Replication of vaccinia DNA in mouse L cells I. In vivo DNA synthesis

VIROLOGY

78,

57-75

(1977)

Replication

of Vaccinia

DNA in Mouse

L Cells

I. In Viva DNA Synthesis MARIANO Department

ESTEBAN

of Microbiology,

College

of Medicine Piscataway, Accepted

JOHN

AND

A. HOLOWCZAK’

and Dentisty of New New Jersey 08854 December

Jersey,

Rutgers

Medical

School,

19,1976

Replication of vaccinia DNA was analyzed in lysates of intact, vaccinia-infected L cells prepared under conditions which preserved the structure of replicating and mature viral DNA molecules. The techniques employed permitted the separation of viral and host DNA, as confirmed by DNA-DNA hybridization. Sedimentation analysis in alkaline sucrose gradients showed that, during the period of maximum vaccinia DNA replication in L cells [2-3 hr postinfection (h.p.i.11, IO-12 S viral DNA fragments were preferentially labeled by short pulses (0.5-10 min) of [3H]thymidine. About 20-30% of the pulse-labeled DNA was hydrolyzed by nuclease S,. These results support the view that viral DNA replication was discontinuous and may involve single-stranded DNA intermediates. Pulse-chase experiments showed that the lo-12 S fragments elongated into 30-50 S “intermediate-sized” DNA species and finally into 70-72 S (full length) viral DNA in about 30 min, which would require the incorporation of 6500 nucleotides/min. The conversion of mature viral DNA (70-72 S) into mature, cross-linked DNA (which sedimented at 92-94 S and 102-106 S in alkaline sucrose gradients) occurred late in infection (5-6 h.p.i.), when virion assembly had begun. Replicating viral DNA molecules were pulse-labeled with [3Hlthymidine and chased with bromodeoxyuridine (BrdU); the labeled DNA species were analyzed by equilibrium density centrifugation in CsCl. Hybrid (HL) molecules (p = 1.77 g/cm31 were detected, demonstrating that viral DNA replication was semiconservative. Analysis of replicating viral DNA molecules in ethidium bromide-CsCl gradients at equilibrium failed to show the presence of circular or superhelical duplexes. This result and the fact that no viral DNA molecules of greater than unit length were labeled during long or short pulses suggest that viral DNA replication is symmetrical. INTRODUCTION

tion analysis (Berns and Silverman, 1970; Parkhurst et al., 197% Geshelin and Berns, 1974; Holowczak, 1976) has been estimated to range from 2120 X lo6 to 170 x 106. Examination of vaccinia virus-infected cells by autoradiography (Cairns, 1960), fluorescent antibody staining (Kato et al., 1959, 1964), and electron microscopy (Dales and Siminovitch, 1961) has indicated that poxvirus DNA replicates in large cytoplasmic inclusions or “factories.” Replicating viral DNA, as well as the parental viral DNA, have been found associated with large cytoplasmic structures that require magnesium ions for their stability (Joklik and Becker, 19641, which may represent the vaccinia “virosomes”

Vaccinia virus contains a single, linear, double-stranded DNA molecule whose strands are naturally complementary cross-linked (Berns and Silverman, 1970; and Parkhurst et al., 1979; Geshelin Berns, 1974; Holowczak, 1976). Each molecule has been shown to contain two crosslinks, one located at or near each end (Geshelin and Berns, 1974). The molecular weight of vaccinia DNA as determined by measurements in the electron microscope (Easterbrook, 1967; Geshelin and Berns, 1974; McCrea and Lipman, 1967; Sarov and Becker, 1967) and by gradient sedimenta’ To whom

requests

for reprints

should

be sent. 57

Copyright All

rights

0 1977 by of reproduction

Academic in any

press, Inc. form

reserved.

ISSN

0042-6622

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AND

identified by autoradiography and electron microscopy. Virosomes can be recovered under appropriate ionic conditions as rapidly sedimenting DNA-protein complexes (Dahl and Kates, 1970a,b). A number of enzymatic activities which may be related to the replication of viral DNA have been identified in cytoplasmic cell extracts from infected cells. These are early enzymes coded for by the parental genome and include thymidine kinase (McAuslan, 1963; Kit and Dubbs, 19651, DNA polymerase (Jungwirth and Joklik, 1965; Magee and Miller, 19671, polynucleotide ligase (Sambrook and Shatkin, 1969), and a number of nucleases (Jungwirth and Joklik, 1965; McAuslan, 1965; McAuslan and Kates, 1966, 1967). It was initially shown that, in cytoplasmic fractions from chick embryo fibroblasts, viral DNA was synthesized in small fragments which elongated with time during a pulse-chase, but the length of the newly synthesized DNA was only 20% of that of mature complementary strands of vaccinia DNA (Magee and Levine, 1970). Holowczak and Diamond (1976) have recently analyzed the replication of vaccinia DNA in cytoplasmic fractions prepared from infected HeLa cells. During short pulses with [3H]thymidine, label was incorporated into small fragments (-20 S) of viral DNA. During a chase, part of the population of short strands was converted into mature full-length molecules (72-75 S) and into cross-linked genomes (102-106 S). The replicating viral DNA was shown to contain ssDNA* regions (Holowczak and Diamond, 1976). While the main site of viral DNA replication appears to be in the cytoplasm of infected cells, the host cell nucleus may also be involved, as suggested by early experiments employing metabolic inhibitors such as mitomycin C (Reich and Franklin, 1961) and vinblastin (Marcus 2 Abbreviations used: dsDNA, double-stranded DNA; ssDNA, single-stranded DNA; EB, elementary bodies; EDTA, ethylenediaminetetraacetate; pME, 2-mercaptoethanol; PFU, plaque-forming units; h.p.i., hours postinfection; HL, hybrid; TCA, trichloroacetic acid; SSC, 0.15 M NaCl, 0.015 M SOdium citrate.

HOLOWCZAK

and Robbins, 19631, studies employing autoradiography (Walen, 1971), and, most recently, by molecular hybridization, which has demonstrated viral sequences in the DNA associated with nuclei isolated from infected cells (La Colla and Weissbath, 1975; Bolden et al., 1975; Gafford and Randall, 1976). The aims of the studies to be presented here were threefold. First, a technique was developed to permit analysis of all the replicating viral DNA in the host cells. This allowed us to examine not only the steps in viral DNA replication which occurred in the cytoplasm of infected cells, but also the events which may be taking place in the nucleus of the infected cell. Second, whole cells were lysed directly on the surface of gradients, avoiding the manipulations required for preparing cytoplasmic and nuclear fractions from infected cells. This minimized mechanical damage to the intracellular forms of viral DNA and the activation of nucleases which could alter the structure of the replicating viral DNA molecules. Finally, we wished to confirm and extend the observations of Holowczak and Diamond (1976) that were made with infected HeLa cells in a second host cell system, the mouse L cell. We will show that the semiconservative replication of vaccinia DNA in intact infected L cells was discontinuous and involved the formation of small fragments which were subsequently ligated together into larger molecules. Full-length viral DNA molecules (Type I, 70-72 S) were synthesized in 30 min (2-2.30 h.p.i.1, while full conversion of the small fragments synthesized in a short pulse into mature, cross-linked molecules (Type II, 92-94 S with single cross-link, or Type III, 102-106 S with two cross-links) was a multistep process which involved a series of intermediate molecules whose complementary strands were not completely sealed and closed at or near each end until late in infection (5-6 h.p.i.1. MATERIALS

AND

METHODS

Virus and cells. Vaccinia virus, strain WR, was propagated in mouse L cells and purified as described previously (Joklik,

