Poxvirus DNA

VIROLOGY

72,

134-146 (1976)

Poxvirus iI. Replication

of Vaccinia Virus DNA in the Cytoplasm of HeLa Cells

J. A. HOLOWCZAK’ Department

of Microbiology,

DNA

College

of Medicine Piscataway, Accepted

AND LEE DIAMOND and Dentistry of New New Jersey 08854

February

Jersey,

Rutgers

Medical

School,

27,1976

Virions, purified by Method I (containing genomes which lack cross-links and appear fragmented when analyzed in alkaline sucrose gradients) or by Method II (containing cross-linked genomes which sedimented at -102 S in alkaline sucrose gradients with no evidence of fragmentation) were found to be equally efficient in replicating their DNA in the cytoplasm of HeLa cells. Replication of vaccinia DNA in the cytoplasm of infected cells was discontinuous; that is, short pulses of 13Hlthymidine were incorporated into fragments that sedimented at about 20 S in alkaline sucrose. As the pulse length increased, label was found in material which sedimented faster than 20 S but more slowly than the strands of mature viral DNA. Molecules sedimenting at 30-40 S in alkaline sucrose appeared to accumulate with longer pulses. During a “chase” in cold medium, part of these molecules were converted to full-length molecules sedimenting at 72-75 S and into mature, cross-linked genomes sedimenting at 102-106 S in alkaline sucrose. Chromatography on BNDcellulose and hydroxyapatite columns demonstrated that replicating viral DNA had single-stranded regions, but such molecules could not be separated from mature genomes by sedimentation in CsCl gradients. No nascent strands of greater than unit length were detected indicating that replication of the vaccinia genome probably does not involve continuous concatemers or the addition of new material to parental strands. This suggests that polymerization may be symmetrical. Evidence for a circular intermediate, with nicks in both strands, was obtained by the analysis of “pulse” and “pulsechase” labeled replicating viral DNA molecules by centrifugation in neutral sucrose gradients. DNA species sedimenting 12-15% more rapidly than parental genomes, at 8286 S, appropriate for such a “nicked” circular intermediate were found, in which label accumulated during a chase before the appearance of mature genomes at 68-72 S. INTRODUCTION

Green et al., 1964; Joklik and Becker, 1964). The exact time course depends on the multiplicity of infection. A number of enzymatic activities which are clearly involved or can be implicated in the replication of the poxvirus genome have been detected in the cytoplasm of infected cells. These are early enzymes, coded for by parental genomes and include thymidine kinase (McAuslan, 1963; Kitt and Dubbs, 1965), DNA polymerase (Jungwirth and Joklik, 1965; Magee and Miller, 19671,polynucleotide ligase (Sambrook and Shatkin, 19691, and a number of nucleases (Jungwirth and Joklik, 1965; McAuslan, 1965; McAuslan and Kates, 1966; Mc-

The replication of poxvirus DNA proceeds in the cytoplasm of infected cells in association with cytoplasmic inclusions or “factories” (Cairns, 1960; Dales and Siminovitch, 1961; Dales, 1963; Kato et al., 1964). In HeLa or L cells, infected with vaccinia virus, DNA synthesis begins at about 1.5 hr after infection, reaches a maximum at about 2.5-3 hr and is 90% or more complete by 4-5 hr post infection (Magee et al., 1960; Salzman, 1960; Hanafusa, 1961; Green, 1962; Shatkin and Salzman, 1963; 1 Author to whom requests for reprints addressed.

should be 134

Copyright All rights

0 1976 by Academic F’rees, Inc. of reproduction in any form reserved.

REPLICATION

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VACCINIA

Auslan and Kates, 1967). There is also experimental evidence for a protein (or proteins) in addition to those described above, coded for by relatively short-lived messenger RNA species, which is also required for DNA replication but its exact function is not known (Kates and McAuslan, 1967). Soon after replication of the viral DNA is complete or perhaps during replication, progeny genomes become associated with .virus-specific polypeptides and can be recovered under the appropriate ionic conditions as rapidly sedimenting DNA-protein complexes or virosomes (Dahl and Kates, 1970). The exact relationship between these complexes and the very large aggregates of DNA which require magnesium ions for their stability described by Joklik and Becker (1964) remains to be established. Few studies have addressed themselves to the mechanism of poxvirus DNA replication in uiuo. Magee and Levine (19701, in studying the effects of interferon on vaccinia virus replication in chick embryo fibroblasts, demonstrated that viral DNA was synthesized in short segments which elongated with time during pulse-chase experiments. The largest ssDNA segments detected in these studies after extended chase periods sedimented at about 40 S in alkaline sucrose gradients, which correspond to about 20% of the length of a mature complementary strand of vaccinia DNA. We have studied the replication of vaccinia DNA genomes in the cytoplasm of HeLa cells. DNA synthesized in short pulses and in pulse-chase experiments was analyzed by sedimentation in neutral and alkaline gradients, by equilibrium-density centrifugation in CsCl and chromatography on hydroxyapatite and BND-cellulose columns. The results of these experiments will demonstrate that the genome of vaccinia virus is synthesized discontinuously, with the newly synthesized segments of the DNA being tightly associated with their template. Pulse-chase experiments show that the DNA segments synthesized in a short pulse can be chased into mature, cross-linked DNA molecules which sediment at about 102-106 S in alkaline sucrose gradients. Evidence for the presence

