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Studies on the replicating pool of viral DNA in cells infected with pseudorabies virus
VIROLOGY
Studies
24, 19-25
(1964)
on the
Replicating
Department
Pool
of Viral
DNA
Pseudora
bies
ALBERT
S. KAPLAN
of Microbiology, Center,
Research Philadelphia, Accepted
Infected
with
Virus’
Laboratories, Albert Pennsylvania May
in Cells
Einstein
Medical
8, 1964
Analysis by centrifugation in density gradients of cesium chloride revealed that not all of the pseudorabies virus DNA synthesized early in the infectious cycle replicates. Only 407’ of the DNA synthesized during the first 4.5 hours after infection and 25% of the DNA made up to 7 hours replicates thereafter, despite continuing active DNA synthesis. The inability of most of the DNA to replicate is not due to its integration into mature virus particles. It may, however, be ascribed, in part, to the rapid development within the cell of a viral DNA-containing structure in which the DNA is insensitive to the action of deoxyribonuclease. It is suggested that this deoxyribonuclease-resistant form may be a precursor of mature virus. INTRODUCTION
In rabbit kidney (RK) cells infected with pseudorabies (Pr) virus, the rate of viral DNA synthesis increases with time after infection (Kaplan and Ben-Porat, 1963; Ben-Porat and Kaplan, 1963). This increasing rate can be interpreted as being the reflection of the exponential mode of synthesis of viral DNA within the cell. However, when viral DNA synthesis in the infected cells is inhibited by 5fluorouracil and the number of viral DNA molecules produced is therefore greatly reduced, the cells nevertheless acquire the capacity for accelerated viral DXA synthesis, so that upon release of the inhibition of DNA synthesis by thymidine, these cells start synthesizing viral DNA at the same rate as control cells in which viral DNA has accumulated (Kaplan, 1962; unpublished results). Thus, the 1 This investigation was supported by grants from the National Institutes of Health (AI02432-05 and AI-00362-04) and from the National Science Foundation (GB-1386) and by a Public Health Service research career program award (AI-K3-19335.01) from the National Institut,e of Allergy and Infectious Diseases. 19
increasing rate of DNA synthesis cannot be due to the increasing number of DNA templates available for replication, and the rate-limiting factor in viral DNA synthesis is not the availability of DNA templates; instead, it is probably the enzymes and precursors necessary for the synthesis of DNA. The role of progeny viral DNA as a template for further replication remained, therefore, to be established. Pr virus DNA is of the double-stranded type and replicates semiconservatively (Kaplan and Ben-Porat, 1964). Moreover, infected cells in the stationary phase of growth synthesize viral DNA in excess, so that of the total amount of viral DlYA synthesized by these cells, approximately only 20% is eventually incorporated into mature virus particles (Ben-Porat and Kaplan, 1963). If the remaining 80 % of the viral DNA is in a form in which the DNA is available and competent to act as a template for its own replication, and if viral DNA multiplies exponentially, most of the DNA molecules present in the cell during the early stages of the infective process should replicate thereafter. The present
KAPLAN
I
I.706
t
1.732
I
1
1.745 1.757
DENSITY FIG. 1. Replication of Pr virus DNA. Reading from left to right, the peaks represent RK DNA, Pr virus thymine-DNA, Pr virus hybrid-DNA, and Pr virus BUDR-DNA.
experiments were designed to whether or not this is the case. MATERIALS
establish
AND METHODS
Virus and cell culture. The properties of Pr virus and the cultivation of RK monolayers, as well as the procedure of virus assay, have been described previously (Kaplan, 1957; Kaplan and Vatter, 1959; Ben-Porat and Kaplan, 1962). Since only viral DNA is synthesized by virus-infected RK cells in the stationary phase of growth (Ben-Porat and Kaplan, 1963), cells in this phase of their growth cycle were used in all the experiments. Media. ELS consists of Earle’s saline, 94.5 %; lactalbumin hydrolyzate (Nutritional Biochemicals), 0.5 % ; and dialyzed bovine serum, 5%. EDS medium contains Eagle’s synthetic medium (Eagle, 1959), plus 3 % dialyzed bovine serum. TBSA has the following composition: NaCl, 0.15 M; KCl, 2.7 X lo-* M; CaC12.2Hz0, 8.8 X 1O-4 M; MgC12.6Hz0, 4.9 X LOU4M; tris(hydroxymethyl)aminomethane, 0.1 M (adjusted with HCI to pH 7.5) ; and 1% crystalline bovine serum albumin. Chemicals.Cesium chloride, optical grade, was purchased from the Harshaw Chemical
Company, thymidine from the Mann Research Laboratories, 5-bron~od~xyuridine (BUDR) from the Caiifornia Corporation for Biochemical Research, and crystalline deoxyribonuclease (DNase) from the Worthington Biochemical Corporation. 5-fluorouracil (FU) was a gift from HoffmanLaRoche Incorporated. Thymidine-d-G4 (specific activity, 62 pc/mg) and adenine8-Q4 (specific activity, 25 pc/mg) were purchased from the New England Nuclear Corporation. Pur~~~at~~ of Pr value DNA. DNA was extracted and purified as described previously (Kaplan and Ben-Porat, 1964). Density gradient centrifugation. This procedure has been described previously (Kaplan and Ben-Porat, 1964). In brief, CsCl solutions containing DNA were introduced slowly with a syringe and needle into a 12-mm Kel-F cell with a 1” negative wedge window. The density of the DNA-C&l solutions was approximately 1.70 g/cm”. The densities of the solutions were determined from their refractive indexes by the equation of Ifft et al. (1961). Sedimentation equilibrium was performed with a Spinco model E analytical centrifuge at 25” for at least 20 hours at 44,770 rpm, and the ultraviolet absorption bands were photographed (the exposure time was such that the photographic response was proportional to the concentration of viral DNA). Tracings of the ultraviolet absorption bands were made with a Joyce-Loebl double-beam microphotodensitometer. The DNA of RK cells (1.706 g/cm3) served as a density marker. RESULTS
Proportion I$ V&l DNA That Replicates in Infected Cells
Cultures in the stationary phase of growth were incubated with FU (10 ~g/mI) for 18 hours prior to infection in order to exhaust the intraceIlular supply of thymine and to stop all synthesis of cellular DNA (Kaplan and Ben-Porat, 1961). They were then infected and incubated in EDS containing FU, 5 Mg of BUDR, and 5 rg of thymidine per milliliter. Four and one-half hours after infection, the BUDR-containing medium was replaced by EDS containing thymidine
REPLICATING TABLE REPLICATION OF BUDR-DNA THE FIRST 4.5 HOURS Time of harvest after infection (hours) 7 8 9
Time after addition of thymidine (hours)
POOL
1 SYNTHESIZED AFTER INFECTION” Earlv DNA rep&atedh (?a
2.5 3.5 4.5
FOR
iEzs,v% total viral DNA<
25 38 41
2.0 3.1 4.4
a See text for details. * The areas under the peaks were measured with a planimeter. Since peak B of Fig. 1 contains DNA composed of two strands, one of which consists of BUDR-DNA and thus comes from the DNA in peak C, and one of which consists of normal viral DNA, the percentage of BUDR-DNA that has undergone replication will be equal to:
0.5B ____x 100 C + 0.5B c Calculated as C) = relative
(0.5B +
follows: increase
(A + B in total viral
+ C)/ DNA.
