Vol. 17, No. 3 Printed in U.S.A.

JouRNAL OF VIROLOGY, Mar. 1976, p. 841-853 Copyright 0 1976 American Society for Microbiology

Multiplication of Parvovirus LuIII in a Synchronized Culture System Ill. Replication of Viral DNA GUNTER SIEGL* AND MARKUS GAUTSCHI Institute of Hygiene and Medical Microbiology, University of Berne, CH 3008 Berne, Switzerland

Received for publication 11 September 1975

The replication of the single-stranded DNA (ssDNA) of parvovirus LuIII was studied in synchronized HeLa cells. After infection of the cells in early S phase, synthesis of a replicative form (RF) DNA became detectable as early as 9 h postinfection, i.e., after display of the cellular helper functions) indispensable for the replication of LuIII virus. According to digestion with nuclease S1, hybridization studies, and electron microscopy, RF DNA is a linear, doublestranded molecule comparable in length to mature ssDNA. It sedimented around 15S in neutral solution and banded at 1.714 g/ml in CsCl. Moreover, replication of LuIIl DNA obviously includes a further replicative intermediate DNA which sedimented in front of RF DNA and bore single-stranded side-chains. Newly synthesized DNA disappeared from pools containing both RF DNA and replicative intermediate DNA within 5 min and reappeared in progeny virions only after 15 min. Intranuclear accumulation of significant amounts of progeny ssDNA could not be detected. It was postulated, therefore, that newly synthesized ssDNA is immediately enclosed in a stable maturation complex and resists extraction by the method of Hirt (1967). Parvovirus LuWE is a member of a subgroup of parvoviruses capable of multiplying in susceptible cells without the presence of a potent helper virus. Replication of these viruses, however, is strictly dependent on cellular physiology. For Kilham rat virus (RV), H-i, and minute virus of mice (MVM), as well as for LuIII virus, evidence has been presented suggesting that the cellular functions) favoring viral replication occur in late S phase of the cell cycle (10, 14, 16, 19, 20). As far as LuIII virus is concerned, the latter statement is based on experimental observations in HeLa cells synchronized for DNA synthesis and infected at various times during S phase (16, 17). Independent of whether infection occurred simultaneously with the onset of cellular DNA synthesis or up to 4 h later at mid-S-phase, both the intranuclear accumulation of viral antigen and the production of hemagglutinating and infectious progeny virus became regularly detectable as early as 8 to 10 h after release of cells from the synchronization block. Preliminary experiments (G. Siegl, abstract in Pathol. Microbiol. 40: 202-203, 1974) indicated that replication of the single-stranded DNA (ssDNA) of parvovirus LulH involves the formation of a linear double-stranded DNA

molecule (dsDNA). A similar dsDNA replicative form (RF) has been identified in extracts of cells infected with either H-1 or MVM (2, 11, 19). According to Rhode (11, 12), synthesis of detectable amounts of H-1 dsDNA coincides with the display of the cellular helper function late in S phase. Experimental data reported here suggest that the same is true for the synthesis of the RF form of LuIII virus dsDNA in synchronized HeLa cells. Moreover, gradient sedimentation and electron microscopy of DNA extracted from infected cells at the height of synthesis of progeny viral DNA revealed the presence of at least one additional nucleic acid species. It is a linear double-stranded molecule bearing single-stranded branches of various length and, therefore, is assumed to represent a replicative intermediate (RI) form in the synthesis of progeny viral DNA. (A preliminary report of part of this work was presented at the workshop on Parvoviruses at the National Institute of Health, Bethesda, Md., in February 1974). MATERIALS AND METHODS Virus and cells. The physicochemical and biological properties of parvovirus LuIII as well as the growth characteristics of the HeLa cells used through-

841

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SIEGL AND GAUTSCHI

out these studies have been previously described (3, 4, 15). The same also applies for the determination of hemagglutination and infectivity titers, immunofluorescence staining, and the techniques adapted for synchronization of cell growth either in monolayer or in spinner cultures (4, 16, 17). Isolation and purification of labeled virus. LuIII virus labeled with [methyl-1H ]thymidine (specific activity, 30 Ci/mmol) or with [2-"4CJthymidine (specific activity, 21 mCi/mmol) was prepared using essentially the same procedures as described previously (15). Briefly, HeLa cells were infected at the time of seeding at a multiplicity of infection of 10. After 24 h, they were incubated in a medium containing either 5 ,Ci of [3H JTdR or 0.5 MCi of [14C JTdR per ml. Virus was harvested at the time of maximum cytopathic effect by subjecting the cultures to three cycles of freezing and thawing. Crude viral harvests were treated with receptor-destroying enzyme, DNase, RNase, trypsin, and deoxycholate. Finally, the virus was concentrated and purified by differential centrifugation and by two successive runs in CsCl density gradients. Isolation of nuclei. Cells grown in monolayer cultures were trypsinized and, like those collected from spinner cultures, were suspended in spinner medium at a concentration of 5 x 105 cells/ml. Cells were sedimented by centrifugation for 5 min at 1,200 x g, washed with 5 ml of ice-cold reticulocyte standard buffer (8), resuspended in the same buffer and, after incubation at 0 C for 15 min, broken by 25 strokes with a tight-fitting pestil in a Dounce homogenizer. Nuclei were sedimented at 2,000 x g for 5 min and washed a second time with the same amount of reticulocyte standard buffer. Extraction of viral DNA from infected cells. Low-molecular-weight DNA was selectively extracted from infected cells following the method described by Hirt (5). Cell monolayers in 75-cm2 plastic flasks were washed twice with cold Hanks solution. Four milliliters of 0.6% sodium dodecyl sulfate in 0.01 M Tris-0.01 M EDTA (pH 7.5) was added per flask. Cells were lysed at room temperature within 30 min. The viscous lysate was carefully transferred into centrifuge tubes and brought to a concentration of 1 M NaCl, and the contents of the tubes were mixed by slowly inverting the tubes 10 times. After incubation at 4 C for at least 16 h, cellular protein, RNA, and highmolecular-weight DNA were precipitated by centrifugation for 30 min at 15,000 x g at 4 C. In some experiments viral DNA was extracted from cells or isolated nuclei suspended in 0.01 M Tris (pH 7.5) by careful mixing the suspensions with a 0.1 volume of 6% sodium dodecyl sulfate in 0.1 M EDTA (pH 7.5). The Hirt supernatant was usually dialyzed extensively against 0.01 M Tris-0.001 M EDTA (pH 7.8), whereas the Hirt pellet had to be dissolved in 0.1 N NaOH during 20 h at room temperature previous to the determination of acid-insoluble radioactivity. Pulse-chase experiments. To study the time dependence of the synthesis of RF DNA in correlation with the incorporation of progeny virus particles, synchronized infected HeLa cell cultures and mockinfected controls were labeled for 30 min in the