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of noncovalently closed circular molecules which may be intermediates involved in the replication or maturation of vaccinia virus DNA will be presented. MATERIALS

AND

METHODS

Virus and cells. The WR strain of vaccinia virus and HeLa S3 cells propagated in spinner culture were employed in these experiments. Cell growth and infection of cells were carried out as described previously (Becker and Joklik, 1964; Oda and Joklik, 1967; Holowczak, 1972). Purification of vaccinia uirions. The virus preparations used in these studies were purified by the two methods described previously (Holowczak, 1975). For the majority of experiments a large stock of virus, purified by Method I, containing 2 x 1O1’ EB (elementary bodies) per ml and having an EB/PFU ratio of 4O:l when titrated on primary chick embryo tibroblasts was employed. Release

of DNA

from

mature

virions.

Aliquots of virus preparations were diluted into 1O-3 M Tris-HCl, pH 7.2, containing 0.001 M EDTA so that the final concentration of viral DNA was 0.3-0.5 kg/ml. The viral suspension was treated with Sarkosyl NL-97 (2%), deoxycholate (O.l%), and 2-mercaptoethanol (0.01 M), and incubated for 15 min at room temperature before sedimentation analysis. Preparation of cytoplasmic samples for analysis of viral DNA. The procedure of

Dahl and Kates (1970) was employed to prepare cytoplasmic extracts from infected or mock-infected, control cells. Briefly, cells were harvested and washed in 0.15 M NaCl containing 0.05 M Tris-HCl, pH 8.0, and EDTA, 0.005 M. The cells were resuspended in 0.01 M Tris-HCl, pH 8.0, containing 0.005 M EDTA and 0.01 M KC1 at a concentration of 3.5 x 10’ cells/ml. After standing at 2” for 5 min, the cell suspension was Dounced or the cells lysed in Triton X-100 (final concentration 1%). The cell lysate was then centrifuged at 800 g for 2 min to collect nuclei and the cytoplasmic fraction removed. It should be noted that when the cells were broken by Dounce homogenization it was essential to determine the number of strokes necessary to disrupt cells without breaking nuclei. This

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was accomplished by Douncing uninfected cells prelabeled with 13H]thymidine and monitoring the breakage of cells microscopically and following the release of labeled DNA from nuclei. As demonstrated by Dahl and Kates (1970) and reproduced in this laboratory endogenous nuclease degradation of labeled vaccinia DNA was minimized under the conditions described above. Velocity gradient centrifugation of DNA samples. Neutral DNA buffer consisted of 1 M NaCl, 0.01 M EDTA, 0.15% Sarkosyl NL-97 in 0.05 M Tris-HCl, pH 7.5. Alkaline DNA buffer consisted of 0.7 M NaCl, 0.3 N NaOH, 0.01 M EDTA, and 0.15%

Sarkosyl NL-97. For analysis, 16 ml, 520% (w:w) gradients in the appropriate buffer were prepared and samples containing 2-8 x lo6 cytoplasmic equivalents treated with Sarkosyl (2%), deoxycholate (O.l%), and 2-mercaptoethanol (0.01 M) were loaded onto the gradient. To denature the DNA in cytoplasmic samples, the samples after detergent treatment were rendered 0.3 M in respect to NaOH and incubated at room temperature for 15 min. Gradients were centrifuged in the Spinco SW27.1 rotor, 25,000 rpm, 20”, 5-6 hr, fractionated, the fractions were precipitated with TCA, the precipitates were collected on Millipore filters, dried, and the distribution of radioactivity was determined by liquid scintillation spectrometry in a toluene-based scintillator. The sedimentation coefficients assigned to the various DNA species detected in these studies were calculated according to the methods of Studier (1965) and Burgi and Hershey (1963), using adenovirus type 2 DNA as a sedimentation marker (Burlingham and Doerfler, 1971). Analysis of DNA samples on hydroxyapatite and BND-cellulose columns. DNA

was purified from the cytoplasmic fraction of infected cells as described previously (Oda and Joklik, 1967; Sarov and Becker, 1967). DNA samples were dissolved in 0.01 M phosphate buffer, pH 6.8, containing 0.1% Sarkosyl NL-97 and analyzed on hydroxyapatite columns (Bernardi, 1971; Holowczak, 1975) or were dissolved in 0.3 M NET buffer (0.3 M NaCl, 0.01 M Tris

AND DIAMOND

buffer, pH 7.5, and 1O-4 M EDTA) containing 0.1% Sarkosyl and analyzed on benzoylated napthoylated DEAE-cellulose columns (Kiger and Sinsheimer, 1969; Schlegel and Thomas, 1972; Bellet and Younghusband, 1972; Holowczak, 1975). Conditions for pulse-chase experiments.

