Initiation and termination of vaccinia virus DNA replication

JOS, 241-248 (1981)

VIROLOGY

Initiation

and Termination

of Vaccinia

B. G. T. POG0,2 M. O’SHEA, Centerfor

Experimental

Cell

Biology,

Mount

Accepted

Sinai

AND P. FREIMUTH

School

September

Virus DNA Replication’

of Medicine,

Nelc

York,

New

York

10029

22, 1980

The initiation and termination sites of replication of vaccinia DNA has been studied by determining the radioactivity in restriction fragments of the pulse-labeled newly synthesized molecules. The results indicate a bimodal distribution of radioactivity in the molecules completed during a pulse shorter than the completion time of synthesis, implying termination at both ends. The symmetry of replication was studied by hybridization of an intermediate in replication (34 S) to the isolated strands of virion DNA followed by restriction analysis. The frequency of hybridization of the fragments to the light or heavy strand shows a unimodal distribution of radioactivit,y indicating asymmetric replication. Together, these observations suggest that initiation and termination sites are located at both ends of the chromosome. Changes observed in the conformation of parental DNA at the time of replication, such as high degree of single-strandness, are compatible with strand displacement during synthesis”

Vaccinia virus RNA synthesis occurs in the cytoplasm of infected cells as a wave between 2 and 5 hr after infection and is completed before mature particles are formed. The viral DNA replicates discontinuously by means of single-stranded (ss) segments that contain covalently linked ribonucleotides (Esteban and Holowczak 1977; Pogo and O’Shea, 1978). These segments become integrated first into a 84 S intermediate and finally into the 68 S full size virus DNA. The 96 S and 106 S crosslinked molecules detected in alkaline sucrose gradients are observed late in infection, suggesting that the acquisition of crosslinks is a postreplicative process occurring at the time that virus maturation prevails (Esteban and Holowczak, 1977; Pogo, 1977). Based on electron microscopic observations, Esteban et al, (1977) have proposed that DNA replication is initiated at and proceeds from one end of the viral DNA molecule. However, the presence at both ends of the molecule of similar inverted repetitive sequences, makes this possibility unlikely (Wittek et al., 1978; Garon et al., 1 Supported in part by Grants AI 12133 and AI 15953 from the National Institutes of Health. * To whom reprint requests should be addressed. 241

1978). The studies presented here were undertaken to detect the sites at which replication starts and terminates. For this purpose labeled replicating DNAs of different sizes were digested with restriction enzymes and the relative yield of each fragment determined. In this manner, it can be established whether a particular region of the DNA is synthesized first or last. This approach has been used to determine the origin and terminus of DNA replication of SV, (Danna and Nathans, 1972) adenovirus (Horwitz, 1976), and adeno-associated virus (Hauswirth and Berms, 1977). More recently herpes virus DNA replication was also studied, following a similar type of analysis (Ben-Par& and Veach, 1980). The results, reported here, suggest that vaccinia DNA replication is initiated and terminated at both termini in a manner reminiscent of adenovirus DNA replication. L cells and vaccinia virus strain IHD-W were used in all experiments in accordace with procedures previously de caft;lts, 1968). Preparation of [l*C]th~~e-~~l~d virus and [3H]thymidine replicating virus DNA were carried out as in previous publications (Pogo, 1977, 198%; Pogo and O’Shea, 1978). Replicating DNA of different 0042-6822/81/010241~~208$02.0010 Copyright 9 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