(1 mg/ml) to stop the incorporation of BUDR into DNAz; at periodic intervals thereafter, the cells were harvested, the DNA extracted, and the banding pattern of the DNA in CsCl density gradients was determined in the analytical centrifuge. The tracings of the UV absorption bands of the DNA obtained from these samples are given in Pig. 1. Immediately after BUDR is replaced by thymidine, only one peak of viral DNA is observed; this BUDRcontaining viral DNA has a buoyant density of 1.757 g/cm3. One hour later, a peak of DNA with a density of 1.745 g/cm3 makes its appearance, and thereafter a third peak of DNA with a density corresponding to normal viral DNA (1.732 g/cm3) appears. The peak of intermediate density consists of DNA containing one strand of normal, thymine-containing DNA and one of BUDR-containing DNA and results from the semiconservative mode of replication * Under these conditions the incorporation of thymidine-2-CY4 into DNA is effectively stopped when this label is present in the EDS medium containing BUDR in which the cells are incubated for the first 4.5 hours after infection.
OF
VIRAL
21
DNA
of Pr virus DNA (Kaplan and Ben-Porat,. 1964). The relative amounts of material present in the BUDR-DNA band and in the band of intermediate density can be used as a measure of the amount of BUDR-DNA that has replicated, and the proportion of the BUDRcontaining DXA that has not undergone replication can be calculated (see footnotes to Table 1). These calculations show (Table 1) that by 4 hours after the replacement of BUDR by thymidine, 41% of the BUDRDNA has replicated while there has been an approximately fourfold increase in total viral DNA. Replication oj Viral DNA Synthesized up to Various Times during the Infective Process To determine whether the same proportion of DNA synthesized up to any stage of the infective process replicates thereafter, the following experiment was performed. RK cells were infected and incubated in BUDR-containing EDS (6 pg/ml BUDR + 4 pg/ml thymidine). At various times after infection, the incorporation of BUDR into DNA was arrested by replacing the incubation medium with EDS containing 1 mg of thymidine per milliliter. At the end of 11 hours of incubation (end of cycle), the cultures (cells and virus) were harvested and the DNA was extracted and analyzed in CsCl gradients in the analytical centrifuge. Tracings were made of the UV absorption bands, the areas under the peaks and the proportion of were measured, TABLE AMOUNT
2
OF CONVERSION OF BUDR-VIRAL T O THE HYBRID FORMS
DNA synthesis to the following times (hours)
Conversio;7?
(L See text for * See footnotes
hybrid* 0 41 40 41 31 28 25
4.5 5 5.5 G 6.5 7 details. to Table
1.
DNA
22
KAPLAN
loo .
I& 5 60 !E I: 60 z I-
. l
6Pq
0 IOuq
BUDA
+ 4,uq
Thymldine/ml
Thymidinehl
40
a %
20
0
I
\
1
0
2
4
6
6
10
BUOR
10
6
6
4
2
0
THYHIMNE
Pq/ml pWnl
FIG. 2. Growth of Pr virus in the presence of BUDR. Monolayers of RK cells were incubated with FU (25 pg/ml) for 24 hours prior to infection. The cultures were infected, virus was allowed to adsorb for 60 minutes, unadsorbed virus was removed by washing, and EDS containing FU, plus varying amounts of BUDR and thymidine was added to different sets of cultures. The cultures were harvested 18 hours after infection and the amount of infectious virus present in each culture was assayed by the plaque method.
14
FIG. 3. Incorporation of adenine&CY4 into the DNA of virus-infected cells incubated in the presence of thymidine or BUDR. Monolayers of RK cells were incubated with EDS containing FU (10 pg/ml) for 18 hours. The cultures were infected (adsorbed multiplicity = IO) ; to one set of cultures was added EDS containing thymidine (10 Mg/ml) and FU (10 .g/ml), and to another EDS containing BUDR (6 pg/ml) and thymidine (4 pg/ml). Adenine&Cr4 (0.4 pc/ml) was added to the cultures 1 hour after infection. At periodic intervals thereafter, the cultures were harvested and the amount of adenine-8.Cl4 incorporated into DNA was determined as described previously (Kaplan and Ben-Porat, 1961).