Short pulses with radioisotopes were terminated by pouring the cell culture over an equal volume of crushed frozen buffer which was to be employed in the washing of cells. In pulse-chase experiments, the pulse was arrested by the addition of 2-2.5 x 10e3 M. cold thymidine, the cells collected by centrifugation, and resuspended in fresh, warm medium containing 2-2.5 x 10m3 M cold thymidine, Groyon and Kniazeff (1967) have demonstrated that vaccinia virus DNA synthesis in synchronized pig kidney cells was blocked in the presence of 7 x 10m3M thymidine but not 2 x 1O-3 M thymidine. Reagents and radioisotopes. Pancreatic ribonuclease and electrophoretically purifled deoxyribonuclease were obtained from the Worthington Biochemical Corp., New Jersey. Nonidet P-40 was a gift of the Shell Oil Chemical Co., Ltd., England; Sarkosyl NL-97 was obtained from the Geigy Chemical Co., Ardsley, New York. The [14Clthymidine (56.5 mCi/mmole) and 13Hlthymidine (20 Cilmmole) were purchased from the New England Nuclear Corp., Boston, Massachusetts. RESULTS

Kinetics of Vaccinia DNA Infected HeLa Cells

Synthesis in

In Fig. 1, the kinetics of DNA synthesis in HeLa cells infected with virions purified by Method I or Method II have been compared. No major differences in the kinetics of DNA synthesis were observed. It was concluded that both methods of purification produced virus stocks with comparable infectivity as measured by their ability to replicate their DNA. In the experiments to be described, the virus stock purified by Method I, described in Materials and Methods, was used, and cytoplasmic DNA synthesis between 1.75-3.5 hr postinfection, the period of maximum viral DNA

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each containing 5 x 10’ cells. The subcultures were then pulsed for various periods at the rate of 3.0-5.0 &i [3Hlthymidine/ lo6 cells. The pulse was terminated by pouring the culture over 100 ml of frozen, Analysis of Viral DNA Synthesized in finely crushed buffer (0.15 M NaCl; 0.005 Short-Pulses M Tris-HCl, pH 8.0; EDTA, 0.005 M). CyHeLa S3 cells were infected with vac- toplasm was prepared by lysis of the cells cinia virus strain WR at a multiplicity of with Triton X-100, aliquots of the cytoplasm were removed and treated with Sar1000 EB/cell. Two hours after infection, kosyl (2%); DOC (sodium deoxycholate) the culture was divided into subcultures (0.1%); 2-mercaptoethanol (0.01 M); and NaOH (0.3 M) for 15 min at room temperature. To some samples, [14C]thymidine-labeled Ad2 DNA was added as an internal sedimentation marker and the samples were analyzed by sedimentation in 5-20% alkaline sucrose gradients. The results of such experiments are shown in Fig. 2. After a 30-60 set pulse DNA species sedimenting at about 15-20 S (major peak) and 28-34 S (minor peak) were detected (Fig. 2A). With a five-minute pulse the major peak of radioactivity had shifted to 25-28 S with some species still sedimenting at 1520 S and a minor peak at about 34 S. With longer pulses a peak of 13H]thymidine-labeled material sedimenting at 30-40 S was dominant and only after extended pulses of 40-60 min (Fig. 2B) were DNA species sedimenting at 65-70 S detected. The 65-70 S species would represent molecules 50-60 0123456 x lo6 daltons in molecular weight (Burgi Time (Iid and Hershey, 1963) and could therefore FIG. 1. Kinetics of DNA synthesis in the cyto- represent mature, but not cross-linked geplasm of HeLa cells infected with virions purified by nomes (Geshelin and Berns, 1974; HolowMethod I as compared to virions purified by Method czak, 1976). However, such molecules repII. Virions were purified by Method I or Method II as resented only 5-8% of the total radioactivdescribed previously (Holowczak, 1975). HeLa cells ity incorporated during these long pulses. (2 x lOa) were infected at a multiplicity of 1000 EB/ of infected cells for 16 hr after cell as described in Materials and Methods, and Labeling infection with [3H]thymidine followed by were diluted into complete medium at a concentraanalysis of the cytoplasm from such cells tion of 1 x lo6 cells/ml. Mock-infected cultures were in alkaline sucrose gradients to determine similarly established. At the time points indicated, 2 x 10’ cells were removed from each culture and the nature of the genomes present, propulsed for 20 min with [3H]thymidine (1 $i/106 duced the result shown in Fig. 3. Here, as cells), the pulse terminated, and the cytoplasmic in the case of the long pulse experiments, fraction from cells prepared by lysis with Triton XDNA species sedimenting at 30-40 S made 100 as described in Materials and Methods. Aliquots up the largest percentage of the total TCA (0.2 ml) of the cytoplasmic fraction were removed precipitable radioactivity (65-70%). The and radioactivity incorporated into TCA-precipitaremainder sedimented as mature viral ble material assayed for by liquid scintillation specDNA strands (60-75 S) and mature, crosstrometry. O-O, Cells infected with virions purilinked DNA genomes (102-106 S). The posfied by Method I; -0, cells infected with virions purified by Method II; W-4, mock-infected cells. sibility that the low molecular weight replication, was analyzed (Fig. 1). In all experiments, mock-infected control cultures were established and were treated in the same manner as the infected cells.