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SHORTCOMMUNICATIONS

sizes were isolated from neutral-sucrose gradients. The fractions containing the 12 S, 34 S, and 68 S species were dialyzed against a solution containing 0.1 M Tris-HCl buffer, pH 7.6, 1 n&f EDTA, and 4 mM NaCl (TES) and concentrated by placing the dialyzing tubing in Carbowax. These DNAs were mixed with [14C]thymidinelabeled virion DNA, digested with Hind111 for 3 hr and electrophoresed in 0.8% agarose gels. (Pogo, 1980b). Separation of the vaccinia DNA strands was accomplished by equilibrium density sedimentation in alkaline C&SO4 gradients (Van der Vliet and Sussenbach, 1972). For this purpose, noncrosslinked replicating DNA sedimenting in neutral sucrose gradients at 68 S, was alkali denatured with 0.1 N NaOH at 37” for 10 min and then mixed with saline citrate solution containing 0.65 g/ml of Cs,S04, 0.01 M EDTA, 0.1% sarkosyl brought to pH 12.8 with 3 N NaOH, and centrifuged to equilibrium for 67 hr at 35,000 rpm in a SW65 rotor. The gradients were fractionated from the bottom and the aliquots of the fractions were counted to localize the peaks. The fractions containing the heavy and the light part of the peak, respectively, were pooled and dialyzed against TES and then subjected to a second centrifugation in Cs,S04. After one or two recyclings, two distinctive peaks could be observed; a heavy, banding at 1.47 g/cm3 and a light at 1.45 g/cm3. Reannealing experiments between the two isolated strands or between the isolated strands and the different species of replicating DNA were carried out following the conditions described by Bolden et al. (1979). Afterwards the hybridized material was further digested with S, or Neurospora crassa nuclease. The separation of hybridized restriction fragments was accomplished by agarose gel electrophoresis as described above. However, due to the presence of enzyme, buffers, and salts the migration of fragments in the gels was different than during regular electrophoretic conditions. To identify the fragments, 3H-labeled 68 S was digested with HindIII, incubated under the same conditions except that the N. crassa or S, nuclease was heat inactivated, and electrophoresed in a separate gel.

Conditions for infection with labeled virus and extraction of parental DNA were carried out as described previously (Pogo, 1977, 198Ob). The separation of double-stranded (ds) and ss DNA was performed by density gradient centrifugation in Cs, and SO, with Hg2+ ions (Nandi et al., 1965) and by hydroxylapatite chromatography (Pogo, 1980a). Pulse-labeled replicating rH]DNA of different sizes, isolated from neutral sucrose gradients, was mixed with virion [‘*C]DNA digested withHind and subjected to agarose gel electrophoresis (Fig. 1). As expected, most of the restriction fragments of 34 S and 68 S species, synthesized during 30and 6Omin pulse, respectively, were labeled. In addition, it was observed that digestion of 12 S species synthesized during a 5-min pulse resulted in radioactivity across the entire gel and mainly in fragments that correspond to both ends of the molecule (fragments B and C) and also in fragment A. This result suggests that the 12 S species represents population of molecules. To 6nd out the specific activity of each fragment, the ratio 3H/14C was determined and the results of these experiments are summarized in Fig. 2. The restriction fragments are drawn to scale following the map published for strain IHD-W using Hind111 by Schumperli et al. (1980). In (a) the ratio 3H/14C of the fragments synthesized during a 5min pulse is shown. Although the 12 S was mainly labeled during this interval, some radioactivity was also detected in the 34 S and 68 S species. In all three size molecules, the fragments with the highest specific activity were those localized at the ends of the genome. The lowest specific activity (ratio 1) was found near the center of the molecule. In (b) analysis of the restriction fragments of the molecules labeled during 30-min pulse shows that all fragments were equally labeled in the 34 S; however, in the 68 S the end fragments were still highly radioactive. In (c) the ratio 3H/14C in the restriction fragments of the 68 S labeled during a 60-min pulse indicates that differences in the specific activity of the individual fragments have disappeared. The reason for the bimodal distribution of the radioactivity shown in Fig. 2 is not clear.

24:s

SHORT COMMUNICATIONS 12s

2m

500

34s

4000

,200o

3000

t 2 a n :

2000

1000

88s

6000

1 2000

4500

0

20

40

60

80

100

FRACTION NUMBER FIG. 1. Fractionation of replicating vaccinia DNA restriction fragments by Agatha gel electrophoresis. Aliquots of 3H-labeled replicating DNA isolated from neutral sucrose gmdb& were mixed with ‘C-labeled virion DNA, digested with HimdIII, and electrophoresed in 0.8% gels as described in the text. The 12 S was labeled during a 5-min p&e and the 34 S and 68 S dtiaing Wand W-min pulses, respectively. After correcting for spillover the disintegrations per minute for each isotope was calculated. Efficiency of counting wm 20% for 3H and 50% for W.

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C WK

F

E 01

GLJH

D

A

0

C YNK

F

E 01

GLJH

D

A

0

FIG. 2. Distribution of radioactivity in restriction fragments of replicating DNA as a function of time. Experimental conditions as in Fig. 1. The ratio of 3H-replicating DNA to virion [‘%]DNA of each restriction fragment is plotted against its location in the genome. The results are the average of three independent experiments. (a) 5 min pulse, (Cl) 12 S DNA; (b) 36-min pulse, (0) 34 S DNA, (c) 66-min pulse, (A) 68 S-DNA.