BUDR-DNA that had replicated was calculated as described above. The results show (Table 2) that as the infectious cycle proceeds, an increasingly smaller proportion of the viral DNA within the cell replicates. Thus, for example, of the DNA made up to 5.5 hours after infection 41% replicates, whereas only 25% of the DNA made up to 7 hours replicates thereafter, despite the fact that there is active Dn’A synthesis up to ,the time of harvest (see Fig. 3). These 1. The experiment described above, sumresults cannot be due to the withdrawal of marized in Table 1, was repeated except that viral DNA and its integration into mature thymidine (10 pg/ml) was added for the virus particles since, as shown previously first 4.5 hours (so that the early DNA con(Ben-Porat and Kaplan, 1963), a maximum sisted of normal viral DNA) and was then of only 20% of the total viral DNA is in- replaced by medium containing BUDR (600 tegrated into particles which behave upon pg/ml BUDR + 400 pg/ml thymidine). differential centrifugation and in gradients The cells were harvested, and the DNA was of CsCl like infectious virus. extracted and banded in CsCl gradients. The fact that a large proportion of the Essentially the same results were obtained DNA does not replicate is not due to the as in the reverse experiment (as summarized experimental conditions used. That the in Table 1) and only 38 % of the early degree of substitution used in these experi- thymine-containing DNA had replicated ments of BUDR for thymidine in the DXA by 9.5 hours after infection. 2. The rate of incorporation of adeninedoes not interfere with the replication of viral DNA, although it reduces the number g-Cl4 into the DNA of cells incubated with of infective centers by approximately 50% thymidine and with BUDR was deter(Fig. 2), is shown by two methods: mined. The results (Fig. 3) show that the
REPLICATING
POOL
BUDR does not interfere with adenine incorporation into DNA and that therefore it is presumably without effect on the rate of synthesis of DNA. Another possible source of interference with DNA synthesis in our experiments may reside in the use of medium containing a high concentration of thynlidine (1 mg/ml). It is known that large amount of this nucleoside arrest the synthesis of DNA by interfering with the formation of deoxycytidylic acid (Reichard et al., 1961; Morris et al., 1963). However, although DNA synthesis in noninfected RK cells is indeed inhibited by a high concentration of thymidine, it remains without effect on the rate of DNA synthesis in Pr virus-infected cells (unpublished results). Thus, neither the fact that BUDR is substituted for thymidine in viral DNA nor the presence of high concentrations of nucleosides in the medium seem to interfere with the rate of DNA synthesis in the infected cells, and the relatively small proportion of viral DKA which undergoes replication in our experiments is therefore not the result of the experimental conditions used.
Since an increasingly smaller proportion of the viral DNA replicates as the infective process proceeds (see Table 2), the possibility was considered that even though viral DNA is not yet part of and/or cannot be isolated with mature virus particles, a major part of it is rapidly transformed into a no~eplicative state. That this may be the case is shown in the next experiment in which the sensitivity of viral DNA to the action of DNase was tested. RK cultures in the stationary phase of growth were infected and incubated in ELS at 37”. To part of the cultures thymidined-C4 (0.04 1~c/ml) was added at 3 hours, and to part at 4 hours after infection. One hour later, incorporation was arrested by the addition of a large excess of unlabeled thymidine (1 me/ml). At various times thereafter, cultures were harvested and sonicated. Part of the samples was used to
OF
VIRAL
23
DNA
determine the fraction of radioactive vi& DNA insensitive to the action of DNase (50 pg/ml), and part to determine the fraction of radioactive viral DNA banding in CsCl density gradients in the position characteristic of infectious virus particles. The results of this experiment are illustrated in Fig. 4. Xost of the radioactive DNA, i.e., the viral DNA synthesized up to 5 hours after infection, is sensitive to DNase up to approximately 5 hours after infection. Thereafter, it rapidly becomes insensitive to the action of the enzyme and by 8 hours after infection, approximately 60% of the radioactive viral DNA is no longer sensitive to the enzyme; at this time, however, only 5-6 % of this DNA can be isolated in a CsCI gradient in the same position as infectious virus. Thus, soon after its synthesis a large part of the viral DNA is converted into a form that is resistant to the action of DNase, even though it does not band with mature virus particles in CsCl gradients. Loss of DNA jTorn> Virus nipulatiolz
Particles
by Ma-
Since the kinetics of the formatZion of DBase-insensitive viral DNA, and of the incorporation of viral DNA into material banding with the virus in C&l gradients are quite different and, in fact, suggest a precursor relationship between the two, it is unlikely that the two forms are identical and that the difference in the total amount of radioactive DNA found in each is due to a loss of DNA from the virus particles during the isolation procedure in the CsCl gradients. Nevertheless, it seemed advisable to deterl~~ine the proportion of DNA recovered in the CsCl band when extrac~llular mature .? virus particles and DNase-insensitive material isolated from the cells are centrifuged in CsCl gradients. Mature virus particles, the DNA of which was labeled with thymidine-2-C’*, were prepared as follows. Infected cells were incubated with thymidine-,?-C14 (0.04 ~c/mI), and at the end of the growth cycle the virus in the supernatant fluid was collected. The cellular debris was removed by centrifugation, and the supernatant fluid was treated with DNase (50 fig/ml) to
24
KAPLAN TABLE
FRACTION THE
OF RADIOACTIVE VIRUS BAND IN CSCL
1 2 3
RECOVERED GRADIENTS”
DENSTY
Extracellular mature virus (% local input)
Experiment
a See text
3 DNA
Intracellular DNase-insensitive material (% total input)
65 91 61 for
IN
6 7 5
details.