J. VIROL. presence of 40 MCi of [3HJTdR (specific activity, 47

Ci/mmol) per ml. The cultures were then washed twice with minimal essential medium at 4 C and incubated at the same temperature in the presence of 6 volumes of minimal essential medium containing 5 x 10-6 M cold TdR. After 60 min the cultures were rapidly warmed to 37 C in a water bath and, subsequently, were held at 37 C for 10, 30, and 60 min in an incubator. After thorough washing, the cells were trypsinized and collected by centrifugation at 4 C. Finally, the cells were suspended in 0.01 M Tris (pH 7.5). Extraction of one-half of each cell sample by means of the described modification of the Hirt technique yielded low-molecular-weight DNA, whereas the second half was used for the isolation and quantitation of labeled progeny virus. For this purpose the cells were broken by three cycles of freezing and thawing. The homogenate was digested for 15 h at 37 C with DNase, RNase, and receptor-destroying enzyme at concentrations of 300 Mg/ml, 20 IAg/ml, and 10%, respectively. Cell debris were spun down at 5,000 x g for 30 min, and virus particles were separated from digestion products by centrifugation through a 10 to 25% linear sucrose gradient in a Beckman SW27.1 rotor at 25,000 rpm for 5 h at 4 C. Sedimentation analysis and equilibrium centrifugation of DNA. Labeled DNA from the Hirt supernatant was analyzed by velocity sedimentation in CsCl (9, 15). Samples of 0.2 ml were layered on 3 ml of CsCl in 5-ml cellulose nitrate tubes. The CsCl solution had a density of 1.500 g/ml and was made up in 0.01 M Tris-1 mM EDTA (pH 8.0). DNA was sedimented at 100,000 x g for 3.5 h at 25 C in a Beckman SW50L or SW50.1 rotor. Fractions of 2 drops were collected from the punctured bottom of the tubes either directly into scintillation counting vials or, for determination of acid-insoluble radioactivity, into test tubes containing 1 ml of 0.01 M Tris-1 mM EDTA (pH 8.0) plus 100 ,ug of salmon sperm DNA as a carrier. For determination of the buoyant density, DNA samples dialyzed against Tris-EDTA were adjusted to a density of 1.7 g/ml by addition of solid CsCl. Samples of 4 ml were centrifuged in an SW50.1 rotor at 30,000 or 35,000 rpm for 40 h at 18 C. On some occasions density analysis was carried out in samples of 7 ml in a type 50 fixed-angle rotor at 42,000 rpm for 40 h. CsCl-ethidium bromide gradients were centrifuged under the same conditions. The gradients had a mean density of 1.56 g/ml and contained ethidium bromide at a concentration of 200

Mg/iml. DNA-DNA hybridization. Viral DNA was extracted from purified LuIII virus with 0.75% sodium dodecyl sulfate in Tris-EDTA buffer (pH 7.5) at 72 C for 90 min as described by Siegl (15) and subjected to an additional treatment with phenol. The concentration of DNA was estimated by absorption at 260 nm using the Eno' of 28 mg/ml found to be characteristic for the ssDNA of phage 4X174 (18). 'H-labeled Hirt supernatant DNA from infected HeLa cells was fractionated by sedimentation in CsCl, and the individual fractions (see Fig. 4A) as well as the DNA from