VACCINIA

DNA

1962a,b; Holowczak and Joklik, 1967). Five different preparations of purified virus were used for these experiments, immediately or shortly after virus purification, to avoid the introduction of “nicks” in the viral DNA which can occur upon repeated freezing and thawing (Holowczak, 1976). Sonication was used as an aid in resuspending pelleted viral particles at two stages of viral purification: (1) after pelleting through a cushion of 40% sucrose (sonication was for 60 set at 40-50 W, position 2, using Sonifer Cell Disruptor Model W185D, Branson Sonic Power Co.) and (2) after collecting the virus band from a second sucrose gradient (sonication was for 15-30 set). The EB/PFU ratio was 30-50:1, based on titrations in primary chick embryo fibroblast cell cultures. Virions labeled with [3H]thymidine in their DNA were prepared as described previously (Joklik, 1962a). When [3Hlthymidine (20-50.8 Cilmmol) was used at the level of 2 &i/l x lo6 infected cells, the specific activity of the purified virion preparations was 3 x lo4 to 1 x lo5 cpm/pg of DNA. Adenovirus type 2 DNA labeled with 13Hlthymidine was purified as described by Burlingham and Doerfler (1971) and had a specific activity of 3 x lo4 cpmlpg of DNA. Mouse L cells were grown in suspension in Eagle’s MEM (Joklik’s modified), supplemented with 5% fetal calf serum. Preparation of Zysates. Conditions for the infection of cells (m.o.i. = 1000 EB/ cell) have been described (Becker and Joklik, 1964; Esteban and Metz, 1973). At the end of the 15-min adsorption period, designated as zero time, cells were diluted into complete medium at a concentration not exceeding 1 x lo6 cells/ml. Mock-infected cultures were established in a similar way. Infected or mock-infected cells were labeled beginning 2 hr postinfection with lo20 $X/ml of [3H]thymidine for the times indicated in the legends to the appropriate figures. At the end of the labeling time, 1.0 ml of cells was removed from the cultures, poured into ice-cold or crushed, frozen buffered-saline solution (50 n&f Tris-HCI buffer, pH 7.6, 150 mM NaCl, 5 mM

59

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EDTA), and collected by centrifugation at 1000 rpm for 2 min. The cells were washed with 4.0 ml of buffered saline, harvested in 1.0 ml of again, and resuspended buffered saline, and aliquots containing 1 to 2 x lo5 cells were loaded onto either neutral or alkaline sucrose gradients overlayed with 0.8 ml of neutral or alkaline lysis solution. The compositions of “neutral” and “alkaline” lysis solutions are indicated below. Cells were allowed to complete lysis for 16-20 hr at 4” in the dark. For CsCl isopycnic gradient analysis, lysate from 5 x lo5 cells was used. Velocity sedimentation tral sucrose gradients.

anaZyysis in neu-

Neutral sucrose gradients [ll.O ml, 15-30% (w/v) sucrose] containing 1 M NaCl, 0.01 M EDTA, 0.15% Sarkosyl NL-97, 0.05 M Tris-HCl buffer, pH 7.6, were overlayed with 0.8 ml of lysis solution (1 M NaCl, 0.01 M EDTA, 1% Sarkosyl NL-97, 0.03 M /3ME in 0.05 M Tris-HCl buffer, pH 7.6). An aliquot of cells was applied and lysis was allowed to proceed as described above. The gradients were then centrifuged for 3 hr at 39,000 rpm at 20” (Spinco SW-41 rotor, L3-50 ultracentrifuge). The gradients were fractionated, 100 pg of carrier (BSA) was added to each fraction, and the samples were precipitated with 10% TCA (1 hr at 0”). The precipitates were collected on GF/ C filters, washed three times with 5% TCA, and dried. The samples were counted in a toluene-based scintillation mixture. Sedimentation coefficients were calculated according to Studier (19651, using adenovirus type 2 DNA and simian virus 40 EcoRI form (III) DNA as sedimentation markers (Burlingham and Doerfler, 1971; Morrow and Berg, 1972). Alkaline-sucrose gradients. Linear sucrose gradients [15-30% (w/v> containing 0.7 M NaCl, 0.3 N NaOH, 0.01 M EDTA, and 0.15% Sarkosyl NL-971 were overlayed with 0.8 ml of lysis solution (0.7 M NaCl, 0.5 N NaOH, 0.01 M EDTA, 1% Sarkosyl) and processed under conditions identical to those used for neutral sucrose gradients.

Analysis in neutral

by equilibrium CsCl gradients.

sedimentation

About 5 x lo5 cells in buffered saline were solubilized in 1% Sarkosyl NL-97 and 30 mA4 PME. After

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AND HOLOWCZAK

standing 10 min at room temperature, the lysate was digested for 2 hr at 37” with 1 mg/ml of pronase (self-digested for 2 hr at 37”). The extract was mixed with a concentrated CsCl solution (prepared in 0.01 M Tris-HCl buffer, pH 7.6, 0.2 M NaCl, 0.005 M EDTA, 0.15% Sarkosyl NL-97) and the density was adjusted to p = 1.736 g/cm3. The mixture was overlayed with mineral oil. The samples were centrifuged at 35,000 rpm for 64 hr at 20” (Spinco SW-56 rotor). Fractions, collected by piercing the bottom of the centrifuge tube with a needle, were processed for determination of radioactivity as indicated above. The refractive index of every fifth fraction, beginning with fraction number 2, was measured at room temperature. Analysis by equilibrium sedimentation in ethidium bromide-&Cl gradients. CsCl

solutions containing 200 pg/ml of ethidium bromide, 0.01 M Tris-HCI buffer, pH 7.6, 0.2 M NaCl, 0.005 M EDTA, 0.15% Sarkosyl NL-97 and 4 pg/ml of calf thymus DNA were prepared and samples were analyzed under the conditions described above for equilibrium sedimentation analysis in neutral CsCl. Efficacy of the conditions used for analysis. The conditions described above for

lysing intact cells and sedimentation analysis in alkaline sucrose gradients have been found to be effective for identifying the intracellular forms of adenovirus DNA (Burger and Doerfler, 1974). To determine if these conditions would allow us to analyze replicating vaccinia DNA in the presence of host DNA, a number of experiments were performed. Mouse L fibroblasts were labeled for 48 hr with [14C]thymidine (0.2 &!ilml), and the cells were collected by centrifugation, washed, and resuspended in fresh medium. Sixteen to twenty hours later, the prelabeled cells were infected (1000 EB/cell) and [3H]thymidine was added to label the replicating viral DNA. In some experiments, a mixture of radioactive amino acids was added to label the proteins. Aliquots of the cell culture were then removed and subjected to analysis as described above. It was determined that, when a maximum of 2-3 x lo5 cells were analyzed, 90% of

[14Clthymidine-labeled host cell DNA molecules pelleted after centrifugation with little or no trapping of viral DNA molecules. Less than 10% of the labeled host cell DNA was released in the form of molecules which sedimented at 5 120-140 S and were therefore resolved in the gradients used for analysis. In experiments in which labeled amino acids were added to infected cells, it could be demonstrated that proteins associated with DNA were completely released by the treatment described and remained at the top of the gradient. DNA-DNA hybridization. Vaccinia virus DNA was isolated and purified using methods described by Sharp et al. (1974). Virions from purified vaccinia virus were lysed in a solution containing 0.01 M TrisHCl buffer, pH 7.6, 0.2 M NaCl, 0.005 M EDTA, 0.03 M /3ME, 1% Sarkosyl NL-97. After standing for 10 min at room temperature, pronase (1 mg/ml) was added and the incubation was continued at 37” for 2 hr. The virus lysate was extracted once at room temperature with an equal volume of redistilled phenol saturated with 0.1 M Tris-HCl buffer, pH 7.6, and twice with 2 vol of chloroform:isoamylalcohol (24:l). The aqueous phase was mixed with 2 vol of cold ethanol and stored at -20” for at least 16 hr, and the nucleic acid was collected by centrifugation at 10,000 rpm for 15 min. The pellet containing the viral DNA was dried, dissolved in 0.3 N NaOH, incubated at 37” for 4 hr, diluted with 2 vol of 0.05 M Tris-HCl buffer, pH 7.6, neutralized with 3 N HCI, precipitated again with cold ethanol, and collected by centrifugation, and the dry pellet was dissolved in 0.1 x SSC (SSC is 0.15 M NaCl, 0.015 M sodium citrate). Immobilization of viral DNA on nitrocellulose filters and DNA-DNA hybridization were performed according to Denhardt (1966). Samples from alkaline-sucrose velocity gradients were neutralized with 3 N HCI, diluted 1:l with distilled water, sonicated, and heat denatured (100” for 10 mid. From each sample, 0.2 ml was added directly to 1.0 ml of hybridization mixture containing 0.02% Ficoll (Pharmacia Fine Chemicals, Inc.), 0.02% polyvinyl-pyrrolidone, and