. i5 _ m 6o I 1 50 -
2g l-
40-
&
JO-
5
20r
Infected cells were incubated with thymidine-MY4 (0.04 /*c/ml) between 3 and 4 hours after infection. They were then incubated for an additional 3 hours in the presence of an excess of C12-thymidine (1 mg/ml). The samples were harvested by scraping the cells into the supernatant fluid and sonicated to disrupt the cells. The samples were treated with DNase (50 pg/ ml), centrifuged, and sonicated again, as described above for the virus preparations. Both sets of samples were assayed for total radioactivity in the DNA, and the proportion of total counts banding with infectious virus in gradients of CsCl was determined. Table 3 summarizes the results. A relatively large variation in recovery was found between different runs and, depending upon the experiment, between 91% and 61% of the radioactivity was found in the band in CsCl of infectious virus; however, only 6 % of the total DNase-insensitive intracellular radioactive DNA was found associated with this band. Thus, even if we allow for a maximum loss of DNA of 50% because of the process of banding the virus in CsCI, only 12% of the DNase-insensitive DNA present intracellularly can be accounted for as mature virus. DISCUSSION
6
TIME
8
IO
I2
14
AFTER INFECTION (HOURS)
FIG. 4. DNase sensitivity of viral DNA and incorporation of viral DNA into mature virus. The fraction of counts associated with DNA which is DNase-insensitive was determined by treating the samples with DNase, by precipitating them with acid, and after appropriate washings, by determining the number of counts in the acid-precipitable material. Thymidine-&-Cl” present between 3 and 4 hours (0 and A); thymidine-2-CY4 present between 4 and 5 hours (0).
remove any contaminating DNA. The virus was sedimented in the Spinco model L (no. 30 head) at 25,000 rpm for 1 hour, was resuspended in TBSA, and was sonicated for 1 minute to break up clumps. Preparations containing DNase-insensitive viral DNA were prepared as follows.
The experiments in this paper demonstrate that within the infected cell filial viral DNA can serve as a template for its own replication. If the total population of viral DNA molecules that are not yet associated with mature virus were part of a pool of replicating DNA, from which a particular DNA molecule had a random chance to act as a template for its own replication, most of the molecules synthesized, for example, up to 4.3
hours
after
infection
should
replicate
in
the following 4 hours. This would be expected, since only a small part of this early DNA is withdrawn and integrated into mature virus and there is during this time a fourfold increase in total viral DXA. However, only approximately 40 % of this DNA replicates. The rapid and extensive integration of viral DNA into a form insensitive to the action of DNase may be responsible for the
REPLICATING
POOL
failure of the major part of the DXA to replicate. The kinetics of formation suggest that this form bears a precursor relationship to mature virus, since between 8 and 12 hours after infection there is an increase of approximately only 8 % in total DNaseinsensitive DNA, while there is an incorporation of approximately 13 % of DiYA into mature virus (see Fig. 4). The DNase insensitive form may be an incomplete form of virus, which is intermediate in the formation of mature virus, the kind of precursor, perhaps, envisaged for T2 phage by Hershey and Melechen (1957). However, it may also represent mature virus contained within membranous structures of the kind described by Horne and Kagington (1959) for poliovirus. Thus, it is possible that virus is synthesized in close contact with such structures, from which it cannot be released by sonication, thereby acquiring different sedimentation and density characteristics from those of released virus; during the later stages of the growth cycle, virus would be freed slowly from these structures. We are now engaged in determining the identity and characteristics of this DBase-insensitive form. ACKNOWLEDGMENT The ckyj
excellent is greatly
assistance appreciated.
of Mr.
Bohdan
Kerny-
REFERENCES BEN-P• RAT, T., and K.~PLSN, A. S. (1962). The chemical composition of herpes simplex and pseudorabies viruses. Virology 16, 261-266. BEN-PoRBT, T., and KAPLAN, A. S. (1963). The synthesis and fate of pseudorabies virus DNA in infected mammalian cells in the stationary phase of growth. Virology 20,310-317. EAGLE, H. (1959). Amino acid metabolism in mammalian cell cultures. Science 130,432-437.
OF
VIRAL
DNA
25
HERSHEY, A. D., and MELECHEN, N. E. (1957). Synthesis of phage-precursor nucleic acid in the presence of chloramphenicol. Virology 3, 207236. HORNE, R. W., ~~~NAGINGTON, J. (1959). Electron microscope studies of the development and structure of poliomyelitis virus. J. MoZ. Biol. 1, 333-338. IFFT, J. B., POET, D. H., and VINOGRAD, J. (1961). The determination of density distributions and density gradients in binary solutions at equilibrium in the ultracentrifuge. J. Phys. Chem. 65, 11381145. K.IPL.IN, A. S. (1957). A study of the herpes simplex virus-rabbit kidney cell system by the plaque technique. Virology 4, 435457. KAPLAN, A. S. (1962). Analysis of the intracellular development of a DNA-containing mammalian virus (pseudorabies) by means of ultraviolet light irradiation. Virology 16, 305313. KAPLAN, A. S., and BEN-PORST, T. (1961). The action of 5-fluorouracil on the nucleic acid metabolism of pseudorabies virus-infected and noninfected rabbit kidney cells. Virology 13, 78-92. KAPLAN, A. S., and BEN-P• RAT, T. (1963). The pattern of viral and cellular DNA synthesis in pseudorabies virus-infected cells in the logarithmic phase of growth. Virology 19, 205-214. K.IPL.IN, A. S., and BEN-PoR.~T, T. (1964). Mode of replication of pseudorabies virus DNA. Virology in press. 23, 90-95. KAPL~~N, A. S., and VATTER, A. E. (1959). A comparison of herpes simplex and pseudorabies viruses. Virology 7, 394407. MORRIS, N. R., REICHARD, P., and FISCHER, G. A. (1963). Studies concerning the inhibition of cellular reproduction by deoxyribonucleosides. II. Inhibition of the synthesis of deoxycytidine by thymine, deoxyadenosine and deoxyguanosine. Biochim. Biophys. Acta 68, 93-99. REICHARD, P., CANELLAKIS, Z. N., and CANELLAKE, E. S. (1961). Studies on a possible regulatory mechanism for the biosynthesis of deoxyribonucleic acid. J. Biol. Chem. 236, 2514-2519.