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DNA species present in such preparations arose due to the action of nucleases in the cytoplasmic samples cannot be completely excluded. Under the conditions of analysis used in these studies degradation of labeled DNA to acid-soluble material was minimal (Dahl and Kates, 1970) but “nicking” of DNA could occur. When DNA from virions isolated by Method II was added as a marker to cytoplasmic samples it was not degraded to low molecular weight fragments. We concluded from these experiments that vaccinia DNA was synthesized dis-

i-

/

B

1 FIG. 2. Alkaline sucrose gradient sedimentation analysis of replicating viral DNA labeled in short and long pulses with r3H]thymidine. HeLa S3 cells were infected with vaccinia virus (1000 EB/cell) and 2 hr after infection the culture was divided into subcultures each containing 5 x 10’ cells. Beginning at 2.5 hr after infection, the cultures were pulsed with [3Hlthymidine (4.5 &i/lo6 cells) and the pulse terminated by rapidly mixing the culture with 100 ml of frozen, finely crushed buffer (0.15 M NaCl; 0.05 M Tris-HCl, pH 8.0; EDTA, 0.005 Ml. Cells were harvested by centrifugation, cytoplasm prepared as described in Materials and Methods, and 4-8 x 10” cytoplasmic equivalents treated with Sarkosyl (2%), DOC (O.l%), 2-mercaptoethanol (0.01 M), and NaOH (0.03 M) for 15 min at room temperature. To some samples [‘4C]thymidine-labeled vaccinia virions purified by Method II or purified [*4Clthymidinelabeled Ad-2 DNA were added to provide sedimentation markers. The insert, Panel A shows the incorporation of 13H]thymidine into TCA-precipitable material with increasing “pulse” times. Experiment I, A-A; Experiment II, O-O. Panel A: Samples pulsed for: O-O, 1 min; O--O, 5 min; x-x, 15 min; O-0, 30 min. The results from

0

IO

20 Fraction

30

FIG. 3. Analysis of vaccinia virus DNA in the cytoplasm of infected cells labeled with 13Hlthymidine, 0.15 to 16-hr postinfection. HeLa cells were infected at a multiplicity of 1000 EB/cell and 0.15 hr after infection [3H]thymidine (1 &i/lo6 cells) was added, and 16 hr later the cells were harvested and cytoplasm was prepared by lysis with Triton X-100 as described in Materials and Methods. Aliquots (5 x lo6 cytoplasmic equivalents) were treated with detergents, 2-mercaptoethanol, and 0.3 M NaOH, and analyzed by sedimentation in alkaline sucrose gradients as described in Materials and Methods.

four separate gradients have been plotted together. Panel B: Cytoplasmic sample prepared from infected cells, pulsed for 40 min with [3H]thymidine, beginning 2.5-hr p.i. was mixed with [‘“Clthymidine-labeled virions purified by Method II before addition of detergents, 2-mercaptoethanol, and NaOH. O-0, [‘CjThymidine-labeled DNA from virions; O-O, DNA species labeled in 40-min pulse; x-x, mock-infected control sample pulsed for 40 min and analyzed in a separate gradient.