VOL. 17, 1976

REPLICATION OF PARVOVIRUS LuIII DNA

purified virions was dialyzed against 0.01 M Na2HPO4 (pH 7.0). Samples of 320 gl containing 100 Ml each of the individual fractions of the Hirt supernatant DNA and 0 to 500 ng of cold LuIII viral DNA were heated to 100 C for 2 min, quenched in an ice bath, and adjusted to 0.8 M NaCl by addition of 80 ul of 4 M NaCl. Reannealing in the presence of the unlabeled viral DNA was allowed to take place at 65 C for 40 h. The samples were then diluted to a volume of 1 ml, and ssDNA was digested with 10 units of S1 nuclease from Aspergillus-oryzae during 4 h at 45 C. In addition to the 0.01 M Na,HPO4, the S1 reaction mixture contained 0.32 M NaCl, 1 mM ZnSO4, 0.03 M sodium acetate, and 5% glycerol and had a pH of 4.7 (21). Trichloroacetic acid-insoluble DNA was collected on Whatman GFC glass-fiber filters in the presence of 100 Mg of salmon sperm DNA per ml as a carrier. Electron microscopy of DNA. DNA was prepared for electron microscopy using the microdroplet modification of the Kleinschmidt technique (6) as described previously (15). The cytochrome C/DNA film developing by diffusion at the surface of the droplets was transferred to collodion-carbon-coated grids, and the preparations were rotary shadowed with a PT-Au-Ag alloy under an angle of 8°. Micrographs were taken at a direct magnification of between x9,000 and 15,000 in a Philips EM200 electron microscope calibrated with a grating replica. Materials. Radiochemicals were obtained from the Radiochemical Centre. The single-strand-specific endonuclease S1 purified from crude a-amylase powder from Aspergillus oryzae (Sigma Chemicals) according to Vogt (21) was a generous gift of 0. HagenbUchle, University of Berne, Berne, Switzerland. All other enzymes as well as cytochrome c (type VI, from horse heart) were purchased from Calbiochem.

RESULTS Synthesis of DNA in infected cells. Experiments described elsewhere (G. Siegel, abstract in Pathol. Microbiol. 40:202-203, 1974) had provided strong evidence for the occurrence of a RF dsDNA in infected HeLa cells at the time of maximum synthesis of LuII1 virus (13 to 15 h postinfection [p.i. ]). It was attempted to correlate the synthesis of this RF DNA with the overall synthesis of DNA in infected cells. For this purpose HeLa cell monolayer cultures were synchronized for DNA synthesis and infected at a multiplicity of infection of 10 immediately after release of cells from the synchronization block. Both virus-infected and mock-infected cultures were pulsed with 10 uCi of [3HJTdR in regular 2-h intervals up to 15 h postadsorption. At the end of each pulse period monolayers were either trypsinized and assayed for total incorporation of label or subjected to the Hirt extraction for isolation and characterization of low-molecular-weight DNA.

843

Figure 1 illustrates the time-dependent variation in acid-insoluble radioactivity within the Hirt supernate. Peak amounts of low-molecular-weight DNA were recovered from both virusinfected and control cells at 5 h p.i., i.e., at mid-S-phase of the synchronized cultures. Moreover, the overall shape of the graph describing the appearance of label in the Hirt supernate of mock-infected cells proved indistinguishable from the curve traced on the basis of incorporation of [3H]TdR into total cellular DNA (see also reference 16). The incorporation of [3H ]TdR into total DNA as well as into Hirt supernatant DNA of infected cultures was indistinguishable from the respective values recorded for mock-infected cells up to mid-S-phase. Between 5 and 9 h, however, the rate of synthesis of both total and Hirt supernatant DNA (Fig. 1) appeared slightly reduced. The observed differences could be reproduced in three separate experiments. From 9 h onward, the rate of synthesis of total DNA approached again the value characteristic for

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hours p.i. FIG. 1. Incorporation of ['H]TdR into low-molecular-weight DNA in both LuIII virus-infected (0) and mock-infected (0) synchronized HeLa cells. HeLa cell monolayer cultures synchronized for DNA synthesis were infected with LuIII virus at a multiplicity of infection of 10 at the time of release from the synchronization block and pulse labeled with 10 MCi of [3H]TdR at regular intervals. DNA of low molecular weight was selectively extracted at the end of each pulse according to Hirt (5) and dialyzed against 0.01 M Tris-O.001 M EDTA (pH 7.8), and 1.0-ml aliquots of the extracts were assayed for acid-insoluble radioactivity.

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mock-infected cultures, but incorporation of label into low-molecular-weight DNA increased almost linearly up to 15 h p.i. Centrifugation of Hirt supernatant DNA through neutral CsCl revealed that the samples collected from virus-infected and control cells up to 8 h p.i. contained DNA molecules of identical sedimentation characteristics (mostly > 26S) (Fig. 2). In parallel with the linear increase of low-molecular-weight DNA in virusinfected cells subsequent to 9 h p.i. (Fig. 1), the sedimentation spectra of the respective Hirt extracts then developed a clear-cut peak at 14 to 16S. It is evident from Fig. 2 that at 13 to 14 h p.i. additional new species of DNA molecules accumulated around 24S and below lOS, whereas the number of molecules sedimenting at > 26S decreased drastically. 24

8

Characterization of low-molecular-weight DNA extracted from LuIII-infected cells at the height of virus synthesis. (i) Presence of prelabeled cellular DNA. In a first attempt to characterize the DNA selectively extracted from LuII1 virus-infected HeLa cells, we tried to show that these molecules were not produced artificially by disruption of cellular DNA during extraction. For this purpose HeLa cell monolayer cultures were grown in the presence of [14C]TdR (1 ,uCi/ml) for 24 h. Subsequently, the cultures were washed, synchronized for DNA synthesis with excess thymidine (16), and infected as described. At 13 h p.i. they were pulsed with [3H]TdR (10 ACi/ml) for 1 h, and low-molecular-weight DNA was isolated. Sedimentation analysis in neutral CsCl (Fig. 3A) yielded convincing evidence that the DNA sedi-

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LuIlf virus-infected (0) synchronized HeLa cells at various times p.i. Aliquots (0.2 ml) of Hirt supernatant

DNA labeled and isolated as described in the legend to Fig. 1 were sedimented through CsCI (p = 1.500 g/ml, pH 8) at 100,000 x g for 3.5 h and 25 C in an SW50 rotor. Fractions of 2 drops were collected from the bottom of the tubes into scintillation vials and assayed for radioactivity.