VACCINIA

DNA REPLICATION

0.02% bovine serum albumin in 3x SSC. Membrane filters with immobilized vaccinia virion DNA (5 pg/filter) were previously incubated for 6 hr at 65”. Hybridization was for 18-24 hr at 65”. Filters were washed five times with 10.0 ml of 3 mM Tris-HCl buffer, pH 9.2, dried, and counted. Pooled fractions from CsCl (neutral) equilibrium density gradients were dialyzed against two changes (1.5 liters each) of 10 r&f Tris-HCl buffer, pH 7.6, 1.0 mM EDTA for 24 hr at 4” and aliquots were hybridized as described above. S-l nuclease assay. S-l nuclease was obtained from Sigma Chemical Co. One unit of enzyme degraded 10 pg of radioactively labeled, heat denatured vaccinia or L-cell DNAs to acid-soluble products in 10 min at 45”. The reaction mixtures contained enzyme, DNA in 0.03 M sodium acetate buffer, pH 4.5, 0.1 M NaCl, 1 mM ZnS04. Chemicals and radioisotopes. Pronase was purchased from Calbiochem Corp., California. A solution of 10 mglml was prepared in 10 mM ‘Iris-HCl buffer, pH 7.6, 10 n-&f EDTA and self-digested at 37 for 2 hr. Sarkosyl NL-97 was obtained from Geigy Chemical Co., Ardsley, New York. [‘4ClThymidine (56.5 mCi/mmol) and VHlthymidine (20 to 50.8 Cilmmol) were purchased from the New England Nuclear Corp., Boston, Massachusetts. RESULTS

Kinetics of DNA synthesis in infected cells. The rate of incorporation of

13Hlthymidine into acid-precipitable radioactivity in intact virus-infected cells is several times greater (5 to 20-fold) than that measured in uninfected cells (Fig. 1). In infected cells, viral DNA synthesis began at about 1 h.p.i., reached a peak at around 2 h.p.i., and then decreased by 4 h.p.i. Similar patterns of DNA synthesis have been observed in intact infected HeLa cells and in cytoplasmic fractions prepared from infected HeLa and L cells (Magee et al., 1960; Salzman, 1960; Hanafusa, 1961; Green, 1963; Shatkin and Salzman, 1963; Green et al., 1964; Joklik and Becker, 1964). Synthesis of viral

DNA

ceZZs.The nature of replicating

in infected L

viral DNA

61

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x 2 ”

FIG. 1. Kinetics of DNA synthesis in intact infected cells. Conditions for virus infection of the cells (m.o.i. = 1000 EB/cell) were as described previously (Esteban and Metz, 1973). Cultures of mock-infected cells were established and analyzed in parallel with the infected cells. At the times indicated after virus infection, 2 x lo6 cells were removed from the cultures and pulse-labeled with 2 @X/ml of [3H]thymidine for 15 min. Cells were collected by centrifugation in buffered saline and washed two times in the same solution, and the radioactivity incorporated into TCA-precipitable material was determined by liquid scintillation spectrometry. 0, infected cells; 0, mock-infected cells.

labeled with [3Hlthymidine for varying periods of time was examined during the period of maximal viral DNA synthesis in infected cells (see Fig. 1). Beginning 2 hr after infection, cells were pulse-labeled with [3Hlthymidine and, for each time point, 2 x lo5 infected or mock-infected cells were lysed on top of an alkaline-sucrose gradient and processed as described under Materials and Methods. In vaccinia virus-infected L cells, the majority of viral DNA molecules labeled during a lo-min pulse sedimented at 10 to 40 S relative to adenovirus type 2 DNA, which sedimented at 34 S under these conditions of analysis (Fig. 2A). With prolonged pulses of 30 and 60 min, the size of

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D

FRACTION

FRACTION

FIG. 2. Alkaline-sucrose sedimentation analysis of pulse-labeled viral DNA synthesized in L cells. Infected or mock-infected cell cultures were prepared as described under Materials and Methods. At 2 h.p.i., lO.O-ml aliquots of cells from each culture were labeled with 10 &i/ml of 13H1thymidine for different lengths of time. At the times indicated, 1.0 ml of cells was removed and washed in buffered saline, and 2 x lo5 cells were loaded onto 15-30% alkaline-sucrose gradients overlayed with a 0.8-ml layer of lysis solution. Lysis of the cells, centrifugation, fractionation of gradients, and determination of the distribution of radioactivity in each fraction were as described under Materials and Methods. (A) Cells pulse-labeled for 10 min. (B) Cells pulse-labeled for 30 min. (C) Cells pulse-labeled for 60 min. (A-C) 0, infected cells; 0, mock-infected cells. (D) Superimposed profiles of pulse-labeled DNA from infected cells expressed as the percentage of total radioactivity: A, 10 min; A, 30 min; 0, 60 min. The fractions indicated in C were pooled and hybridized to virion DNA immobilized on nitrocellulose filters as described under Materials and Methods. The height of the horizontal bars in the graph indicates the percentage of the 3H-labeled DNA from infected cells that hybridized to virion DNA. The percentages of total radioactivity recovered in the pellet fraction of the gradients were, respectively: A, 4 (0) and 9% (0); B, 4 (0) and 19% (0); C, 10 (0) and 50% (0).

VACCINIA

DNA

the replicating DNA increased, and DNA species sedimenting at 30 to 50 S and 40 to 70 S were observed, respectively (Fig. 2B). DNA molecules sedimenting at 70-72 S (Type I) could be detected after a 30-min but not after a lo-min pulse (Fig. 2B). Labeled molecules which sedimented at 92-94 S (Type II) and 102-106 S (Type III) were labeled significantly only after a 60min pulse (Fig. 2C). The sedimentation behavior of molecules labeled during pulses of various lengths is summarized in Fig. 2D. The viral nature of the DNA synthesized in infected cells was shown by hybridization of pooled fractions from the gradients to virion DNA immobilized on nitrocellulose filters. DNA molecules which hybridized with viral DNA were detected throughout the gradient (Fig. 2C), in good agreement with the distribution of newly synthesized 13Hlthymidine-labeled viral DNA species. Discontinuous synthesis of vaccinia DNA. In the experiments described above

(Fig. 2), the molecules synthesized during a short pulse (10 min) were predominantly of low molecular weight. With prolonged pulses, there was an increase in the size of the newly replicated viral DNA. This would suggest that viral DNA replication was discontinuous and involved the formation of small fragments which were subsequently ligated together into larger molecules, as first described for the replication of Escherichia coli by Okazaki et al. (1968). To test whether or not vaccinia virus DNA synthesis proceeded through a discontinuous intermediate, DNA synthesized after very short labeling times (10 set) was chased with 5 mM cold thymidine and the products were analyzed in alkaline-sucrose gradients (Fig. 3). After a loset pulse, more than 70% of the labeled DNA species sedimented at 8-12 S. With prolonged chases (30 and 60 min), molecules sedimenting at about 30-50 S and 4070 S, respectively, made up the bulk of the newly synthesized DNA. Since nearly all of the TCA-precipitable radioactivity was found in small fragments after the shortest pulses, discontinuous synthesis may occur on both template strands at the replication

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fork. The average sedimentation coefflcient of the initially labeled material, which may consist of the growing and completed short chains, was 8 to 12 S (Fig. 3E), suggesting that the length of the “unit” of the discontinuous synthesis was 1000-2000 nucleotides. Formation cross-linked replication.

of intermediate strands and molecules during viral DNA

The experiments depicted in Figs. 2 and 3 showed that, at the time of maximal DNA synthesis in infected cells (2 to 3 h.p.i.), a large proportion (60-70%) of the DNA molecules synthesized had a sedimentation coefficient between 30 and 70 S, while only about 20-30% of the molecules were found to be the size of, or larger than, an intact single strand of complementary viral DNA (70-72 S). The time required for conversion of small fragments into mature ssDNA was about 30 min (Figs. 2 and 3). The rate of DNA synthesis as measured by autoradiography in mammalian cells (Huberman and Riggs, 1968) has been reported to be about 1 pmlmin, corresponding to a polymerization of about 3000 nucleotide pairslminlfork. Vaccinia virus DNA has a contour length of about 62 pm, equivalent to 132 2 6 x lo6 daltons as determined by electron microscopy (Esteban, Flores, and Holowczak, in preparation), in good agreement with the results of Geshelin and Berns (1974). The data presented here indicate a polymerization rate of about 6500 nucleotide pairs/min for replicating vaccinia DNA or twice the rate at which host cell DNA would be replicated. The viral DNA molecules synthesized during a 30- to 60-min pulse or pulsechase (see Figs. 2 and 3) were very heterogeneous in size (30 to 70 S). This would suggest that, even though most of the molecules have finished elongation, the elongation of some molecules may be limited by the availability of ligase activity. Alternatively, it may be that synthesis occurred with, different frequencies in the replicon(s). The latter possibility appears to be unlikely since the rate of synthesis was maximal between 2 and 3 h.p.i. Polynucleotide ligase activity has been demonstrated in cytoplasmic cell extracts from vaccinia virus-infected HeLa cells (Sam-