In order to determine whether (1) initiation of growth starts in the middle proceeding toward each end of the molecule or (2) beginning of replication is at each terminus and proceeds to the other end, experiments were designed to determine the gradient of labeling in each of the complementary strands. Attempts to separate the strands of the restriction fragments by electrophoresis under denaturing conditions were not successful. Instead, experiments were designed in which 3H-labeled replicative molecules, 34 S and 68 S, were hybridized with an excess of isolated strands of 14Clabeled vaccinia DNA, followed by digestion with Hind111 and separation of the fragments. The 12 S species were not used in these experiments because of their heterogeneity. The degree of hybridization was measured by resistance to a ss nuclease (N. crassa). The results of such an experi-

ment are summarized in Table 1. It can be seen that isolated strands self-reannealed between 10 and 16% indicating a high degree of purity and that 34 S and 68 S hybridized to the isolated strands. The strands reassociated with the 68 S with a CJ,,, of 0.060; close to that reported for virion DNA by Pedrali-Noy and Weissbath (1977). The results, obtained when the 34 S intermediate was hybridized to heavy and light strands, digested with HindIII, ss nuclease, and the fragments separated by electrophoresis, are illustrated in Fig. 3. The values are expressed as frequency of hybridization of each fragment to one of the strands, as indicated by the formula: No. times Rf present x 1oo No. of experiments ’ Since some hybridization within the 34 S

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molecules took place (Table l), only those restriction fragments containing both 3H and 14C radioactivity were considered. It is evident that one end of the replicating molecules hybridized preferentially to one end of the strand. The frequency of which the fraction of the 34 S molecule containing fragments A and B hybridized with the fragments A and B of the heavy strand is 100%. The same is true for fragment C with the complementary strand L. By contrast, when the 68 S replicating DNA was hybridized against the 68 S, no gradient was observed. All the fragments were present in the gels with the same frequency (data not shown). The unimodal distribution of radioactivity in each of the replicating 34 S strands with the highest specific activity near the termini suggests bidirectional replication; i.e., beginning at both termini. The results of the experiments described above suggest that vaccinia DNA replicates bidirectionally, beginning at both ends. A mechanism called “displacement synthesis” has been described in adenovirus replication. This concept implies that during DNA synthesis, one of the parental strands is displaced by the growth of the replicating fork in the complementary strand, thus generating regions of ss DNA (Ellens et al., 1974). To find out whether changes in parental DNA occur during DNA synthesis, the conformation of labeled parental DNA was studied by density gradient centrifugation and hydroxylapatite chromatography. The results of experiments in which labeled parental DNA extracted 1 and 3 hr after infection was banded in a gradient of Cs, SO, containing Hg2+ ions are shown in Fig. 4. This procedure allows the separation of ds DNA from the DNA with ss regions or gaps. It can be seen that parental DNA extracted 1 hr after infection, before DNA synthesis started, banded mostly at 1.50 g/cm3 while parental DNA extracted after 3 hr of infection banded at 1.59 g/cm3 indicating a higher degree of single-strandness. Similarly, analysis by hydroxylapatite chromatography showed that after 3 hr of infection, 45% of the parental DNA eluted as ss DNA (Table 2).

TABLE

1

HYBRIDIZATION OF ISOLATED STRANDS DNA WITH REPLICATIVE 34 S AND 68

OF VAC~INIA

S MOLECULES

Percentage hybridized Conditions ‘%-labeled H strand + 3H-labeled 34 S ‘%-labeled L strand + “H-labeled 34 S ‘%-labeled H strand + 3H-labeled 68 S W-labeled L strand + “H-labeled 68 S W-labeled H strand alone W-labeled L strand alone

1°C

‘H

40

100

60

100

80

80

80

80

10

-

-...

16

Note. Aliquots of heavy (2.75 pg) and light (2.25 *gl strands of vaccinia [*‘C]DNA, prepared as indicated in the text, were mixed with 0.5 pg of 3H-labeled 34 S or 68 S molecules in a solution containing 0.05 M HEPES buffer, pH 7.1,0.5 M NaCl, and 1 mkf EDTA, heated for 5 min at loo”, and incubated for 20 hr at 65”. When heavy or light strands were incubated alone the concentration of DNA was 5.5 Kg and 4.50 pg, respectively. Hybrid formation was de&&d by digestion of the samples with 10 units of N. croaau endonuclease in 0.1 M Tris- HCl, pH 8,lOmAf Mg Cl,, and 0.15 M Na Cl, for 2 hr at 3’7’. Samples incubated without the enzyme were used as blanks to determine the input radioactivity. The reaction was terminated by addition of cold 10% trichloroacetic acid, the precipitates were collected, and the arid insoluble radioactivity measured. The results are expressed as the percentage of rH]- or [W]DNA resistant to endonuelease digestion.