REPLICATION OF PARVOVIRUS LuIll DNA

VOL. 17, 1976

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FIG. 3. Sedimentation analysis of double-labeled DNA of low molecular weight extracted from (A) LuILI virus-infected and (B) mock-infected HeLa cells. HeLa cell monolayer cultures grown in the presence of 1 ACi of ["4C]TdR per ml for 24 h were synchronized for DNA synthesis with excess thymidine and infected as described (16). At 13 to 14 h p.i. the cultures were pulsed with 10 A.Ci of ['H]TdR (specific activity, 30 Ci/mmol) per ml, Hirt supernatant DNA was isolated, and 0.2-ml aliquots were sedimented under standard conditions. Fractions of 2 drops were collected from the bottom of the tubes into scintillation vials and assayed for 14C (0) as well as for 3H (0) specific radioactivity in a Packard Tricarb spectrometer fitted with an absolute activity analyzer.

menting between 14 and 16S contained almost exclusively the 'H label and, hence, consisted of de novo synthesized molecules. On the other hand, distribution of 'H and 14C label was parallel in fractions containing DNA of more than 26S. It can be assumed, therefore, that such DNA molecules are of cellular origin. The identity of the sedimentation spectra of "4Clabeled molecules recovered from LuIII-infected as well as from mock-infected monolayers lends further support to these conclusions (Fig. 3B). (ii) Digestion with nuclease SI and hybridization studies. As evident from Fig. 2, the relative amount of DNA sedimenting at more than 26S decreased steadily in parallel with progressing virus synthesis. An additional reduction in the amount of this DNA species was achieved when the Hirt supernatant was separated from cellular DNA, RNA, and proteins by centrifugation at 0 C instead of at 4 C. Further

845

experiments made use of these observations, and DNA samples almost completely free of molecules > 26S were regularly obtained. Figure 4A, for example, represents the sedimentation profile of low-molecular-weight DNA extracted from LuIll-infected cells which, 13 h p.i., had been labeled for 120 min in the presence of 40 lCi of [3H]TdR per ml. Individual fractions of 12 identical sedimentation runs of this DNA were pooled as indicated and dialyzed against 0.01 M NaHPO, (pH 7.0). Incubation of equal volumes of pools I to X with 10 units of the single-strand-specific nuclease S1 under standard conditions for 4 h revealed a disproportionate distribution of digestible DNA sequences within individual pools (Fig. 4B, Table 1). Molecules sedimenting between 13 and 16S and constituting the majority of Hirt supernatant DNA contained only 1.9 to 6.9% of the DNA accessible to the action of S1. DNA running in front of the main peak (18-23S, pools IV and V) had about 34% of the digestible sequences, whereas fractions with 24-25S yielded 45% of the acid-soluble radioactivity. The dsDNA of pools I to X was finally assayed for the presence of virus-specific base sequences by displacement hybridization as described above. Experiments were run with 0, 50, 100, and 500 ng of purified, unlabeled LuIII virus DNA. In the case of pool VI, however, additional concentrations of 5 and 10 ng were included. Figure 5 depicts the displacement of radioactivity from dsDNA of the latter pool by various amounts of unlabeled viral DNA. It is evident that 100 to 500 ng proved sufficient to achieve maximum displacement. Comparable results have been obtained with all the other pools. Therefore, the data recorded in the presence of 500 ng of LuIll DNA (Table 1) would suggest that, with the exception of pools I and II, all DNA molecules resistant to digestion by S1 nuclease contained about 50% of the base sequences also present in mature viral DNA. The data given in Table 1, however, are mean values of three separate experiments and, as indicated in Fig. 5, individual results may deviate by about ± 10%. Y The reliability of the results of both S1 digestion and displacement hybridization experiments may be judged on the basis of control experiments conducted in parallel. First, it can be seen from Table 2 that the S1 preparation used was highly specific for ssDNA. Second, 10 units of S1 proved sufficient to reach a plateau of digestion within four h. Third, mature viral ssDNA behaved differently, depending on the treatment previous to digestion. Viral DNA added to the S1 reaction mixture without any

846

SIEGL AND GAUTSC.HI

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Fractions FIG. 4. Fractionation by sedimentation in neutral CsCl (A) and digestion with the single-strand-specific nuclease Si (B) of Hirt supernatant DNA extracted from LuIlI virus-infected HeLa cells. HeLa cell monolayer cultures synchronized and infected as described were pulsed with 40 ACi of [3H]TdR (specific activity, 47 Ci/mmol) per ml at 13 h p.i. for 120 min. Hirt supernatant DNA was extracted and sedimented under standard conditions. Individual fractions of 12 identical sedimentation runs were collected in pools I to X (A). After dialysis against 0.01 M NaHPO4 (pH 7.0), each of the pools was assayed for acid-insoluble radioactivity before (dsDNA plus ssDNA) and after (dsDNA, dotted columns) digestion of aliquots with 10 units of the single-strand-specific endonuclease Si.

pretreatment behaved as if about 40% of its structure were of double-stranded configuration. After heat denaturation and incubation for 40 h at 65 C under conditions of displacement

hybridization, the Si-resistant fraction of LuIII DNA was reduced to about 20%. (More detailed

data concerning this phenomenon and its significance in respect to the configuration of mature viral DNA will be reported in a separate paper.) Fourth, preincubation of labeled viral ssDNA with 500 ng of unlabeled viral DNA under reannealing conditions of displacement hybridi-