Studies
24, 19-25
(1964)
on the
Replicating
Department
Pool
of Viral
DNA
Pseudora
bies
ALBERT
S. KAPLAN
of Microbiology, Center,
Research Philadelphia, Accepted
Infected
with
Virus’
Laboratories, Albert Pennsylvania May
in Cells
Einstein
Medical
8, 1964
Analysis by centrifugation in density gradients of cesium chloride revealed that not all of the pseudorabies virus DNA synthesized early in the infectious cycle replicates. Only 407’ of the DNA synthesized during the first 4.5 hours after infection and 25% of the DNA made up to 7 hours replicates thereafter, despite continuing active DNA synthesis. The inability of most of the DNA to replicate is not due to its integration into mature virus particles. It may, however, be ascribed, in part, to the rapid development within the cell of a viral DNA-containing structure in which the DNA is insensitive to the action of deoxyribonuclease. It is suggested that this deoxyribonuclease-resistant form may be a precursor of mature virus. INTRODUCTION
In rabbit kidney (RK) cells infected with pseudorabies (Pr) virus, the rate of viral DNA synthesis increases with time after infection (Kaplan and Ben-Porat, 1963; Ben-Porat and Kaplan, 1963). This increasing rate can be interpreted as being the reflection of the exponential mode of synthesis of viral DNA within the cell. However, when viral DNA synthesis in the infected cells is inhibited by 5fluorouracil and the number of viral DNA molecules produced is therefore greatly reduced, the cells nevertheless acquire the capacity for accelerated viral DXA synthesis, so that upon release of the inhibition of DNA synthesis by thymidine, these cells start synthesizing viral DNA at the same rate as control cells in which viral DNA has accumulated (Kaplan, 1962; unpublished results). Thus, the 1 This investigation was supported by grants from the National Institutes of Health (AI02432-05 and AI-00362-04) and from the National Science Foundation (GB-1386) and by a Public Health Service research career program award (AI-K3-19335.01) from the National Institut,e of Allergy and Infectious Diseases. 19
increasing rate of DNA synthesis cannot be due to the increasing number of DNA templates available for replication, and the rate-limiting factor in viral DNA synthesis is not the availability of DNA templates; instead, it is probably the enzymes and precursors necessary for the synthesis of DNA. The role of progeny viral DNA as a template for further replication remained, therefore, to be established. Pr virus DNA is of the double-stranded type and replicates semiconservatively (Kaplan and Ben-Porat, 1964). Moreover, infected cells in the stationary phase of growth synthesize viral DNA in excess, so that of the total amount of viral DlYA synthesized by these cells, approximately only 20% is eventually incorporated into mature virus particles (Ben-Porat and Kaplan, 1963). If the remaining 80 % of the viral DNA is in a form in which the DNA is available and competent to act as a template for its own replication, and if viral DNA multiplies exponentially, most of the DNA molecules present in the cell during the early stages of the infective process should replicate thereafter. The present
KAPLAN
I
I.706
t
1.732
I
1
1.745 1.757
DENSITY FIG. 1. Replication of Pr virus DNA. Reading from left to right, the peaks represent RK DNA, Pr virus thymine-DNA, Pr virus hybrid-DNA, and Pr virus BUDR-DNA.
experiments were designed to whether or not this is the case. MATERIALS
establish
AND METHODS
Virus and cell culture. The properties of Pr virus and the cultivation of RK monolayers, as well as the procedure of virus assay, have been described previously (Kaplan, 1957; Kaplan and Vatter, 1959; Ben-Porat and Kaplan, 1962). Since only viral DNA is synthesized by virus-infected RK cells in the stationary phase of growth (Ben-Porat and Kaplan, 1963), cells in this phase of their growth cycle were used in all the experiments. Media. ELS consists of Earle’s saline, 94.5 %; lactalbumin hydrolyzate (Nutritional Biochemicals), 0.5 % ; and dialyzed bovine serum, 5%. EDS medium contains Eagle’s synthetic medium (Eagle, 1959), plus 3 % dialyzed bovine serum. TBSA has the following composition: NaCl, 0.15 M; KCl, 2.7 X lo-* M; CaC12.2Hz0, 8.8 X 1O-4 M; MgC12.6Hz0, 4.9 X LOU4M; tris(hydroxymethyl)aminomethane, 0.1 M (adjusted with HCI to pH 7.5) ; and 1% crystalline bovine serum albumin. Chemicals.Cesium chloride, optical grade, was purchased from the Harshaw Chemical
Company, thymidine from the Mann Research Laboratories, 5-bron~od~xyuridine (BUDR) from the Caiifornia Corporation for Biochemical Research, and crystalline deoxyribonuclease (DNase) from the Worthington Biochemical Corporation. 5-fluorouracil (FU) was a gift from HoffmanLaRoche Incorporated. Thymidine-d-G4 (specific activity, 62 pc/mg) and adenine8-Q4 (specific activity, 25 pc/mg) were purchased from the New England Nuclear Corporation. Pur~~~at~~ of Pr value DNA. DNA was extracted and purified as described previously (Kaplan and Ben-Porat, 1964). Density gradient centrifugation. This procedure has been described previously (Kaplan and Ben-Porat, 1964). In brief, CsCl solutions containing DNA were introduced slowly with a syringe and needle into a 12-mm Kel-F cell with a 1” negative wedge window. The density of the DNA-C&l solutions was approximately 1.70 g/cm”. The densities of the solutions were determined from their refractive indexes by the equation of Ifft et al. (1961). Sedimentation equilibrium was performed with a Spinco model E analytical centrifuge at 25” for at least 20 hours at 44,770 rpm, and the ultraviolet absorption bands were photographed (the exposure time was such that the photographic response was proportional to the concentration of viral DNA). Tracings of the ultraviolet absorption bands were made with a Joyce-Loebl double-beam microphotodensitometer. The DNA of RK cells (1.706 g/cm3) served as a density marker. RESULTS
Proportion I$ V&l DNA That Replicates in Infected Cells
Cultures in the stationary phase of growth were incubated with FU (10 ~g/mI) for 18 hours prior to infection in order to exhaust the intraceIlular supply of thymine and to stop all synthesis of cellular DNA (Kaplan and Ben-Porat, 1961). They were then infected and incubated in EDS containing FU, 5 Mg of BUDR, and 5 rg of thymidine per milliliter. Four and one-half hours after infection, the BUDR-containing medium was replaced by EDS containing thymidine
REPLICATING TABLE REPLICATION OF BUDR-DNA THE FIRST 4.5 HOURS Time of harvest after infection (hours) 7 8 9
Time after addition of thymidine (hours)
POOL
1 SYNTHESIZED AFTER INFECTION” Earlv DNA rep&atedh (?a
2.5 3.5 4.5
FOR
iEzs,v% total viral DNA<
25 38 41
2.0 3.1 4.4
a See text for details. * The areas under the peaks were measured with a planimeter. Since peak B of Fig. 1 contains DNA composed of two strands, one of which consists of BUDR-DNA and thus comes from the DNA in peak C, and one of which consists of normal viral DNA, the percentage of BUDR-DNA that has undergone replication will be equal to:
0.5B ____x 100 C + 0.5B c Calculated as C) = relative
(0.5B +
follows: increase
(A + B in total viral
+ C)/ DNA.