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Evidence for ssDNA Species as Intermedicontinuously. Although their exact size reates in the Replication of Vaccinia mains to be critically determined, labeled DNA molecules sedimenting at about 15-20 S were detected after the shortest pulses Infected HeLa cells were pulse-labeled with [3Hlthymidine. Such molecules as described in Fig. 2, and 13Hlthymidinewould be larger than the classical Okazaki labeled vaccinia DNA was purified from fragments (Okazaki et al., 1968; Sugino the cytoplasmic fraction as described in and Okazaki, 1972; Hirose et al., 1973) and Materials and Methods and in Fig. 2. The the intermediates which occur during the replication of Simian Virus 40 (Fareed et purified viral DNA samples were then analyzed by chromatography on hydroxyapaal., 1973) and polyoma virus DNA (Magtite and BND-cellulose columns and the nusson et al., 1973; Francke and Hunter, 1974). Magee and Levine (1970) reported results are shown in Figs. 4 and 5. In Fig. molecules of the size detected in these ex4A, the elution pattern obtained when periments, labeled after short-pulses, in heat-denatured [~~Hlthymidine-labeled vaccinia DNA, isolated and purified from their studies of vaccinia DNA replication in CEF. When the replication of DNA in vii-ions, was chromatographed on hydroxyMouse P-815 cells was examined, Gautschi apatite is shown. Greater than 90% of the and Clarkson (1975) detected lo-20 S DNA radioactivity, adsorbed to the column, was segments which were labeled after pulses eluted with 0.14-o. 16 M salt, characteristic of 60 sec. They were able to demonstrate of ssDNA species (Bernardi, 1971). Purithat these arose by joining of “Okazaki” fied native viral DNA eluted with 0.24fragments, 2.5-4.5 S in size, during the 0.26 M salt, characteristic of dsDNA (Fig. pulse. It remains to be established if the 4B). When viral DNA, labeled in a l-3 min molecules described here arise by a similar pulse, isolated and purified from infected mechanism. cell cytoplasm, was analyzed on hydroxyThe molecules sedimenting at 30-40 S apatite, the elution pattern shown in Fig. which appear to accumulate during longer 4C was obtained. About 20-30% of the total pulses are similar to the “replicon” sized TCA precipitable radioactivity adsorbed to intermediates reported in studies of DNA the column eluted with 0.14-0.18 M salt, replication in mammalian cells (Gautschi characteristic of ssDNA species, the rest et al., 1973; Gautschi and Clarkson, 1975). of the adsorbed DNA eluted with 0.24-0.28 Further, the data indicate that during M salt, characteristic of dsDNA. When shorter pulse times, incorporation of infected cells were pulsed with [:%Hlthy13Hlthymidine occurs preferentially into midine and then chased for 30 min shorter DNA strands, in agreement with with cold thymidine as described in Mateprevious reports showing a correlation be- rials and Methods, the amount of ssDNA tween the length of the pulse and the size recovered after hydroxyapatite chromatogof the labeled DNA strands (Tsukada et raphy was found to decrease while the raal., 1968; Habener et al., 1969; Schandl and dioactivity recovered in the dsDNA fracTaylor, 1969; Bellett and Younghusband, tion increased proportionately (Fig. 4D). 1972) in those eukaryotic systems where To further characterize the pulse-labeled DNA appears to be replicated in a disconviral DNA species, chromatographic analtinuous fashion. Finally, the growing ysis on BND-cellulose was carried out as strands of vaccinia DNA sedimented more described in Fig. 5. No pulse-labeled DNA slowly than those of the mature DNA in species were eluted with 1.0 M NET buffer; alkaline sucrose. This would argue against where dsDNA would be expected to elute methods of replication involving continu(Iyer and Rupp, 19711, 20-30% of the laous concatemers or the addition of new beled molecules eluted with 2% caffeine in material to parental strands as in the roll1.0 M NET buffer characteristic of ssDNA ing circle model (Gilbert and Dressler, or dsDNA containing significant ssDNA 1968). sequences. The major portion of the pulse-

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40

60

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FIG. 4. Chromatographic analysis on hydroxyapatite columns of pulse-labeled replicating viral DNA, purified from the cytoplasm of infected cells. Vaccinia DNA was released from [3H]thymidine-labeled virions and purified by phenol extraction as described in Materials and Methods. Ethanol precipitated viral DNA samples were collected by centrifugation and dissolved in 0.01 M phosphate buffer, pH 6.8, containing 0.1% Sarkosyl at a concentration of about 3.1 pg/ml. Part of the preparation was sonicated and heated at 100” for 2 min and flash-cooled in an acetone-dry ice bath to denature the DNA. HeLa cells infected with vaccinia virus were pulse-labeled for 3 min at 2.5-hr postinfection, with [3H]thymidine (5 &i/lOG cells) or pulsed for 3 min and chased with “cold medium” for 30 min. The cytoplasmic fraction from such cells was prepared as described in Fig. 2. Conditions for the chase with “cold medium” are detailed in Materials and Methods and in Fig. 6. Pulse or pulse-chase labeled DNA from 5 x 10’ cytoplasmic equivalents was solubilized by treatment with Sarkosyl (2%), DOC (O.l%), and 2-mercaptoethanol (0.1 M) and purified by phenol extraction. Ethanol-precipitated DNA samples were dissolved in 0.01 M phosphate buffer, pH 6.8, containing 0.1% Sarkosyl. Samples were analyzed on hydroxyapatite columns as described in Materials and Methods. Panel A: Heat-denatured viral DNA, Panel B: Native viral DNA; Panel C: Replicating viral DNA, pulse-labeled for 3 min; Panel D: Replicating viral DNA, pulse-labeled for 3 min followed by a 30-min chase with “cold medium.” Greater than 90% of the DNA applied to the column (in terms of radioactivity) adsorbed to the column and was not eluted by the preliminary wash with 0.01 M phosphate buffer, pH 6.8.