VOL. 17, 1976

REPLICATION OF PARVOVIRUS LuIII DNA

zation indicated formation of double-stranded structures by interaction of individual mature viral DNA molecules is very unlikely to occur. Moreover, the reaction kinetics of S1 nuclease was not significantly reduced in the presence of an additional 500 ng of unlabeled viral ssDNA per test sample. Finally, only about 5% more label remained acid insoluble when viral ssDNA was incubated in the presence of 500 ng of heatdenatured HeLa DNA under conditions of displacement hybridization. The latter observation obviously suggests that interaction between viral ssDNA and cellular DNA sequences which TABLE 1. Digestion by S1 nuclease and displacement hybridization or Hirt supernatant DNA isolated from LuIOH virus-infected cultures and fractionated via sedimentation in neutral CsCIa % DNA

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% Sl-resistant DNA displaced by 500 ng of LuIII DNA

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847

might have been present in pools I to X influenced the outcome of the displacement experiments only to a limited degree. (iii) Electron microscopy. To isolate DNA of low molecular weight for electron microscopy, synchronized LuIII virus-infected as well as mock-infected HeLa cell monolayers were labeled for 2 h in the presence of 10 gCi of [3H]TdR per ml starting 13 h p.i. DNA was subsequently extracted by the Hirt method, and samples were concentrated about 20-fold by vacuum dialysis. Sedimentation of concentrated DNA from LuIII-infected cells in neutral CsCl revealed a small yet distinct peak at about 100 loo 80 0

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LuN-DNA FIG. 5. Displacement of labeled molecules from the dsDNA of pool VI (Fig. 4) by denaturation and reannealing in the presence of unlabeled viral ssDNA. Aliquots of pool VI DNA was heated to 100 C for 2 min in the presence of indicated amounts of cold LuOIH DNA, quenched in an ice bath, and adjusted to 0.8 M NaCI. Reannealing took place at 65 C for 40 h. The reported values represent the percentage of pool VI dsDNA-associated label recovered as acid-insoluble radioactivity after digestion of the reannealing product with 10 units of nuclease SI. ng

TABLE 2. Percentage of total radioactivity of various DNAs rendered acid soluble by 10 units of Si nuclease during incubation at 45 C for the time indicated HeLa DNA

Digestion time (min.)

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32.1 41.3 62.4 77.2 76.7 83.1

35.9 37.0 49.1 53.8 54.1 57.4

LuIllI virus DNA Preincubated "Hybridized" "Hybridized" at 65"C for to 500 ng of to 500 ng of 40 hd LuIII DNA' HeLa-DNA' 39.5 53.7 71.7 76.3 81.2 79.0

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a ['H]HeLa DNA isolated by chromatography on benzoylated-naphthoylated-DEAE-cellulose as described by Siegl (15). 'DNA described in footnote a heat denatured at 100 C for 2 min. ['HJTdR-labeled native viral DNA. dDNA described in footnote c heat denatured for 2 min and incubated under conditions of displacement hybridization. 'DNA described in footnote d incubated in the presence of 500 ng of undiluted native viral DNA. 'DNA described in footnote d incubated in the presence of 500 ng of heat-denatured HeLa DNA (HeLa dsDNA molecules sedimented at about 16S in neutral solution).

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24S, and the majority of the DNA sedimented around 15S (Fig. 6). Fractions belonging to the individual peaks as well as those containing 18 to 22S DNA were collected in pools I to III (see Fig. 6). Corresponding pools were obtained from gradients loaded with Hirt supernatant DNA of mock-infected cells. When DNA of the various pools was prepared for electron microscopy by means of the microdroplet diffusion technique of Lang (6), it became evident that the main peak sedimenting around 14.7S contained linear molecules of rather uniform size. Measurement of 112 molecules yielded a mean value of 1.55 0.15 Mm. About 8 to 10% of the molecules bore short branches at one or at both ends (Fig. 7A). DNA sedimenting with 18 to 22S (pool II) consisted of similarly sized molecules; however, many of them appeared to have side chains (Fig. 7B). The number of side chains per molecule as well as their relative length could not be determined to a reliable degree since only 14 out of more than 200 molecules were spread well enough to allow measurement. Experiments are now under way to concentrate sufficient amount of 18 to 22S molecules for spreading under conditions of partial denaturation. On the basis of studies concerned with the characteristics of mature viral DNA (15), it was expected that the minor peak sedimenting around 24S would be made up of linear viral ssDNA about 1.6,um in length. Electron microscopy revealed single-stranded molecules of that size; however, they only constituted about 10 to 15% of the total DNA of pool I. The large majority consisted of double strands which-according to tracing of 67 individual molecules0.4 ,m in length. About 35% of these were 4.7 DNA strands bore small side chains (Fig. 7C) at a relative regular distance of about 1.5 Am, suggesting that the strands represent some kind of an association product of individual 14.7S dsDNA. In addition, the 24S pool contained a few relaxed circular molecules comparable in size to mitochondrial DNA. These circles disappeared selectively when DNA was extracted from purified nuclei of infected cells only (see below). A main characteristic of pools I to III isolated from the Hirt supernate of mock-infected cells

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FIG. 6. Fractionation of Hirt supernatant DNA extracted from LuIlf virus-infected (0) and mockinfected (0) HeLa cells for electron microscopy. Hirt supernatant DNA labeled in the presence of 10 ,Ci of ['H]TdR (specific activity, 30 Ci/mmol) for 2 h was isolated at 15 h p.i. from both virus-infected and mock-infected synchronized HeLa cells, concentrated by vacuum dialysis, and sedimented in neutral CsCl under standard conditions. Fractions of individual sedimentation runs were collected in pools I to III as indicated.