The experiments described here suggest that vaccinia DNA replication is initiated and proceeds toward the ends in a bWrectional manner, similar to adenovimc DNA. It is important to emphasize that in these studies, DNA synthesis was not completely synchronized and this can explain in part, the heterogeneous pattern of labeling of the 12 S species. The use of inhibitors of DNA synthesis was avoided because ofthe they are known to cause and only thymidine starvation was used to increase Meled thymidine incorporation.

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SHORT COMMUNICATIONS

mr m

60

!lea -60 eo

I OLJH

C YIK

F

EOI

6LJH

D

A

RESTRICTION FRAGMENT FIG. 3. Frequency of hybridization of replicating DNA with isolated strands of vaccinia DNA. Aliquots of 3H-labeled 34 S synthesized during a 20-min pulse were mixed with heavy or light strands of W-labeled 63 S DNA; they were heat denatured for 5 min at loo” and incubated in annealing buffer for 18 hr at 65”. Afterward they were digested with Hind111 for 2 hr followed by incubation with N. crassa or S, endonuclease for 2 hr and electrophoresed in agarose gels. The number of times that a fragment hybridized with one strand percent over the number of experiments performed is plotted against their location in the genome. The experiments were performed at least four times. H, heavy strand; L, light strand.

When a radioactive precursor enters the replicating fork of growing molecules it will be located at different sites, depending on the growth of the molecule. Molecules, which are completed during labeling periods shorter than the time of synthesis will be preferentially labeled at the terminus. Thus the 34 S and the 63 S molecules labeled during a “pulse” of 5 min show highest specific activity at the terminus, since the the replication times of these molecules are 20 and 60 min, respectively (Pogo and O’Shea, 1978). The differences between the specific activity of the individual fragments, are expected to disappear with longer periods of labeling. Thus the fragments of the 34 S after 30-min “pulse” are equally labeled as well as are the fragments of the 63 S

after 60-min pulse. The bimodal distribution of radioactivity in the fragments suggests that the growth of the DNA molecules is bidirectional. On the other hand the unimodal gradient of radioactivity of the replicating strands clearly demonstrates that the strands are synthesized asymmetrically. However, there is a higher degree of hybridization than expected between the 34 S and part of the L strand molecule that contains fragments A and B (Fig. 3). This finding can be explained by contamination of the light strand with small amounts of ds DNA or by the fact that there are repetitive sequences in fragment B similar to those in fragment C (Garon et al., 19’78; Wittek et al., 1978). The second possibility seems unlikely since with H

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strand, the frequency of hybridization of fragment C is only 18%. Some of the models of replication of adenovirus DNA can be also applied to vaccinia DNA. Thus changes in parental DNA conformation and a certain degree of singlestrandedness found in replicating DNA can be accounted for by displacement synthesis. The presence of a circular intermediate can be also consistent with this model of replication. Structural variability of the vaccinia genome, such as the identical deletions at both ends, can be explained by a circular intermediate in replication (McFadden and Dales, 1979). However, attempts to isolate such an intermediate using density gradient centrifugation have been negative (unpublished results). Although electron microscopic evidence for the initiation at one end of the molecule have been published, Y-shaped molecules characteristic of displacement synthesis have yet to be described (Esteban et al., 1977; Ellens et aZ., 1974). Whether initia-

I.5

1.7

s

IS FRACTION

2s

%

36

TABLE

DISTRIBUTION OF ss OR ds DNA AS PERCENTAGE OF THE TOTAL ELUTED FROM HYDROXYLAPATITE

IN VIRAL DNA RADIOACTIVITY

Percentage of total cpm Source of DNA

ss

Virus 0 time 45 min p.i. 90 min p.i. 120 min p.i. 180 min pi.