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FIG. 3. Alkaline-sucrose sedimentation analysis of replicating viral DNA synthesized in L cells during short pulse-chases. Infected or mock-infected cells were prepared as described under Materials and Methods. At 2 hr after virus infection, 1 x 10’ cells were removed, labeled with 10 &i/ml of PHlthymidine for 10 set, and then chased with 5 mM cold thymidine. At the times indicated, 1 ml of the labeled cultures was removed, poured onto finely crushed buffered saline, and washed once in buffered saline, and 2 x lo5 cells were loaded onto a 1530% alkaline-sucrose gradient overlayed with a O.&ml layer of lysis solution and analyzed as described in Fig. 2. (A) Cells pulse-labeled for 10 sec. (B) Cells chased for 30 min after a lo-set pulse. (C) Cells chased for 60 min after a IO-set pulse. (A-C) 0, infected cells; 0, mock-infected cells. (D) Superimposed profiles of pulse-chased DNA from infected cells expressed as a percentage of total radioactivity: A, lo-set pulse; A, 30-min chase after a lo-set pulse; l , 60-min chase after a lo-set pulse. The percentages of total radioactivity recovered in the pellet fraction of the gradients were, respectively: A, 1 (0) and 4% (0); B, 7 (0) and 22% (0); C, 8 (0) and 55% (0). (El Cells labeled with 10 $X/ml of VHJthymidine for 1 min at 2 h.p.i. Centrifugation was for 18 hr at 25,000 rpm. Adenovirus type 2 DNA and simian virus 40 EcoRI form (III) DNA were run in parallel gradients.

brook and Shatkin, 19691, and we have detected an increase in ligase activity in cytoplasmic fractions prepared from infected L cells (Estaban and Holowczak, in preparation). To determine the time needed for conversion of the intermediate strands into cross-linked molecules, Type II (92-94 S) and Type III (102-106 S) cells were pulselabeled for 2 min with 13Hlthymidine at 2

hr after infection and chased for extended periods of time with 5 mM cold thymidine, and the viral DNA from 2 x lo5 cells was then analyzed on alkaline-sucrose gradients (Fig. 4). The short DNA strands synthesized in a 2-min pulse C-20 S) grew to large molecules during a 2-hr chase (i.e., at 4 h.p.i.) when about 50% of these molecules were of similar size or larger than intact mature viral ssDNA (Type I,

VACCINIA

5

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FIG. 4. Alkaline-sucrose sedimentation analysis of replicating viral DNA synthesized in L cells during long pulse-chases. Cells were prelabeled with 0.2 &i/ml of [‘Qthymidine for 48 hr. Sixteen hours prior to virus infection, the cells were harvested and resuspended in fresh media. Conditions for virus infection and mock-infection of the cells were as indicated under Materials and Methods. At 2 hr after infection, 15 ml of each culture was removed, labeled with 10 &i/ml of 13Hlthymidine for 2 min, and then chased with 5 n&f cold thymidine. At the times indicated, LO-ml aliquots were removed from the cultures and washed in buffered saline, and 2 x lo5 cells were loaded on a 15-30% alkaline-sucrose gradient overlayed with a 0.8-ml layer of lysis solution. Lysis of the cells and conditions for sedimentation analysis were as described under Materials and Methods. (A) Cells pulse-labeled for 2 min. (B) Cells pulse-labeled for 2 min and then chased for up to 4 h.p.i. (C) Cells pulse-labeled for 2 min and then chased for up to 6 h.p.i. (A-C) 0, infected cells; 0, mock-infected cells. (D) Virion DNA (0.4 wg of DNA). Conditions for lysing purified virus on top of the gradients were the same as those used for intact cells. The percentages of total [~4Clthymidine-labeled host DNA recovered in the pellet fraction ofthe gradients were, respectively: A, 89 (0) and 91% (0); B, 92 (0) and 93% (0); C, 90 (0) and 93% (0). Approximately 6000 cpm of host DNA was analyzed in each gradient.

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70-72 S). With extended chases (at 6 h.p.i.), over 70% of these molecules sedimented faster than mature viral DNA and cosedimented with cross-linked virion DNA (Fig. 4D). The results of these studies showed that formation of mature, cross-linked viral DNA molecules was a multistep, synchronized process which involved synthesis of short fragments, growing of the chains into a pool of intermediate strands, sealing of gaps, and closure of the complementary strands by introduction of cross-links at or near each end of the molecule late in infection. The intermediate molecules were bound very tightly to their template as determined by analysis of replicating molecules in neutral-sucrose velocity gradients (Fig. 5). Replicating molecules were the same size as mature linear double-stranded virion DNA (68-72 S) by 4 and 6 hr after infection. In neutral-sucrose gradients, the short strands synthesized during a 2min pulse pelleted with the bulk of host DNA to the bottom of the centrifuge tube after centrifugation, indicating that they may be part of the large DNA aggregates found in HeLa cells (Joklik and Becker, 1964; Dahl and Kates, 1970a,b: Holowczak and Diamond, 1976). Replication of viral DNA in L cells is semiconservative. At least one of the natu-

rally occurring cross-links in the vaccinia genome would have to be removed if viral DNA replication were semiconservative. To determine if the loss of cross-links did in fact occur, cells were infected with [3H]thymidine-labeled virus (1 x lo5 cpm/ pug of DNA) and the size of the parental DNA in the infected cells was then analyzed by velocity sedimentation (Fig. 6). When DNA from purified virions was analyzed in alkaline-sucrose gradients, two major peaks of radioactivity at about 102-106 S and 92-94 S were observed (Fig. 6A). The sedimentation behavior of the 9294 S molecules would be appropriate for linear ssDNA with twice the molecular weight expected for a single complementary DNA resulting from the removal of one cross-link or introduction of a single nick in molecules having two cross-links. Molecules sedimenting at 102-106 S would

HOLOWCZAK

correspond to intact circular genomes with two cross-links and twice the molecular weight of single-stranded viral DNA (7072 S) (Berns and Silverman, 1970; Parkhurst et al., 1973; Geshelin and Berns, 1974; Holowczak, 1976). By 2 hr after infection, about 30-40% of the cell-associated parental DNA molecules were found to sediment more slowly than the DNA from mature virions (102106 S) (Fig. 6A) and even Type I molecules (70-72 S) (Figs. 6B-6D). This change in sedimentation could occur if the parental DNA molecules had their cross-links removed, if nicks were introduced in one or both DNA strands, or if a combination of these events occurred (we cannot distinguish between these possibilities). This alteration may be due to the activity of the ssDNA nuclease which is associated with the core structure of the virion (Pogo and Dales, 1969). By 4 and 6 h.p.i., the proportion of “nicked” molecules had increased to about 40 and 60%, respectively (Figs. 6C and 6D). The data presented here indicated that removal of the cross-link(s) or “nicking” of the viral DNA does occur after infection, which would then permit semiconservative replication of the strands to proceed. To demonstrate that, in fact, viral DNA replication proceeded in a semiconservative fashion, infected cells were pulse-labeled for 2 min at 2 hr after infection and chased with 5 mM cold BrdU for 30 min; the cells were solubilized and digested with pronase, and the DNA was banded to equilibrium in CsCl equilibrium density gradients as described under Materials and Methods (Fig. 7). Approximately 70% of the DNA synthesized from 2 to 2.30 h.p.i. had its density shifted from p = 1.712 g/cm3 (L) to p = 1.77 g/cm3 (HL), while the density of host DNA was p = 1.705 g/cm3. The density of the replicating viral DNA molecules (p = 1.712) labeled with [3Hlthymidine present before the introduction of BrdU was greater than that reported for purified vaccinia DNA (p = 1.695) obtained from virions (Joklik, 1962a,b). As we will demonstrate below, replicating DNA molecules, detected under the conditions of analysis described

VACCINIA

32 S I

DNA

s

-1

0

5

10

15

20

25

30

35

40

FraCfiOn

FIG. 5. Sedimentation analysis in neutral-sucrose gradients of replicating viral DNA synthesized in L cells. At 2 hr after virus infection, 1 x 10’ cells were labeled with 10 pCi/ml of [3Hlthymidine for 2 min and then chased with 5 m&f cold thymidine. At the times indicated, l.O-ml aliquots were removed and washed in buffered saline, and 2 x IO5 cells were loaded onto a l&30% neutral-sucrose gradient overlayed with a 0.8-ml layer of neutral lysis solution. Lysis of the cells, centrifugation, and determination of radioactivity were as described under Materials and Methods. A: A, infected cells pulse-labeled for 2 min; 0, infected cells pulse-labeled for 2 min and chased for up to 4 h.p.i.; 0, infected cells pulselabeled for 2 min and chased for up to 6 h.p.i.; A, mock-infected cells pulse-labeled for 2 min and chased for up to 6 h.p.i. The percentages of total radioactivity recovered in the pellet fraction of the gradients were, respectively: A, (A.) 92%; (0) 11 and (a) 10%; mock-infected cells (A), 95%. In other experiments when cells were prelabeled with