labeled DNA molecules were not eluted until denatured with 0.1 N NaOH. This elution pattern was similar to that reported for replication CELO virus DNA genomes (Bellet and Younghusband, 1972). The nature of the molecules eluted with 0.1 N NaOH has not been completely determined (Upholt and Borst, 1974; Holowczak, 1976). We concluded from these experiments that ssDNA segments were intermediates in the replication of vaccinia DNA. We were unable to detect pulselabeled DNA species in the cytoplasm of infected cells which banded at a higher density in CsCl than purified viral DNA. Such species, which have been recovered from adenovirus infected cells, (Pettersson, 19731, have been shown to be replicating DNA molecules with significant ssDNA regions. The ssDNA species we have detected may be tightly associated

with their template within the cell and their release may be dependent on the conditions used to extract and purify the DNA (Habener et al., 1975). Analysis of Vaccinia DNA Labeled in Pulse-Chase Experiments

HeLa S3 cells were infected at a multiplicity of 1000 EB/cell with vaccinia virus, and at 2.15 hr after infection the cells were pulsed for 60 set with L3Hlthymidine (2 &i/lo6 cells). Cold thymidine, 2.5 mM, was added to the culture and an aliquot (5 x 10’ cells) was removed and poured over frozen, finely crushed wash buffer. When carried out in this manner, the pulsed sample contained DNA labeled for the period of the pulse and a short 30-60 set chase with cold thymidine before incorporation of label was completely arrested. The remainder of the culture was centri-

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40 S were the major labeled species detected (Fig. 6A). With chasing, the radioactivity was found associated with more rapidly sedimenting species suggesting that the pulse-labeled DNA molecules were elongated with time. With a 40-60 min chase, mature genomes sedimenting at 65-75 S, were present (Fig. 6B). With extended chases of up to 120 min, a significant accumulation of genomes sedimenting at 102-106 S was observed (Fig. 60 which would represent mature, crosslinked vaccinia genomes (Berns and Silverman, 1970; Geshelin and Berns, 1974; Holowczak, 1976). We were unable to find conditions under which all the pulse-labeled DNA species were chased into the 102-106 S material. In the insert, Fig. 6B, the total TCA-precipitable radioactivity 60 recovered after the pulse and during the Fraction chase is plotted, demonstrating that under FIG. 5. Analysis of pulse-labeled, replicating the conditions described further incorporaviral DNA by chromatography on BND-cellulose tion of radioactive precursor into TCA-precolumns. Part of the DNA labeled in a 3-min pulse, cipitable material did not occur. In Fig. 7, purified from the cytoplasm of vaccinia-infected the kinetics of appearance of label in varHeLa cells as described in Fig. 5, was dissolved in ious DNA species as related to the time of 0.3 M NET buffer (0.3 M NaCl, 0.01 M Tris-HCl buffer, pH 7.5, and lo-’ M EDTA) containing 0.1% the chase are plotted. These data suggest Sarkosyl. The sample (5 ml) was applied to a BNDthat a pool of molecules sedimenting at 30cellulose column (1.5 x 5 cm) packed in 0.3 M NET 40 S were present which may be ligated buffer. The column was then eluted with 50-75 ml of together or grow by the addition of “Okathe following buffers in stepwise fashion: 0.3 M NET zaki-fragments” to form genomes of the buffer, 1.0 M NET buffer (1.0 M NaCl, 0.01 M Trissize found in mature visions. HCl, pH 7.5; lo-’ M EDTA); 2% caffeine in 1.0 M NET buffer (warmed to 379, and 0.1 N NaOH in 1.0 M NET buffer. Samples, 3-6 ml in volume, were collected, precipitated with TCA, and counted by liquid scintillation spectrometry.

Analysis of Pulse-Labeled and PulseChased Labeled DNA in Neutral Sucrose Gradients

fuged at room temperature to collect the cells and the cell pellet was resuspended in complete medium, prewarmed to 37”, containing 2.5 mM cold thymidine (total elapsed time of this operation was 6-7.5 min). At the times indicated in Fig. 6, (calculated from the time of the end of the pulse), aliquots of the resuspended cultures were removed, cytoplasm was prepared by lysis of the cells with Triton X100, and the labeled DNA species in the cytoplasm analyzed in alkaline sucrose gradients as described in Fig. 6. In short pulses (with a short chase as described above), DNA molecules sedimenting at 30-

Cells infected with vaccinia virus were labeled as described in Figs. 2 and 6. The cytoplasmic fraction was treated with detergents and 2-mercaptoethanol and aliquots were analyzed by sedimentation in 520% neutral sucrose gradients as described in Fig. 8. These analyses were complicated by the fact that 20-35% of the total radioactivity applied to the gradient was recovered in the pellet fraction. When DNA species labeled in a short pulse (l-3 min) were analyzed, the major labeled species detected sedimented at 110-125 S with a smaller peak at about 90-96 S (Fig. 8A). As can be seen in Fig. 8B, the 110-125 S material was not always detected (seen in four to seven samples analyzed) and may