5-10 10 14 20 25 45

ds

-

95-w

90 86 80 75 55

Note. Aliquots of YH-labeled vaccinia DNA extracted from purified virus particles or from the cytoplasmic fraction of virus-infected cells were adsorbed to hydroxylapatite and eluted with increasing concentrations of the phosphate buffer. Sin&+stranded DNA eluted with 0.15 1cp phosphate buffer and double-stranded with 0.25 M. Between 80 to 90% oft he radioactivity added was recovered.

tion occurs at both ends at the same time is not clear at the moment, nor is the precise site within the terminal fragment where it starts. Both terminal fragments contain inverted repetitive sequences of about 10,500 base pairs and it is attempting to speculate that at least part of these sequences may be recognition sites for the proteins involved in DNA rephcation. A better understanding of the primary structure of the molecule is necessary to clarify this issue.

No.

4. C&SO,-Hg2+ density gradient centrifugation of parental vaccinia DNA. Cells were infected with 13H]thymidine-labeled vaccinia virus and the parental DNA was extracted from the cytoplasmic fraction of virus-infected cells 1 and 3-hr after infection. Aliquots of the DNA were mixed with sodium borate buffer (final concentration 5 mM, pH 9), solid C&SO,, and HgCI, in this order, to give a final density of 1.55 g/cm3 and R, (molar ratio of total Hg*+ to total DNA phosphate) of 0.30. The mixture was centrifuged at 35,000 rpm at 4” for 65 hr in a SW65. Fractions were collected, precipitated with cold 10% trichloroacetic acid, and the radioactivity determined. (X) DNA extracted 1 hr after infection; (0) DNA extracted 3 hr after infection.

2

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FIG.

BEN-P•

RAT,

T. and VEACH,

R. A. (1930).

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of

replication of the DNA of a herpesvirus (pseudorabbies). Proc. Nat. Acad. Sci. USA 77, 172-175. BOLDEN,

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PEDRALI-NOY,

G., and WEISSBACH,

A.

(1979). Vaccinia virus infection of HeLa cells. II Disparity between cytoplasmic and nuclear viralspecific RNA. Virology 94, 138 145. DALES, S. (1963). The uptake and development of vaccinia virus in strain L cells followed with labeled viral deoxyribonucleic acid J. Cell Biol. 18, 51-72. DANNA, K. J., and NATHANS,

replication

D. (1972). Bidirectional of simian virus 40 DNA. Proc. Nat.

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GARON, C. G., BARBOSA, E., and Moss, B. (1978). Visualization of an inverted terminal repetition in vaccinia virus DNA. Proc. Nat. Acad. Sci. USA 75, 4863-4867.

HAUSWIRTH, W. W., and BERNS,K. I. (1977). Origin and termination of adeno-associated virus DNA replication. Virology 78, 488-499. HORWITZ, M. S. (1976). Bidirectional replication of adenovirus type 2 DNA. J. Virol. 18, 307-315. MCFADDEN, D., and DALES, S. (1979). Biogenesis of poxviruses mirror-image deletions in vaccinia virus DNA. Cell 18: 101-108. PEDRALI-NOY, G., and WEISSBACH, A. (1977). Evidence of a repetitive sequence in vaccinia virus DNA. J. Viral. 24, 406-407. POCO, B. G. T. (1977). Elimination of naturally occuring cross-links in vaccinia virus DNA after virus

penetration. Proc. Nat. Acad. Sci. USA 74, 17391742. POGO, B. G. T. (1980a). Terminal cross-linking of vaccinia DNA strands by an in vitro system. Virology 100, 339-347.

POGO, B. G. T. (1980b). Changes in parental vaccinia virus DNA after viral penetration into cells. Virology 101, 520-524.

POGO, B. G. T., and O’SHEA, M. (1978). The mode of replication of vaccinia virus DNA. Virology 84, l-8. NANDI, U. S., WANG, J. C., and DAVIDSON, N. (1965). Separation of deoxyribonucleic acids by Hg (II) binding and CsZ SO, density-gradient centrifugation. Biochemistry 4, 1687-1696. SCH~MPERLI, D., MCFADDEN, G., WYLER, R., and DALES, S. (1980). Location of a new endonuclease restriction site associated with a temperaturesensitive mutation of a vaccinia virus. Virology 101, 281-285.

VAN DER VLIET, P. C., and SUSSENBACH, J. S. (19’72). The mechanism of adenovirus-DNA synthesis in isolated nuclei. Eur. J. Biochem. 30,584-592. WITTEK, R., MENNA, A., MOLLER, H. K., SCHUMPERLI, D., BOSELEY, P. G., and WYLER, R. (1978). Inverted terminal repeats in rabbit poxvirus and vaccinia virus DNA. J. Viral. 28, 171-181.