(1 mg/ml) to stop the incorporation of BUDR into DNAz; at periodic intervals thereafter, the cells were harvested, the DNA extracted, and the banding pattern of the DNA in CsCl density gradients was determined in the analytical centrifuge. The tracings of the UV absorption bands of the DNA obtained from these samples are given in Pig. 1. Immediately after BUDR is replaced by thymidine, only one peak of viral DNA is observed; this BUDRcontaining viral DNA has a buoyant density of 1.757 g/cm3. One hour later, a peak of DNA with a density of 1.745 g/cm3 makes its appearance, and thereafter a third peak of DNA with a density corresponding to normal viral DNA (1.732 g/cm3) appears. The peak of intermediate density consists of DNA containing one strand of normal, thymine-containing DNA and one of BUDR-containing DNA and results from the semiconservative mode of replication * Under these conditions the incorporation of thymidine-2-CY4 into DNA is effectively stopped when this label is present in the EDS medium containing BUDR in which the cells are incubated for the first 4.5 hours after infection.
OF
VIRAL
21
DNA
of Pr virus DNA (Kaplan and Ben-Porat,. 1964). The relative amounts of material present in the BUDR-DNA band and in the band of intermediate density can be used as a measure of the amount of BUDR-DNA that has replicated, and the proportion of the BUDRcontaining DXA that has not undergone replication can be calculated (see footnotes to Table 1). These calculations show (Table 1) that by 4 hours after the replacement of BUDR by thymidine, 41% of the BUDRDNA has replicated while there has been an approximately fourfold increase in total viral DNA. Replication oj Viral DNA Synthesized up to Various Times during the Infective Process To determine whether the same proportion of DNA synthesized up to any stage of the infective process replicates thereafter, the following experiment was performed. RK cells were infected and incubated in BUDR-containing EDS (6 pg/ml BUDR + 4 pg/ml thymidine). At various times after infection, the incorporation of BUDR into DNA was arrested by replacing the incubation medium with EDS containing 1 mg of thymidine per milliliter. At the end of 11 hours of incubation (end of cycle), the cultures (cells and virus) were harvested and the DNA was extracted and analyzed in CsCl gradients in the analytical centrifuge. Tracings were made of the UV absorption bands, the areas under the peaks and the proportion of were measured, TABLE AMOUNT
2
OF CONVERSION OF BUDR-VIRAL T O THE HYBRID FORMS
DNA synthesis to the following times (hours)
Conversio;7?
(L See text for * See footnotes
hybrid* 0 41 40 41 31 28 25
4.5 5 5.5 G 6.5 7 details. to Table
1.
DNA
22
KAPLAN
loo .
I& 5 60 !E I: 60 z I-
. l
6Pq
0 IOuq
BUDA
+ 4,uq
Thymldine/ml
Thymidinehl
40
a %
20
0
I
\
1
0
2
4
6
6
10
BUOR
10
6
6
4
2
0
THYHIMNE
Pq/ml pWnl
FIG. 2. Growth of Pr virus in the presence of BUDR. Monolayers of RK cells were incubated with FU (25 pg/ml) for 24 hours prior to infection. The cultures were infected, virus was allowed to adsorb for 60 minutes, unadsorbed virus was removed by washing, and EDS containing FU, plus varying amounts of BUDR and thymidine was added to different sets of cultures. The cultures were harvested 18 hours after infection and the amount of infectious virus present in each culture was assayed by the plaque method.
14
FIG. 3. Incorporation of adenine&CY4 into the DNA of virus-infected cells incubated in the presence of thymidine or BUDR. Monolayers of RK cells were incubated with EDS containing FU (10 pg/ml) for 18 hours. The cultures were infected (adsorbed multiplicity = IO) ; to one set of cultures was added EDS containing thymidine (10 Mg/ml) and FU (10 .g/ml), and to another EDS containing BUDR (6 pg/ml) and thymidine (4 pg/ml). Adenine&Cr4 (0.4 pc/ml) was added to the cultures 1 hour after infection. At periodic intervals thereafter, the cultures were harvested and the amount of adenine-8.Cl4 incorporated into DNA was determined as described previously (Kaplan and Ben-Porat, 1961).