that nucleic acid molecules appeared about 100-fold less frequently in the respective electron microscope preparations than in the case of pools concentrated from infected cells. Moreover, the few DNA strands found in pool II (18-22S) proved free of any branched structures, and no linear molecules comparable in length to mature viral DNA were present around 24S. Some circular DNA molecules about 5.2 ,m in length were found, yet, as in the case of infected cells, this DNA was absent after extraction of DNA from isolated nuclei. (iv) Density gradient centrifugation. Electron microscopy of Hirt supernatant DNA prepared from whole cells revealed the presence

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FiG. 7. Electron micrographs of DNA isolated by sedimentation in neutral CsCl from the Hirt supernate of LulII virus-infected HeLa cells (compare Fig. 6). (A) RF dsDNA molecules sedimenting at 14.7S and bearing small terminal branches (arrows). (B) One of the few RI DNA molecules showing well-extended side chains, the presence of which was found characteristic for DNA sedimenting at 18 to 22S. (C) A DNA molecule sedimenting at 24S and about three times as long as RF DNA. Arrows indicate the presence of small side chains located at a distance comparable to the length of RF DNA. (Bar represents 0.5 um).

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of circular DNA. The molecules were comparable in size to mitochondrial DNA of HeLa cells and, moreover, could not be detected in extracts of isolated nuclei. To exclude the possibility that these and further, so far undetected, covalently closed circular molecules (cccDNA) play a role in the synthesis of parvovirus ssDNA, Hirt supernates prepared from whole cells as well as from isolated nuclei of both LuIII-infected and mock-infected cultures were centrifuged to equilibrium in CsCl-ethidium bromide gradients. Independent of whether virus-infected or control cultures were used as starting material, DNA extracted from whole cells gave rise to the formation of two distinct peaks with densities of 1.592 and 1.635 g/ml, respectively. According to electron microscopy, the high-density peak contained only one type of covalently closed circular molecules comparable in length to those detected earlier. The total number of counts present in the high-density peak of DNA from virus-infected cultures did not differ significantly from value calculated for control samples. However, whereas the cccDNA-specific label constituted only 4 to 7% of the total counts in extracts of infected cells, it amounted to 20% of the DNA isolated from mock-infected cultures. Centrifugation of DNA prepared from isolated nuclei, on the other hand, always gave rise to only one band with a mean density of 1.59 g/ml. For determination of the buoyant density of the double-stranded RF DNA, aliquots of fraction VI (see Fig. 4A) as well as of pool III (see Fig. 6) which had been characterized by displacement hybridization and electron microscopy, respectively, were centrifuged to equilibrium in CsCl. Mature viral ssDNA (p = 1.725 g/ml) labeled with [4C ]TdR was included as a marker. In both cases the majority of the DNA banded around 1.714 g/ml; however, the peaks appeared something skewed towards lower densities. Evidence for rapid encapsidation of de novo synthesized viral ssDNA. If the dsDNA molecule sedimenting at about 15S is a true and necessary intermediate in the synthesis of progeny viral ssDNA, pulse-chase experiments should reveal the displacement of progeny ssDNA from RF DNA and, perhaps, its accumulation within the nucleus. When LuIIIinfected cells were pulsed with [3H]TdR for 5, 10, 30, 60, 120, and 180 min at the height of viral DNA synthesis, the relative amount of labeled molecules sedimenting around 24S and at about 15S remained constant, although there was a

J. VIROL.

significant increase in extractable labeled molecules in parallel with increasing pulse time. Chase experiments in the course of which infected cells pulsed for either 5, 10, or 30 min in the presence of 40 ,Ci of [3H]TdR per ml were thoroughly washed and incubated in the presence of 5 x 10-5M unlabeled thymidine at 37 C for periods ranging between 5 and 120 min gave similarly inconclusive results. The overall shape of sedimentation spectra of Hirt supernatant DNA extracted at the end of the pulse period and after different chase periods remained indistinguishable. With progressing chase time the total amount of label within DNA sedimenting around 15S still increased steadily, yet, compared to the previous pulse experiments, at a significantly reduced rate. The most logical explanation for the continuing incorporation of [3H ]TdR into RF DNA even in the presence of 103 times the amount of unlabeled TdR in the culture medium, as well as for the lacking accumulation of progeny viral DNA within the Hirt supernate, seemed to be that: (i) the equilibration between extracellular unlabeled TdR and the intracytoplasmic as well as intranuclear pools of tritiated TdR occurred at a rather slow rate, and (ii) viral ssDNA was rapidly encapsidated after synthesis. Therefore, in further studies 60 min at 4 C was allowed for the equilibration between extracellular and intracellular pools of TdR before DNA was chased at 37 C. Moreover, both the amount of label incorporated into RF DNA and into the DNA of progeny mature virus particles was measured. Differentiation between these types of DNA molecules was achieved without difficulties. As control experiments showed, the Hirt technique failed to release ssDNA from [3H ]TdR-labeled virions added to HeLa cells prior to the Hirt treatment. Since mature LuIII virus particles also resist the action of DNase (3), encapsidated DNA could be readily quantitated in the form of virus particles sedimenting at 110S after cell homogenates had been digested with the nuclease (see above). Figure 8 summarizes the results of such a pulse-chase experiment. Synchronized HeLa cells were infected concomitantly with release from synchronization block and, 13 h later, were pulsed for 30 min with [3H]TdR. It is evident that, within 10 min after warming of the equilibrated cultures to 37 C, the amount of labeled DNA sedimenting between 14 and 22S was drastically reduced to about one-third the value found in low-molecular-weight DNA extracted previous to warming. With progressing chase time there was apparently again a slight in-