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67

here, contain significant ssDNA regions which may explain this difference in density (Petterson, 1973). Holowczak and Diamond (1976) failed to detect such molecules in cytoplasmic samples prepared from vaccinia-infected HeLa cells. However, these investigators purified their DNA by phenol extraction before analysis. We have found that such procedures, even under conditions where manipulation of the DNA is minimized (Holowczak, 19761, result in the loss of ssDNA regions associated with the replicating viral DNA (see the legend to Table 1). Substitution of [3H]thymidine with BrdU during synthesis allowed a distinction to be made between preexisting DNA and newly synthesized DNA. The time required for synthesis of a full-length strand of viral DNA was about 30 min (Fig. 2). Within the time period of the experiment described above, only 50% substitution would be expected if semiconservative synthesis occurred, that is, one new and one old strand should be present in double helical molecules. Complete replacement of dTMP by BrdUMP in poly[d(A-T)] results in an increase in the buoyant density of 200 mg/ml (Baldwin and Shouter, 1963); vaccinia DNA containing 64% A-T (Joklik, 1962a,b) would be expected to shift its density (200) (0.64)/2 or an increase of 64 mglml after one complete round of replication. Thus, the density shift expected for a complete substitution of L3Hlthymidine by BrdU after one round of DNA replication would be to p = 1.776 g/cm3, in good agreement with the results of the experiments presented in Fig. 7. The viral nature of the newly synthesized DNA was confirmed by hybridization of pooled fractions from the gradient to virion DNA (Figs. 7A and 7B). The data also show that during the synthesis of vaccinia DNA extensive replication was clearly occurring 2 hr after infection, while repair synthesis appeared to be very limited since replacement of thymidine by [‘Clthymidine for 24-28 hr prior to virus infection, greater than 95% of the total [‘Qthymidine-labeled host DNA was recovered in the pellet fraction. B, virion DNA (0.4 pg of DNA). Conditions for lysing purified virus on top of the gradients were the same as those used for intact cells.

ESTEBAN

AND HOLOWCZAK

1 A

VIRION 93s

ms

ir

i

34s 1

FRACTION

FRACTION

FIG. 6. Fate of [3Hlthymidine-labeled parental vaccinia DNA in infected L cells. Conditions for infection of cells with labeled virus (m.0.i. = 1000 EB/ml; sp act, lo5 cpm/pg of DNA) were as described under Materials and Methods. At the times indicated, 1.0 ml of cells was removed from the cultures and washed in buffered saline, and 2 x lo5 cells were loaded onto a 15-30% alkaline-sucrose gradient overlayed with a 0.8ml layer of lysis solution. Lysis of cells on t,op of the gradients, centrifugation, and determination of radioactivity in each fraction were as described under Materials and Methods. (A) Virion DNA (1.6 pg of DNA); conditions for lsying purified virus on the gradients were the same as those used for intact cells. (3) Infected cells at 2 h.p.i. (0 Infected cells at 4 h.p.i. (D) Infected cells at 6 h.p.i.

BrdU was almost complete. Complete substitution would not be expected because of the endogenous nucleotide pools present in the ceils. These results established that the replication of vaccinia DNA was semiconservative. Replicahg vaccinia DNA contains ssDNA regions. To determine whether or

not the newly synthesized viral DNA contains ssDNA regions, the F3Hlthymidinelabeled viral DNA synthesized at different times postinfection was isolated and then treated with the single-stranded specific enzyme S-l nuclease (Table 1). The two forms of viral DNA, i.e., (L) and HL (30 min), isolated after C&l density centrifu-

VACCINIA

69

DNA REPLICATION

gation (pulse-labeled for 2 min at 2 h.p.i. and then chased for 30 min with thymidine or BrdU, see Fig. 7) were found to contain ssDNA regions to an extent of about 2030% (Table 1). However, when the replication of viral DNA was allowed to continue

for longer times and then isolated either by centrifugation in CsCl density gradient [pulse-labeled for 2 min at 2 h.p.i. and then chased for 60 min, HL (60 min)] or by sedimentation in neutral-sucrose gradients (pulse-labeled from 2 to 4 h.p.i.1, the C

FRACTION

FIG. 7. Analysis of replicating viral DNA by equilibrium density centrifugation in neutral CsCI. Cells were prelabeled with 0.2 &i/ml of [Y!lthymidine for 48 hr. Sixteen hours prior to virus infection, the cells were collected by centrifugation and resuspended in fresh media. Cells were infected or mock-infected as described under Materials and Methods. At 2 hr after virus infection, 2 x LO-ml aliquots were removed from the cultures, pulse-labeled with 20 &i/ml of 13Hlthymidine for 2 min, and then chased with 5 mM of either cold thymidine or bromodeoxyuridine (BrdU) for 23 min. At 2.30 h.p.i., the cells were collected, washed two times in buffered saline, resuspended in 0.4 ml of buffered saline, and dissolved in 1% Sarkosyl in the presence of 30 mM PME. After incubation for 10 min at room temperature, the lysate was digested with 1 mg/ml of pronase for 2 hr at 37”. The pronase-treated lysate from 5 x lo5 cells was analyzed by equilibrium density centrifugation in neutral CsCl as described under Materials and Methods. (A) Infected cells pulsed for 2 min with 13H1thymidine and chased with 5 mM thymidine for 30 min (0) or with (0) [Wlthymidine host DNA. (B) Infected cells pulsed for 2 min with [3Hlthymidine and chased with 5 mM BrdU for 30 min (0) or with (0) ‘*C-prelabeled host DNA, horizontal bars denote the percentage of [3H]thymidine (solid lines)- or [‘*Clthymidine (broken lines)-labeled DNA which hybridized to vaccinia virion DNA immobilized on nitrocellulose filters. (0 Peak fractions from B (fractions 10 to 15) were pooled, dialyzedd, and analyzed in a second CsCl gradient under the same conditions as A and B. (D) Vaccinia virion DNA labeled with 13Hlthymidine released by detergent lysis and pronase digestion of 4 x 10” EB was analyzed by equilibrium density centrifugation in neutral CsCl. The peak fractions from this analysis were pooled, dialyzed, and rerun in a second CsCl gradient under conditions identical to those described for the samples in A and B.

70

ESTEBAN AND HOLOWCZAK TABLE 1

EFFECT

OF S-l NUCLEASE ON SEVERAL FORMS REPLICATING VACCINIA DNA”

Source of DNA

CsCl L HL (30 min) HL (60 min)

Input: counts per minute

854 796 1195

OF

S-l nuclease Counts per minute 698 588 1120

Percentage resistant 81.7 72.0 93.7

Neutral sucrose 2 to 4 h.p.i. Virions Ad-2

3398 3171 93.3 3701 3511 94.8 15346 15029 97.9 a Newly synthesized vaccinia virus DNA from infected cells was analyzed by either isopycnic centrifugation in CsCl or sedimentation in neutral-sucrose velocity gradients. Peak fractions corresponcling to L and HL DNA from infected cells labeled during a 2-min pulse at 2 h.p.i., followed by a chase with cold thymidine or BrdU for 30 and 60 min, respectively, were pooled (see Fig. 7). Peak fractions containing viral DNA analyzed by sedimentation in neutral-sucrose gradients from either infected cells (labeled from 2 to 4 h.p.i.) or purified virus (see Fig. 5) were pooled. Attempts to purify and recover DNA by phenol extraction and ethanol precipitation (Holowczak, 1976) led to significant losses of ssDNA species as measured by S-l nuclease digestion. The following method was therefore employed to prepare DNA samples for S-l nuclease digestion: The pooled fractions were dialyzed against two changes (1.5 liters each) of 10 n&f Tris-HCl buffer, pH 7.6, 1 mM EDTA for 24 hr at 4” and aliquots were used for S-l nuclease assay. Adeno-2 DNA was extracted and purified as described previously (Burlingham and Doerfler, 1971). Samples were digested with S-l (40 units/ml) nuclease for 15 min at 45” under the conditions described under Materials and Methods. L, unlabeled or [3H]thymidine-labelecl viral DNA molecules; HL, viral DNA molecules in which one strand was unlabeled or labeled with [3Hlthymidine while the complementary DNA strand contained significant substitution of thymidine residues with Brau.

