, it was reported that the activity of deoxythymidine kinase was enhanced after infection of green monkey kidney cells by simian adenoviruses, simian papovavirus SV40, and an adenovirus-SV40 “hybrid”. (Kit, S., Dubbs, D. R., deTorres, R. A., and Melnick, J. L., Virology 27,453-457 (1965).) ACKNOWLEDGMENTS The author is indebted to Dr. R. Dulbecco for valuable advice and to Dr. J. Buchanan for his interest and helpful suggestions. This investigation was supported by a Public Health Service Special Research Fellowship from the National Institute of General Medical Sciences to the author, and by a Research Grant from the Public Health Service, National Institutes of Health, No. CA-07592. REFERENCES BALTIMORE, D., and FR.~NKLIN, R. M. (1962). The effect of mengovirus infection on the activity of the DNA dependent RNA polymerase of n-cells. Proc. Natl. Acad. Sci. U.S. 48, 13831390. BRANDON, F. B., and MCLEBN, I. W. (1962). Adenovirus. Advan. Virus Res. 9, 157-193. BRESNICK, E. (1962). Feedback inhibition by deoxyribonucleotides of aspartate transcarbamylase activity in liver preparations. Biochim. Biophys. Acta 61, 598605. BRESNICK, E., THOMPSON, U.B., MORRIS, H.P., and LIEBELT, A. G. (1964). Inhibition of thymidine kinase activity in liver and hepatomas by TTP and d-CTP. Biochem. Biophys. Res. Commun. 16, 278-284. BURTON, K. (1956). A study of the conditions and mechanisms of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-323. CHARG~FF, E., and DAVIDSON, J. N. (1955). In
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