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60 0 10 30 chase time(min) FIG. 8. Encapsidation of newly synthesized viral ssDNA into progeny LuIII virions. Synchronized HeLa cell monolayer cultures were infected with LulII virus concomitantly with release from synchronization block as described (16). At 13 h p.i. the cultures were pulsed with 40 ,Ci of [3H]TdR (specific activity, 47 Ci/mmol) per ml, and labeled DNA was subsequently chased in the presence of 5 x 10- 5M cold TdR. At the end of the indicated chase times, cells were harvested by trypsinization. DNA of low molecular weight was selectively extracted from one-half of the cell suspension, whereas the second half was processed for the isolation of progeny virions as described. In the case of Hirt supernatant DNA, the values included in Fig. 8 represent the integral amount of counts per 1 ml of the original cell suspension which sedimented at 13 to 22S in neutral CsCl. Therefore, they are the total of labeled RF plus RI DNA molecules. Values given for encapsidated DNA were calculated and normalized on the basis of DNase-resistant label sedimenting in a 10 to 25% linear sucrose gradient at 110S, i.e., at the position of mature virions. crease in the incorporation of [3H ]TdR into RF DNA. Nevertheless, the amount of label remained below one-half the value recorded at zero time. On the other hand, incorporation of labeled DNA into virus particles sedimenting at 110S increased linearly in parallel to the disappearance of molecules from the RF plus RI DNA pool. However, whereas displacement of DNA from this pool was completed after 10 min, maximum amounts of progeny ssDNA were found in mature virus particles only after about 30 min. DISCUSSION Mature, infective virions of parvovirus LuIII contain a linear molecule of ssDNA with a molecular weight of 1.59 x 106 (15). According to pulse-chase experiments conducted in a system of synchronized HeLa cells, synthesis of

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progeny molecules of this DNA occurs simultaneously with the production of progeny infectious virions (16). The latter studies also provided evidence for both the replication of viral DNA and the formation of progeny virions depending strictly on cellular events in late S or early G2 phase of the HeLa cell life cycle (16, 17). Since preliminary studies in addition suggested that replication of viral ssDNA proceeds via a linear RF dsDNA (G. Siegl, abstract in Pathol. Microbiol. 40:202-203, 1974), there arose the question of whether or not the obviously necessary cellular functions control the formation of a parental double-stranded RF molecule, its reduplication, or the synthesis of progeny viral ssDNA. According to the pulse-labeling experiments described here, LuIII virus-specific- DNA with characteristics of an RF dsDNA molecule is synthesized effectively only after the infected cells reached the end of the S phase or, in other words, after the cellular helper functions) have been displayed. Comparable results were obtained in studies concerned with the replication of H-1 virus DNA (11, 12). Pulse-labeling experiments, however, allow no discrimination between conversion of the infecting viral ssDNA into an RF dsDNA and the replication of the latter molecule. Moreover, synthesis of a DNA strand complementary to the parental ssDNA may occur at such a low level that it escapes detection by the applied labeling and extraction procedures. Neither the studies with H-1 virus nor those with LuIII virus therefore furnished conclusive evidence as to whether the formation of a parental RF DNA depends on the expression of the cellular helper functions. Using [3H ]TdR-labeled RV, Salzman and White (13) have shown that the parental ssDNA of this parvovirus is converted into a dsDNA within 60 min after infection. Their studies, however, were based on randomly growing cells and, therefore, allowed no interpretation with respect to cellular events. Rhode (11, 12), on the other hand, was unsuccessful in following the fate of the infecting H-1 virus ssDNA by means of the same method. He reported a very low recovery of prelabeled viral DNA (less than 1% of the absorbed virions) from the infected cells and attributed this failure to a high particleinfectivity ratio in the inoculum. Our own results with this experimental approach (data not shown in detail) are comparable to the results of Rhode; we could show that a rather small but constant amount of the DNA of the infecting LuIII virus reached the nuclei of both randomly growing and synchronized HeLa cells

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within 90 to 120 min after adsorption. At the same time the label associated with the nuclei was quantitatively recovered in the Hirt supernate and banded in CsCl gradients at the density position of mature viral DNA (1.725 g/ml). Three to four hours later, i.e., around mid-S-phase of the infected cells, most of the extracted label was found in two peaks with density values comparable to the 1.714 g/ml determined for an isolated, well-characterized RF dsDNA and the 1.700 g/ml recorded for HeLa DNA, respectively. These observations may lend support to the assumption that, in the replication of the LuIII virus DNA, the formation of a parental RF molecule is achieved independently of those cellular helper functions which obviously are indispensable for the production of progeny virus particles. However, since the recovery in our experiments was only slightly higher than in the study reported by Rhode, it proved impossible to characterize the extracted labeled material by methods different from density gradient centrifugation. In consequence, the results must be referred to with reservation. Data concerning the low-molecular-weight DNA synthesized in LuIlI virus-infected cultures subsequent to the display of cellular helper functions are more conclusive. The great majority of this DNA consisted of linear, double-stranded molecules sedimenting around 15S in neutral solution. They were made up of virus-specific base sequences and, calculated on the basis of both sedimentation and length measurements on electron micrographs, had a molecular weight of 2.8 x 106 to 3.1 x 10', corresponding to about twice the figure determined for viral ssDNA. In accordance with the findings for the replication of the parvoviruses RV, H-1, and MVM (2, 11, 13, 19), these double-stranded molecules therefore were assumed to represent the LuIII virus-specific RF DNA. The same dsDNA obviously also constituted the backbone of those branched molecules sedimenting in front of the RF-DNA peak and bearing an elevated amount of base sequences digestable by the single-strand-specific endonuclease S1. Studying the replication of H-1 DNA, Rhode (11) observed that DNA sedimenting in this region was preferentially labeled during short pulses. Moreover, most of the briefly labeled MVM-specific DNA of low molecular weight eluted from benzoylatedDEAE-cellulose under conditions allowing the release of dsDNA with extended single-stranded regions (19). These observations strongly support the existence of a further necessary inter-