proportion of ssDNA decreased to <7%, a value similar to that found for mature virion DNA (~5%) (Table 1). Thus, ssDNA species are intermediates in the replication of vaccinia DNA as determined here by the use of S-l nuclease (Table 1) and as shown by analysis by hydroxylapatite and BND-cellulose chromatography of pulse-

labeled DNA isolated from vaccinia virusinfected HeLa cells (Holowczak and Diamond, 1976). Replication of vaccinia DNA in L cells is symmetrical. When pulse-labeled DNA

(from 10 set to 10 min) synthesized 2 hr after virus infection was analyzed by sedimentation in alkaline-sucrose gradients, molecules with sedimentation coefficients between 10 and 30 S were the major DNA species detected (see Figs. 2, 3, and 4). Very little radioactivity was found in the region of unit length viral DNA Type I (70-72 S), indicating that polymerization may be symmetrical and proceeds via the mechanism proposed by Cairns (1963). Further evidence in favor of this hypothesis was obtained when replicating viral DNA was analyzed in ethidium bromideCsCl equilibrium density gradients. Cells were pulse-labeled for 2 min with [3H]thymidine 2 hr after infection and then chased with cold thymidine or BrdU (5 m&f each) for 30 min. The labeled DNA was then analyzed by equilibrium density gradient centrifugation in neutral CsCl (see Fig. 7). Fractions containing the bulk of the viral DNA were pooled, dialyzed, and analyzed in ethidium bromide-CsCl equilibrium density gradients (Fig. 8). As shown in Fig. 8, all forms of viral DNA had their densities decreased to about p = 1.59-1.60, indicating that they were linear molecules rather than circular or superhelical duplexes whose density would increase under these condition of analysis (Radloff et al., 1967). Replication of vaccinia virus DNA therefore appears to be symmetrical and does not involve covalently bound continuous concatemers or the addition of new molecules to growing strands as in the rolling circle model (Gilbert and Dressler, 1968; Kelley and Thomas, 1969; Ihler and Thomas, 1970). DISCUSSION

By using intact cells to analyze the replication of vaccinia virus DNA, manipulations which could affect the structure of the large viral DNA molecules were avoided. Newly synthesized viral DNA could be readily identified and separated from the bulk of the host DNA by two

VACCINIA

DNA

FIG. 8. Analysis of newly synthesized viral DNA by centrifugation in ethidium bromide-CsCl equilibrium density gradients. Peak fractions of the viral specific DNA isolated by centrifugation in neutral CsCl equilibrium density gradients (Fig. 7) were pooled, dialyzed, and analyzed in preformed CsCl gradients containing ethidium bromide, as described under Materials and Methods. (A) Fractions 18 to 22 from Fig. 7A: 0, 3H-labeled viral DNA labeled during a 2-min pulse and chased with cold thymidine; 0, W-prelabeled host DNA. (B) Fractions 10 to 15 from Fig. 7B: l , 3H-labeled viral DNA labeled during a 2-min pulse and chased for 30 min with cold BrdU; 0, W-prelabeled host DNA. (C) Vaccinia virion DNA. The peak fractions used were those indicated in Fig. 7D.

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71

methods of analysis: (1) sedimentation in both neutral- and alkaline-sucrose velocity gradients and (2) equilibrium density centrifugation of the newly synthesized viral DNA after replacement of thymidine with bromodeoxyuridine (BrdU). Under our experimental conditions of analysis in both neutral- and alkaline-sucrose velocity gradients, more than 90% of the 14C-prelabeled host DNA was pelleted, while about 90% of the newly synthesized viral DNA was resolved in the gradients [note that replicating viral DNA analyzed in neutralsucrose gradients at 2 h.p.i., but not after 4 h.p.i., sedimented as large aggregates with the bulk of host DNA (see the legend to Fig. 511. Infection with vaccinia virus rapidly leads to inhibition of host DNA replication (Kit and Dubbs, 1962; Jungwirth and Lanner, 1968). We have not observed a breakdown of host DNA into molecules <120140 S at 2 h.p.i. after infection of L cells with vaccinia virus. As late as 6 h.p.i., approximately 90% of the [14C]thymidineprelabeled host DNA still sedimented to the bottom of the centrifuge tube under our conditions of analysis (see the legend to Fig. 4). In the case of infected HeLa cells, extensive degradation of host DNA was reported by 2 hr after infection (Parkhurst et al., 1973). It may be that the fates of HeLa and L cell DNAs after vaccinia virus infection are different. A nuclease (ssDNase) which is present within the virion has been implicated in the degradation of host DNA, but its specificity as related to the DNA of the host cell species is unclear (Pogo and Dales, 1973, 1974). Moreover, we have employed an extended period of 16-20 hr after removal of the [14Clthymidine used to label the host cell DNA before infecting the cells. This period allows depletion of cellular pools and a period in which host cell DNA replication can be completed. In their studies, Parkhurst et al. (1973) infected HeLa cells 2 hr after removal of the isotope used to label the host cell’s DNA. Such cells could contain DNA segments still undergoing replication, which may be more susceptible to the putative action of the virion nuclease. Our results show that the replication of

72

ESTEBAN

AND

vaccinia DNA in L cells is discontinuous, with the formation of small fragments (-10 S) which are subsequently ligated to larger molecules (Fig. 3). The time needed for completion of an intact single-stranded mature DNA molecule was found to be about 30 min (Figs. 2 and 3). Full conversion of the short strands (-10 S) to mature cross-link molecules, however, did not occur until late in infection (Fig. 4). These results indicate that maturation of vaccinia DNA is a multistep and highly synchronized process involving discontinuous synthesis of short fragments (-10 S), growing of the chains into, presumably, a pool of intermediate strands, ligation of the intermediate strands, and closure of the completed complementary strands by formation of cross-links at or near each end of the molecule. Since assembly of virus progeny commences at around 5-6 h.p.i. (Joklik and Becker, 1964), maturation into cross-linked molecules may be a prerequisite for virus assembly. By analysis of the fate of cross-linked parental DNA molecules after infection (Fig. 6) and the use of BrdU to density label replicating viral DNA molecules (Fig. 7), we have been able to demonstrate that the appropriate template and replicating intermediates can be detected to demonstrate a semiconservative mode of replication for vaccinia DNA. The “intermediate molecules” detected in these studies were very tightly bound to their template, as indicated by the analysis of pulse-chase-labeled viral DNA in neutral-sucrose gradients at 4 and 6 h.p.i. (Fig. 5), but can be distinguished by their density in CsCl (p = 1.712). At 2 h.p.i., under the conditions of analysis described here, they are recovered as part of large aggregates which sediment with the bulk of host DNA. Such aggregates have been isolated from cytoplasmic extracts prepared from vaccinia virus-infected HeLa cells (Dahl and Kates, 1970a,b; Polisky and Kates, 1972, 1975; Holowczak and Diamond, 1976). Mature viral DNA is not completely released from these aggregates until 3 h.p.i. and a number of intermediates of different lengths can be seen in shorter pulse-chases (Esteban and

HOLOWCZAK

Holowczak, in preparation). In cytoplasmic cell extracts from vaccinia virusinfected HeLa cells, a series of intermediates which may represent circular molecules with nicks in both strands has been described (Holowczak and Diamond, 1976). Whether they are truly replicative intermediates or complexes which arise as a result of the partial disruption of the large viral aggregates when cells are broken is not clear. The replicating viral DNA molecules contain ssDNA regions, as shown by their sensitivity to Sl nuclease (70-80% resistant, see Table 1). Replicating DNA molecules with ssDNA regions (20-30%) labeled after very short pulses have been detected in vaccinia virus-infected HeLa cells (Holowczak and Diamond, 1976). Polisky and Kates (1976) have demonstrated that, of newly replicated viral DNA from virosomes, 40% is ssDNA. Polymerization of the growing strands of viral DNA appears to be symmetrical since no molecules of greater length than the template were observed in pulses of from 10 set to 10 min (Figs. 2-4) when analyzed in alkaline gradients. Further evidence which favors this hypothesis was obtained by analysis of these molecules in ethidium bromide-CsCl equilibrium density gradients (Fig. 8). The results reported here confirm and extend those presented by Holowczak and Diamond (1976), who analyzed the replicating vaccinia DNA molecules in cytoplasmic extracts prepared from HeLa cells. However, several important points of distinction, in addition to those already noted, should be made. First, the techniques described here permitted analysis of the viral DNA molecules present in both the cytoplasm and the nucleus of infected cells. No obvious differences in the “kinds” of viral DNA molecules detected here, as compared to those present in the cytoplasm of infected HeLa cells (Holowczak and Diamond, 1976), were evident. This would suggest that no “unique” events in vaccinia virus DNA replication occur in the host cell nucleus. However, until techniques become available for the preparation of nuclei completely free of cytoplas-