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20 0 M 33 0 0 10 30 40 rrocm FIPmn Rmmn FIG. 6. Sedimentation analysis in alkaline sucrose gradients of replicating vaccinia DNA labeled in pulse-chase experiments. At 2.15hr postinfection, HeLa cells infected with vaccinia virus (1000 EB/cell) were pulsed with 13Hlthymidine (5 &i/ml) or [Wlthymidine (1 &i/ml), cold thymidine (final concentration 2.5 nW was added and 5 x 10’ cells were removed and rapidly mixed with frozen, finely crushed wash buffer (see Fig. 2) to terminate the pulse. The remainder of the culture was centrifuged to collect the cells and the cells were resuspended in fresh, prewarmed (37”) complete medium containing 2.5 mM cold thymidine (“cold medium”). At the times indicated 5 x 10’ cells were collected, cytoplasm was prepared as described in Materials and Methods, and 4-8 x lo6 cytoplasmic equivalents treated with Sarkosyl(2%), DOC (O.l%), 2-mercaptoethanol(O.1 M), and NaOH (0.3 M) at room temperature for 15 min. Ad-2 DNA or vaccinia virions purified by Method II, labeled with [3H]thymidine or [“Clthymidine were added to provide sedimentation markers. Panel A: O-O, DNA species in infected cells labeled with r3H]thymidine in 60-set pulse (followed by 30-60 set chase with cold thymidine before incorporation of label was completely arrested); O-O, DNA species labeled in pulse (as above) followed by a 30-min chase with “cold medium”; x-x, DNA species labeled in a 60-set pulse followed by a 60-min chase with “cold medium.” Panel B: O-O, [3H]thymidine-labeled DNA species in the cytoplasm of infected cells labeled in a 60-set pulse (as in Panel A); x-x, DNA species labeled in a 60-set pulse followed by a 60-min chase in “cold medium.” GO, mock-infected, control cells pulsed for 60 set and chased for 60 min with “cold medium.” Insert shows recovery of TCA-precipitable material from 4 x lo6 cells during the course of the pulse-chase experiment. Panel C: x-x, DNA species labeled in a 60-set pulse with [Wlthymidine; O-O, DNA species labeled in a 60-set pulse with [Wlthymidine followed by a 75-min chase with “cold medium”; O-O, DNA species labeled in a 60-set pulse with [Wthymidine followed by a 120-min chase with “cold medium.” Each sample was analyzed in a separate gradient as described in Fig. 2. The position of the 34 S, Ad-2 marker DNA was identical in those gradients which have been presented together in each panel of the figure. represent an aggregate or complex which could pellet under the conditions of analysis described in Fig. 8. The 90-96 S species

were always detected (Fig. 8A and B). Under the conditions described in Fig. 6, radioactivity was chased from the 110-125 S species (when present) into the 90-96 and 82-84 S species (Fig. 8A) and finally into DNA species which cosedimented with mature viral genomes at 68-72 S (Fig. 8A and B). Careful analysis of the radioactivity recovered in these species and those which pelleted under the conditions of “chasing” described, indicated that during the chase, material which normally pelleted (20-35% in the short pulse) was converted to species which banded in the gradient, so that after a long chase, the pellet represented only 8-12% of the total radio-

activity applied to the gradient. The 82-84 S species sedimented about 13% faster than mature genomes (68-72 S and may represent circular molecules with “nicks” in both strands. No evidence for covalently closed circular molecules was obtained when the labeled replicating viral DNA was analyzed in alkaline gradients (Figs. 3 and 7). Joklik and Becker (1964) first described the association of replicating vaccinia DNA molecules with large cytoplasmic complexes which required magnesium ions for their stability. Fil et al. (1974) have described heterogeneously sedimenting vaccinia viral DNA molecules in HeLa cells after hydroxyurea reversal. The species resolved under the conditions described here may represent the replicative intermediates involved in vaccinia DNA

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FIG. 7. Kinetics of appearance of label in DNA species detected in the cytoplasm of infected cells during the course of pulse-chase experiments. The fate of viral DNA labeled in a 60-set pulse during a chase with “cold medium” was followed as described in Fig. 6. The appearance of label in DNA species with the sedimentation values indicated as related to the time of “chasing” was estimated by summingup the total radioactivity recovered in the appropriate fractions, after samples were analyzed by sedimentation analysis in alkaline sucrose gradients (Figs. 2 and 6).

replication which are normally held together in large complexes in the cytoplasm of infected cells. DISCUSSION

It has been demonstrated that when vaccinia virions were purified under certain experimental conditions (Berns and Silverman, 1970; Parkhurst et al., 1973; Geshelin and Berns, 1974; Holowczak, 1976) the genomes of the virions recovered were continuous and cross-linked, while under other conditions (Holowczak, 1976) virions could be isolated which contained genomes without cross-links and had interruptions in the phosphate-sugar backbone of their DNA. We have shown that both kinds of virions are infectious and appear to replicate their DNA with equal efficiency in the cytoplasm of cells they infect. We have been able to demonstrate that the replication of vaccinia virus DNA in the cytoplasm of infected cells is discontinuous; that is, “Okazaki-type” fragments