BUDR-DNA that had replicated was calculated as described above. The results show (Table 2) that as the infectious cycle proceeds, an increasingly smaller proportion of the viral DNA within the cell replicates. Thus, for example, of the DNA made up to 5.5 hours after infection 41% replicates, whereas only 25% of the DNA made up to 7 hours replicates thereafter, despite the fact that there is active Dn’A synthesis up to ,the time of harvest (see Fig. 3). These 1. The experiment described above, sumresults cannot be due to the withdrawal of marized in Table 1, was repeated except that viral DNA and its integration into mature thymidine (10 pg/ml) was added for the virus particles since, as shown previously first 4.5 hours (so that the early DNA con(Ben-Porat and Kaplan, 1963), a maximum sisted of normal viral DNA) and was then of only 20% of the total viral DNA is in- replaced by medium containing BUDR (600 tegrated into particles which behave upon pg/ml BUDR + 400 pg/ml thymidine). differential centrifugation and in gradients The cells were harvested, and the DNA was of CsCl like infectious virus. extracted and banded in CsCl gradients. The fact that a large proportion of the Essentially the same results were obtained DNA does not replicate is not due to the as in the reverse experiment (as summarized experimental conditions used. That the in Table 1) and only 38 % of the early degree of substitution used in these experi- thymine-containing DNA had replicated ments of BUDR for thymidine in the DXA by 9.5 hours after infection. 2. The rate of incorporation of adeninedoes not interfere with the replication of viral DNA, although it reduces the number g-Cl4 into the DNA of cells incubated with of infective centers by approximately 50% thymidine and with BUDR was deter(Fig. 2), is shown by two methods: mined. The results (Fig. 3) show that the
REPLICATING
POOL
BUDR does not interfere with adenine incorporation into DNA and that therefore it is presumably without effect on the rate of synthesis of DNA. Another possible source of interference with DNA synthesis in our experiments may reside in the use of medium containing a high concentration of thynlidine (1 mg/ml). It is known that large amount of this nucleoside arrest the synthesis of DNA by interfering with the formation of deoxycytidylic acid (Reichard et al., 1961; Morris et al., 1963). However, although DNA synthesis in noninfected RK cells is indeed inhibited by a high concentration of thymidine, it remains without effect on the rate of DNA synthesis in Pr virus-infected cells (unpublished results). Thus, neither the fact that BUDR is substituted for thymidine in viral DNA nor the presence of high concentrations of nucleosides in the medium seem to interfere with the rate of DNA synthesis in the infected cells, and the relatively small proportion of viral DKA which undergoes replication in our experiments is therefore not the result of the experimental conditions used.
Since an increasingly smaller proportion of the viral DNA replicates as the infective process proceeds (see Table 2), the possibility was considered that even though viral DNA is not yet part of and/or cannot be isolated with mature virus particles, a major part of it is rapidly transformed into a no~eplicative state. That this may be the case is shown in the next experiment in which the sensitivity of viral DNA to the action of DNase was tested. RK cultures in the stationary phase of growth were infected and incubated in ELS at 37”. To part of the cultures thymidined-C4 (0.04 1~c/ml) was added at 3 hours, and to part at 4 hours after infection. One hour later, incorporation was arrested by the addition of a large excess of unlabeled thymidine (1 me/ml). At various times thereafter, cultures were harvested and sonicated. Part of the samples was used to
OF
VIRAL
23
DNA
determine the fraction of radioactive vi& DNA insensitive to the action of DNase (50 pg/ml), and part to determine the fraction of radioactive viral DNA banding in CsCl density gradients in the position characteristic of infectious virus particles. The results of this experiment are illustrated in Fig. 4. Xost of the radioactive DNA, i.e., the viral DNA synthesized up to 5 hours after infection, is sensitive to DNase up to approximately 5 hours after infection. Thereafter, it rapidly becomes insensitive to the action of the enzyme and by 8 hours after infection, approximately 60% of the radioactive viral DNA is no longer sensitive to the enzyme; at this time, however, only 5-6 % of this DNA can be isolated in a CsCI gradient in the same position as infectious virus. Thus, soon after its synthesis a large part of the viral DNA is converted into a form that is resistant to the action of DNase, even though it does not band with mature virus particles in CsCl gradients. Loss of DNA jTorn> Virus nipulatiolz
Particles
by Ma-
Since the kinetics of the formatZion of DBase-insensitive viral DNA, and of the incorporation of viral DNA into material banding with the virus in C&l gradients are quite different and, in fact, suggest a precursor relationship between the two, it is unlikely that the two forms are identical and that the difference in the total amount of radioactive DNA found in each is due to a loss of DNA from the virus particles during the isolation procedure in the CsCl gradients. Nevertheless, it seemed advisable to deterl~~ine the proportion of DNA recovered in the CsCl band when extrac~llular mature .? virus particles and DNase-insensitive material isolated from the cells are centrifuged in CsCl gradients. Mature virus particles, the DNA of which was labeled with thymidine-2-C’*, were prepared as follows. Infected cells were incubated with thymidine-,?-C14 (0.04 ~c/mI), and at the end of the growth cycle the virus in the supernatant fluid was collected. The cellular debris was removed by centrifugation, and the supernatant fluid was treated with DNase (50 fig/ml) to
24
KAPLAN TABLE
FRACTION THE
OF RADIOACTIVE VIRUS BAND IN CSCL
1 2 3
RECOVERED GRADIENTS”
DENSTY
Extracellular mature virus (% local input)
Experiment
a See text
3 DNA
Intracellular DNase-insensitive material (% total input)
65 91 61 for
IN
6 7 5
details.