VACCINIA

DNA

mic contamination under conditions which preserve the structural integrity of replicating viral DNA molecules, the question of the exact function of the host cell nucleus in viral DNA replication remains to be determined. Second, when pulse-labeled viral DNA molecules were chased for extended periods with cold thymidine, the majority, if not all, of the short strands could be chased into mature, cross-linked viral DNA molecules (Fig. 4). Holowczak and Diamond (1976; Fig. 6) were unable to find conditions of chasing and analysis to demonstrate such a complete conversion. While this may reflect a difference in the mode of replication occurring in the host cells used, it may also reflect damage to replicating viral DNA molecules caused mechanically or through nuclease activity. By avoiding the preparation of cytoplasmic extracts, we feel that both problems are minimized, permitting a more accurate analysis of the events occurring during vaccinia virus DNA replication. As indicated under Results and in Table 1, the problems of mechanical damage or nuclease activity may have also contributed to the inability of Holowczak and Diamond (1976) to detect ssDNA regions in vaccinia DNA purified from the cytoplasm of infected cells by equilibrium density centrifugation in CsCl. In conclusion, replication of vaccinia virus DNA, as studied in intact infected L cells and in cytoplasmic extracts from infected HeLa cells (Holowczak and Diamond, 1976), appears to closely resemble the mode of DNA replication in mammalian cells (Schandl and Taylor, 1969; Nuzzo et al., 1970; Huberman and Horwitz, 1973; Korken et al., 1975; Gautschi and Clarkson, 1975; Friedman et al., 1975; Berger and Huang, 1974; Tseng and Goulian, 1975). Thus, an understanding of the molecular events during replication of vaccinia DNA may prove to be a useful model for understanding the replication of the intrinsically more complex mammalian chromosome. ACKNOWLEDGMENTS The Bucolo

expert technical is gratefully

assistance acknowledged.

of Ms. Domenica We wish to

73

REPLICATION

thank Dr. K. Raska and Dr. R. Walter Schlesinger for critically reading the manuscript and K. Biron for the gift of adenovirus type 2 DNA. This investigation was supported by Public Health Service Research Grant CA-11027-08 from the National Cancer Institute. J.A.H. is a recipient of Public Health Service Research Career Development Award CA-70458 from the National Cancer Institute. REFERENCES BALDWIN, R. L., and SHOUTER, E. M. (19631. The alkaline transition of BU-containing DNA and its bearing on the replication of DNA. J. Mol. Biol. 7, 511-526. BECKER, Y., and JOKLIK, W. K. (19641. Messenger RNA in cells infected with vaccinia virus. Proc. Nat. Acad. Sci. USA 51, 577-585. BECKER, Y., and SAROV, I. (1968). Electron microscopy of vaccinia DNA. J. Mol. Biol. 34,655-660. BERGER, H., JR., and HUANG, R. C. (1974). Studies on nascent DNA in mouse myeloma. Cell 2, 23-30. BERNS, K. J., and SILVERMAN, C. (19701. Natural occurrence of cross-linked vaccinia virus deoxyribonucleic acid. J. Virol. 5, 299-304. BOLDEN, A., ANCKER, J., and WEISSBACH, A. (1975). Synthesis of Herpes Simplex virus, vaccinia virus, and adenovirus DNA in isolated HeLa cell nuclei. I. Effect of viral-specific antisera and phosphoroacetic acid. J. Virol. 16, 1584-1592. BURGER, H., and DOERFLER, W. (19741. Intracellular forms of adenovirus DNA. III. Integration of the DNA of adenovirus type 2 into host DNA in productively infected cells. J. Virol. 13, 975-992. BURLINGHAM, B. T., and DOERFLER, W. (19711. Three size-classes of intracellular adenovirus deoxyribonucleic acid. J. Viral. 7, 707-719. CAIRNS, J. (19601. The initiation of vaccinia infection. Virology 11, 603-623. CAIRNS, J. (1963). The bacterial chromosome and its manner of replication as seen by autoradiography. J. Mol. Biol. 6, 208-213. DAHL, R., and KATES, J. (1970al. Intracellular structures containing vaccinia DNA: Isolation and partial characterizations. Virology 42, 453-462. DAHL, R., and KATES, J. (1970bl. Synthesis of vaccinia virus “early” and “late” messenger RNA in vitro with nucleoprotein structures isolated from infected cells. Virology 42, 463-472. DALES, S., and SIMINOVITCH, L. (1961). The development of vaccinia virus in Earle’s L strain cells as examined by electron microscopy. J. Biophys. Biothem. Cytol. 10, 475-502. DENHARDT, D. (1966). A membrane filter technique for the detection of complementary DNA. Biothem. Biophys. Res. Commun. 23, 641-646. EASTERBROOK, K. B. (1967). Morphology of deoxyribonucleic acid extracted from cores of vaccinia virus. J. Virol. 1. 643-645.

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AND HOLOWCZAK

M., and METZ, D. H. (1973). Early virus protein synthesis in vaccinia virus infected cells. J. Gen. Virol. 19, 201-216. FAREED, G. E., KHOURY, G., and SALZMAN, N. P. (1973). Self-annealing of 4s strands from replicating simian virus SV-40. J. Mol. Biol. 77, 457-462. FRIEDMAN, C. A., KOHN, D., and ERICKSON, L. (1975). DNA chain growth during replication of asynchronous L 1210 cells. Alkaline sedimentation studies. Biochemistry 14, 40184033. GAFF~RD, L. G., and RANDALL, C. (1976). Virusspecific RNA and DNA in nuclei of cells infected with fowlpox virus. Virology 69, 1-14. GAUTSCHI, J. R., and CLARKSON, J. M. (1975). Discontinuous DNA replication in mouse P-815. Eur. J. Biochem. 50, 403-412. GESHELIN, P., and BERNS, K. I. (1974). Characterization and localization of the naturally occurring cross-links in vaccinia virus DNA. J. Mol. Biol. 88, 785-796. GILBERT, W., and DRESSLER, D. (1968). DNA replication: The rolling circle model. Cold Spring Harbor ESTEBAN,

Symp. GREEN,

Quant.

Biol.

23, 473-484.

M. (1963). Studies on the biosynthesis of viral DNA. Cold Spring Harbor Symp. Quant. Biol. 27, 219-233. GREEN, M., PI&A, M., and CHAGOYA, U. (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. HANAFUSA, T. (1961). Enzymatic synthesis and breakdown of deoxyribonucleic acid by extracts of L cells infected with vaccinia virus. Biken’s J. 4, 97-110. HOL~WCZAK, J. A. (1976). Poxvirus DNA. I. Studies on the structure of the vaccinia genome. Virology 72, 121-133. HOL~WCZAK, J. A., and DIAMOND, L. (1976). Poxvirus DNA. II. Replication of vaccinia virus DNA in the cytoplasm of HeLa cells. Virology 72, 134-146. HOLOWCZAK, J. A., and JOKLIK, W. K. (1967). Studies on the proteins of vaccinia virus. I. Structural proteins of the virion and core. Virology 33, 717725. HUBERMAN, J. A., and HORWITZ, H. (1973). Discontinuous DNA synthesis in mammalian cells. Cold Spring Harbor HUBERMAN, J.

Symp.

Quant.

Biol.

38,233-238.