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are synthesized in short-pulses, which can be demonstrated to elongate in a chase, finally reaching the size of full-length genomes. These results are in agreement with the work of Magee and Levine (1970). However, in their experiments, carried out in CEF, Magee and Levine (1970) were unable to chase the segments synthesized in a short-pulse into molecules which corresponded to full-length genomes. The largest species they could detect after extended chases sedimented at about 40 S in alkaline sucrose gradients. In our studies we have shown that molecules of this size are intermediates in the replication process and appear to accumulate. We would like to suggest that these molecules represent the “intermediate strands” described by Huberman and Horwitz (1973) in their studies of DNA replication in CHO cells and that they grow by the addition of “Okazaki-fragments.” The presence of such a pool of “intermediate strands” in vaccinia-infected cells could help to explain the ease with which poxviruses can be shown to recombine, as well as certain interactions between poxviruses which have been termed “rescue phenomena” (Joklik, 1966). The data obtained by chromatographic analysis of replicating vaccinia DNA on hydroxyapatite and BND-cellulose indicate that such DNA has singlestranded regions, at least after the DNA has been released from infected cells and purified. The ssDNA species detected after short-pulses appear to be rapidly converted to dsDNA forms and must be very closely associated with their template. In the experiments described here, the growing strands of vaccinia DNA were found to sediment more slowly than those of mature viral DNA in alkaline sucrose. When molecules were labeled in a pulse or a pulse followed by a chase and analyzed in alkaline sucrose, no DNA structures in which one or both strands were covalently closed circles were detected. No closed circular molecules could be detected when pulse-labeled viral DNA was centrifuged in CsCl in the presence of ethidium bromide (Esteban and Holowczak, unpublished observations). These results would indicate that the replication of the vacci-

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FIG. 8. Sedimentation analysis in neutral sucrose gradients of replicating viral DNA, labeled in pulsechase experiments. Part of the cytoplasmic samples obtained from vaccinia virus-infected HeLa cells labeled in a short pulse or a pulse followed by “chasing” in cold medium, prepared as described in Fig. 6, were treated with Sarkosyl (2%), DOC (O.l%), and 2-mercaptoethanol (0.1 M). Approximately 4-8 x 108 cytoplasmic equivalents were loaded onto 5-208 linear, neutral sucrose gradients and centrifuged in the Spinco SW27.1 rotor (5 hr; 25,000 rpm; 20”). Gradients were fractionated and the distribution of TCA-precipitable material determined as described in Materials and Methods. Panel A: x-x, DNA species labeled in a 60set pulse (followed by a short, 30-60 set chase with cold thymidine); O-O, DNA species labeled in a 60-set pulse followed by a 60-min chase in “cold medium”; 0 -0, DNA species labeled in a 60-set pulse followed by a 90-min chase in “cold medium.” Panel B: O-O, DNA species labeled in a 3-min pulse (followed by a short chase as in Panel A); O---O, DNA species labeled in a 3-min pulse followed by a 120.min chase in “cold medium.” In Panel A approximately 18,000 total cpm were analyzed of which 25-308 were recovered in the pellet after analysis of the pulse-labeled sample. During the chase, the amount of radioactivity recovered in the pellet was reduced so that after a 90-min chase with “cold medium” the pellet contained 1904 cpm or 7.8% of the total radioactivity applied to the gradient. The positions of 68 S marker DNA from virions purified by Method II and 32 S Ad2 DNA when analyzed under these conditions are indicated. The results from analyses carried out in separate gradients have been plotted together.

genome does not involve covalently continuous concatemers or the addition of new material to parental strands as in the rolling circle model (Kelley and Thomas, 1969; Ihler and Thomas, 1970). However, it has been demonstrated (Holowczak, 1976) that virions purified by Method I (the method used to purify the stock virus employed in these experiments) have interruptions in their genomes. It is possible, therefore, that intermediates which would be of greater length than the parental genome would be “masked” in alkaline sucrose analysis because of the discontinuities in the parental DNA. Since we failed to detect any nascent strands of greater than unit length, polymerization may be symmetrical and proceed via the mechanism proposed by Cairn’s (1963). Evidence for circular intercontaining nicks in both mediates, strands, was obtained when pulse-labeled and pulse-chase labeled replicating DNA

nia

molecules were analyzed in neutral sucrose gradients (Fig. 8). Such circular molecules would be expected to sediment 1315% more rapidly than linear vaccinia DNA (taken to be about 72 S) under these conditions of analysis (Fiers and Sinsheimer, 1962; Rhoades and Thomas, 1968). DNA species sedimenting as a discrete band at about 82-84 S, in which label accumulated after a chase were in fact detected and may represent such inetermediates. The exact nature of these intermediates and the other DNA species resolved by analysis in neutral sucrose gradients remains to be critically determined. ACKNOWLEDGMENTS The expert technical assistance of Domenica Buc010 is gratefully acknowledged. This investigation was supported by Public Health Service Research Grant No. CA-110267, and Training Grant No. CA-05234 from the National Cancer Institute. J.A.H. is a recipient of Public Health Service Research Career Development

REPLICATION Award stitute.

No. CA-70458

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the National

OF Cancer

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REFERENCES

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