. i5 _ m 6o I 1 50 -
2g l-
40-
&
JO-
5
20r
Infected cells were incubated with thymidine-MY4 (0.04 /*c/ml) between 3 and 4 hours after infection. They were then incubated for an additional 3 hours in the presence of an excess of C12-thymidine (1 mg/ml). The samples were harvested by scraping the cells into the supernatant fluid and sonicated to disrupt the cells. The samples were treated with DNase (50 pg/ ml), centrifuged, and sonicated again, as described above for the virus preparations. Both sets of samples were assayed for total radioactivity in the DNA, and the proportion of total counts banding with infectious virus in gradients of CsCl was determined. Table 3 summarizes the results. A relatively large variation in recovery was found between different runs and, depending upon the experiment, between 91% and 61% of the radioactivity was found in the band in CsCl of infectious virus; however, only 6 % of the total DNase-insensitive intracellular radioactive DNA was found associated with this band. Thus, even if we allow for a maximum loss of DNA of 50% because of the process of banding the virus in CsCI, only 12% of the DNase-insensitive DNA present intracellularly can be accounted for as mature virus. DISCUSSION
6
TIME
8
IO
I2
14
AFTER INFECTION (HOURS)
FIG. 4. DNase sensitivity of viral DNA and incorporation of viral DNA into mature virus. The fraction of counts associated with DNA which is DNase-insensitive was determined by treating the samples with DNase, by precipitating them with acid, and after appropriate washings, by determining the number of counts in the acid-precipitable material. Thymidine-&-Cl” present between 3 and 4 hours (0 and A); thymidine-2-CY4 present between 4 and 5 hours (0).
remove any contaminating DNA. The virus was sedimented in the Spinco model L (no. 30 head) at 25,000 rpm for 1 hour, was resuspended in TBSA, and was sonicated for 1 minute to break up clumps. Preparations containing DNase-insensitive viral DNA were prepared as follows.
The experiments in this paper demonstrate that within the infected cell filial viral DNA can serve as a template for its own replication. If the total population of viral DNA molecules that are not yet associated with mature virus were part of a pool of replicating DNA, from which a particular DNA molecule had a random chance to act as a template for its own replication, most of the molecules synthesized, for example, up to 4.3
hours
after
infection
should
replicate
in
the following 4 hours. This would be expected, since only a small part of this early DNA is withdrawn and integrated into mature virus and there is during this time a fourfold increase in total viral DXA. However, only approximately 40 % of this DNA replicates. The rapid and extensive integration of viral DNA into a form insensitive to the action of DNase may be responsible for the
REPLICATING
POOL
failure of the major part of the DXA to replicate. The kinetics of formation suggest that this form bears a precursor relationship to mature virus, since between 8 and 12 hours after infection there is an increase of approximately only 8 % in total DNaseinsensitive DNA, while there is an incorporation of approximately 13 % of DiYA into mature virus (see Fig. 4). The DNase insensitive form may be an incomplete form of virus, which is intermediate in the formation of mature virus, the kind of precursor, perhaps, envisaged for T2 phage by Hershey and Melechen (1957). However, it may also represent mature virus contained within membranous structures of the kind described by Horne and Kagington (1959) for poliovirus. Thus, it is possible that virus is synthesized in close contact with such structures, from which it cannot be released by sonication, thereby acquiring different sedimentation and density characteristics from those of released virus; during the later stages of the growth cycle, virus would be freed slowly from these structures. We are now engaged in determining the identity and characteristics of this DBase-insensitive form. ACKNOWLEDGMENT The ckyj
excellent is greatly
assistance appreciated.
of Mr.
Bohdan
Kerny-
REFERENCES BEN-P• RAT, T., and K.~PLSN, A. S. (1962). The chemical composition of herpes simplex and pseudorabies viruses. Virology 16, 261-266. BEN-PoRBT, T., and KAPLAN, A. S. (1963). The synthesis and fate of pseudorabies virus DNA in infected mammalian cells in the stationary phase of growth. Virology 20,310-317. EAGLE, H. (1959). Amino acid metabolism in mammalian cell cultures. Science 130,432-437.
OF
VIRAL
DNA
25
HERSHEY, A. D., and MELECHEN, N. E. (1957). Synthesis of phage-precursor nucleic acid in the presence of chloramphenicol. Virology 3, 207236. HORNE, R. W., ~~~NAGINGTON, J. (1959). Electron microscope studies of the development and structure of poliomyelitis virus. J. MoZ. Biol. 1, 333-338. IFFT, J. B., POET, D. H., and VINOGRAD, J. (1961). The determination of density distributions and density gradients in binary solutions at equilibrium in the ultracentrifuge. J. Phys. Chem. 65, 11381145. K.IPL.IN, A. S. (1957). A study of the herpes simplex virus-rabbit kidney cell system by the plaque technique. Virology 4, 435457. KAPLAN, A. S. (1962). Analysis of the intracellular development of a DNA-containing mammalian virus (pseudorabies) by means of ultraviolet light irradiation. Virology 16, 305313. KAPLAN, A. S., and BEN-PORST, T. (1961). The action of 5-fluorouracil on the nucleic acid metabolism of pseudorabies virus-infected and noninfected rabbit kidney cells. Virology 13, 78-92. KAPLAN, A. S., and BEN-P• RAT, T. (1963). The pattern of viral and cellular DNA synthesis in pseudorabies virus-infected cells in the logarithmic phase of growth. Virology 19, 205-214. K.IPL.IN, A. S., and BEN-PoR.~T, T. (1964). Mode of replication of pseudorabies virus DNA. Virology in press. 23, 90-95. KAPL~~N, A. S., and VATTER, A. E. (1959). A comparison of herpes simplex and pseudorabies viruses. Virology 7, 394407. MORRIS, N. R., REICHARD, P., and FISCHER, G. A. (1963). Studies concerning the inhibition of cellular reproduction by deoxyribonucleosides. II. Inhibition of the synthesis of deoxycytidine by thymine, deoxyadenosine and deoxyguanosine. Biochim. Biophys. Acta 68, 93-99. REICHARD, P., CANELLAKIS, Z. N., and CANELLAKE, E. S. (1961). Studies on a possible regulatory mechanism for the biosynthesis of deoxyribonucleic acid. J. Biol. Chem. 236, 2514-2519.