A., and RIGGS, A. D. (1968). On the mechanism of DNA replication in mammalian chromosomes. J. Mol. Biol. 32, 327-341. IHLER, G. M., and THOMAS, C. A. (1970). Equal incorporation of both parental bacteriophage T7 deoxyribonucleic acid strands into intracellular concatemeric DNA. J. Virol. 6, 877-879. JOKLIK, W. K. (1962a). Some properties of poxvirus DNA. J. Mol. Biol. 5, 265-274. JOKLIK, W. K. (1962b). The preparation and characteristics of highly purified radioactivity labeled

poxvirus. Biochim. Biophys. Acta 61, 290-301. W. K., and BECKER, Y. (1964). The replication and coating of vaccinia DNA. J. Mol. Biol. 10, 452-474. JUNGWIRTH, C., and JOKLIK, W. K. (1965). Studies on “early” enzymes in HeLa cells infected with vaccinia virus. Virology 27, 80-93. JUNGWIRTH, C., and LAUNER, J. (1968). Effect of poxvirus infection on host cell deoxyribonucleic acid synthesis. J. Virol. 2, 401-409. KATO, S., and OGAWA, M., and MYAMOTO, H. (1964). Nucleocytoplasmic interaction in poxvirus infected cells. I. Relationship between inclusion formation and DNA metabolism of the cells. Biken’s JOKLIK,

J. 7, 45-56. KATO, S., TAKAHASHI, MAHORA, J. (1959).

M., KAMEYAMA, S., and KAA study on the morphological and cytoimmunological relationship between the inclusions of variola, cowpox, rabbitpox, vaccinia (variola origin) and vaccinia IHD and a consideration of the term “Guarineri body.” Biken’s J. 2, 353-363. KELLY, T. J., JR., and THOMAS, C. A. JR. (1969). An intermediate in the replication of bacteriophage T7 DNA molecules. J. Mol. Biol. 44, 459-475. KIT, S., and DUBBS, D. K. (1962). Biochemistry of vaccinia infected mouse fibroblasts (Strain L-M). II. Properties of the chromosomal DNA of infected cells. Virology 18, 286-297. KIT, S., and DUBBS, D. K. (1965). Properties of deoxythymidine kinase partially purified from noninfected and virus infected mouse fibroblast cells. Virology 26, 16-27. KROKEN, H., COOKE, L., and PRYDZ, H. 11975). DNA synthesis in isolated HeLa cells nuclei. Evidence for in vitro initiation of synthesis of small pieces of DNA and their subsequent ligation. Biochemistry 14, 4233-4337. LA COLLA, P., and WEISSBACH, A. (1975). Vaccinia virus infection of HeLa cells. I. Synthesis of vaccinia DNA in host cell nuclei. J. Viral. 15, 305-315. MAGEE, W. E., and LEVINE, S. (1970). The effects of interferon on vaccinia virus infection in tissue culture. Ann. N.Y. Acad. Sci. 173, 362-378. MAGEE, W. E., and MILLER, W. (1967). Immunological evidence for the appearance of a new DNA polymerase in cells infected with vaccinia virus. Virology 31, 64-69. MAGEE, W. E., SHUK, M. R., and BURROWS, M. J. (1960). The synthesis of vaccinia deoxyribonucleic acid. Virology 11, 296-299. MAGNUSSON, G., PIGIET, U., WINNACKER, E. L., ABRAMS, R., and REICHARD, P. (1973). RNAlinked short DNA fragments during polyoma replication. Proc. Nat. Acad. Sci. USA 70,412-415. MARCUS, P. I., and ROBBINS, E. (1963). Viral inhibition in the metaphase-arrest cell. Proc. Nat. Acad. Sci. USA 50, 1156-1164. MCAUSLAN, B. R. (1963). The induction and repres-

VACCINIA

DNA REPLICATION

sion of thymidine kinase in poxvirus infected HeLa cells. Virology 21, 383-389. MCAUSLAN, B. R. (1965). Deoxyribonuclease activity of normal and poxvirus infected cells. Bio&em. Biophys. Res. Commun. 20, 586-591. MCAUSLAN, B. R., and KATES, J. R. (1966). Regulation of virus induced deoxyribonuclease. Proc. Nut. Acad. Sci. USA 55, 1581-1587. MCAUSLAN, B. R., and KATES, J. R. (1967). Poxvirus induced deoxyribonuclease; regulation of synthesis; control of activity in uiuo; purification and properties of the enzyme. Virology 33, 709-716. MCCREA, J. F., and LIPMAN, M. B. (1967). Strandlength measurements of normal and S-iodo-2deoxy-uridine-treated vaccinia virus deoxyribonucleic acid released by the Kleinschmidt method. J. Viral. 1, 1037-1044. MORROW, J. F., and BERG, P. (1972). Cleavage of simian virus 40 DNA at a unique site by a bacterial restriction enzyme. Proc. Nut. Acad. Sci. USA

69, 3365-3369.

Nuzzo, F., BREGA, A., and FALASCH~, A. (1970). DNA replication in mammalian cells. I. The size of newly synthesized helices. Proc. Nat. Acud. Sci. USA 65, 1017-1024. OKAZAKI, R., OKAZAKI, T., SAKABE, K., SUGIMOTO, K., and SUGINO, A. (1968). Mechanism of DNA chain growth. I. Possible discontinuity and unusual secondary structure of newly synthesized chains. Proc. Nut. Acud. Sci. USA 59, 598-605. PARKHURST, R. J., PETERSON,A. R., and HEIDELBERGER, C. (1973). Breakdown of HeLa cell DNA medicated by vaccinia virus. Proc. Nut. Acud. Sci. USA

70, 3200-3204.

PETTERSSON, LJ. (1973). Some unusual properties of replicating adenovirus type 2 DNA. J. Mol. Biol. 81, 521-527. POGO, B. G. T., and DALES, S. (1969). Two deoxyribonuclease activities within purified vaccinia virus. Proc.

Nat.

Acud.

Sci.

USA

63, 820-827.

B. G. T., and DALES, S. (1973). Biogenesis of poxvirus: Inactivation of host DNA polymerase by a component of the invading inoculum particle. Proc. Nut. Acad. Sci. USA 70, 1726-1729. POGO, B. G. T., and DALES, S. (1974). Biogenesis of poxviruses: further evidence for inhibition of host and virus DNA synthesis by a component of the invading inoculum particle. Virology 58, 377-386. POLISKY, B., and KATES, J. R. (1972). Vaccinia virus intracellular DNA-proteins complex: Biochemical characteristics of associated protein. Virology 49, POGO,

68-79.

75

POLISKY, B., and KATES, J. R. (1975). Viral specific polypeptides associated with newly replicated vaccinia DNA. Virology 66, 128-139. POLISKY, B., and KATES, J. (1976). Interaction of vaccinia DNA binding proteins with DNA in vitro. Virology 69, 143-147. RADLOFF, R., BAUER, W., and VINOGRAD, J. (1967). A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: The closed circular DNA in HeLa cells. Proc. Nut. Acud. Sci. USA 57, 1514-1521. REICH, E., and FRANKLIN, R. M. (1961). Effect of mitomycin C on the growth of some animal viruses. Proc. Nat. Acud. Sci. USA 47,1212-1217. SALZMAN, N. P. (1960). The rate of formation of vaccinia deoxyribonucleic acid and vaccinia virus. Virology 10, 150-152. SAMBROOK, J., and SHATKIN, A. J. (1969). Polynucleotide ligase activity in cells infected with simian virus 40, polyoma virus, or vaccinia virus. J. Virol. 4, 719-726. SAROV, I., and BECKER, Y. (1967). Studies on vaccinia virus DNA. Virology 33, 364-373. SCHANDL, E. K., and TAYLOR, J. H. (1969). Early events in the replication and integration of DNA into mammalian chromosomes. Biochem. Biophys. Res. Commun. 34, 291-300. SHARP, P. A., PETTERSSON, U., and SAMBROOK, J. (19741. Viral DNA in transformed cell lines. I. A study of the sequences of adenovirus 2 DNA in a line of transformed rat cells using specific fragments of the viral genome. J. Mol. Biol. 86, 709726. SHATKIN, A. J., and SALZMAN, N. P. (1963). Deoxyribonucleic acid synthesis in vaccinia virus infected HeLa cells. Virology 19, 551-560. STUDIER, F. W. (1965). Sedimentation studies of the size and shape of DNA. J. Mol. Biol. 11, 373-390. TSENG, B. Y., and GOULIAN, M. (1975). DNAsynthesis in human lymphocytes: Intermediates in DNA synthesis, in vitro and in ho. J. Mol. Biol. 99, 317-337. VLAK, J. M., ROZYN, T. H., and SUSSENBACH, J. S. (1975). Studies on the mechanism of replication of adenovirus DNA. IV. Discontinuous DNA chain propagation. Virology 63, 168-175. WALEN, K. H. (1971). Nuclear involvement in poxvirus infection. Proc. Nut. Acud. Sci. USA 68, 165168. WINNACKER, E. L. (1975). Adenovirus type 2 DNA replication. I. Evidence for discontinuous DNA synthesis. J. Virol. 15, 744-758.