J. VIROL.

mediate in the synthesis of the ssDNA of parvoviruses. In analogy to comparable nucleic acid structures found during replication of the ssRNA of picorna viruses (1), we suggest the use of the term RI DNA for these molecules. Subjecting pulse-labeled, MVM-infected cells to digestion with Pronase, Tattersall et al. (19) isolated a Hirt supernatant DNA containing equal amounts of RF dsDNA and singlestranded molecules. Moreover, Dobson and Helleiner (2) were able to chase DNA from the RF peak into single-stranded molecules sedimenting at the position of mature MVM DNA. In the case of LuIII virus, on the contrary, an accumulation of mature viral ssDNA was never observed. Likewise, attempts to chase RF DNAspecific label into free ssDNA and, thus, to establish a direct precursor-product relationship failed. It proved possible, however, to correlate the disappearance of labeled molecules from the RF DNA peak with the incorporation of DNA into mature virus particles sedi' menting at 1lbS (see Fig. 8). A maximum amount of labeled DNA was displaced from the pool containing both RF and RI DNA within 10 min after the addition of an excess amount of unlabeled thymidine. At the same time the incorporation of labeled DNA into mature virus particles increased at a somewhat lower yet nevertheless linear rate. Thirty minutes after the beginning of the chase the incorporation of labeled DNA into virus particles had reached a plateau corresponding to the accumulation of about 80% of label lost from free RF and RI DNA molecules. The data depicted in Fig. 8 allow a rough estimation of the time necessary for both the synthesis of viral DNA and its incorporation into progeny LuIII virions. The half-life time of labeled DNA within the pool of RF and RI DNA can be calculated to be about 5 min. This is in good agreement with the period of 2 to 4 min required for replication of DNA of a molecular weight around 3 x 106 (7, 12). On the other hand, 50% of the displaced label was incorporated into progeny virions after 15 min. Encapsidation of viral ssDNA and maturation of progeny virions therefore proceeds at a much slower rate than does the synthesis of progeny viral DNA. Theoretically, this should lead to the accumulation of mature unencapsidated viral DNA. Since such an accumulation was never observed throughout these studies, we have to postulate the existence of a stable complex into which progeny ssDNA is immediately enclosed after synthesis. This structureperhaps a maturation complex-appears to be

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stable under conditions of the Hirt extraction and, consequently, mature viral DNA is precipitated together with cellular nucleoproteins in the Hirt pellet instead of being recovered in the supernate. ACKNOWLEDGMENTS We wish to thank G. Kronauer, E. Banyai, and L. Lagcher for expert technical assistance and F. Sessiz for preparing the graphs. LITERATURE CITED 1. Bishop, J. M., and L. Levintow. 1971. Replicative forms of viral RNA. Structure and function. Prog. Med. Virol. 13: 1-82. 2. Dobson, P. R., and C. W. Helleiner. 1973. A replicative form DNA of minute virus of mice. Can. J. Microbiol. 19:35-41. 3. Hallauer, C., G. Kronauer, and G. Siegl. 1971. Parvoviruses as contaminants of permanent human cell lines. I. Virus isolations from 1960-1970. Arch. Gesamte Virusforsch. 35:80-90. 4. Hallauer, C., G. Siegl, and G. Kronauer. 1972. Parvoviruses as contaminants of permanent human cell lines. III. The biologic properties of the isolated viruses. Arch. Gesamte Virusforsch. 38:366-382. 5. Hirt, B. 1967. Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26:365-369. 6. Lang, D. 1971. Individual macromolecules: preparation and recent results with DNA. Philos. Trans. R. Soc. London Ser. B 261:151-158. 7. Pearson, G. D. 1975. Intermediate in adenovirus type 2 replication. J. Virol. 16:17-26. 8. Penman, S. 1969. Preparation of purified nuclei and nucleoli from mammalian cells. p. 36-48. In K. Habel and N. P. Salzman (ed.) Fundamental techniques in virology. Academic Press Inc., New York. 9. Petursson, G., and R. Weil. 1968. A study on the mechanism of polyoma-induced activation of the cellular DNA synthesizing apparatus. Synchronization by

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FUDR of virus-induced DNA synthesis. Arch. Gesamte Virusforsch. 24:1-29. Rhode, S. L. 1973. Replication process of the parvovirus H-i. I. Kinetics in a parasynchronous cell system. J. Virol. 11:856-861. Rhode, S. L. 1974. Replication process of the parvovirus H-i. II. Isolation and characterization of H-1 replicative form DNA. J. Virol. 13:400-410. Rhode, S. L. 1974. Replication process of the parvovirus H-i. III. Factors affecting -H-1 RF DNA synthesis. J. Virol. 14:791-801. Salzman, L. A., and W. White. 1973. In vivo conversion of the single-stranded DNA of Kilham rat virus to a double-stranded form. J. Virol. 11:299-305. Salzman, L. A., W. L. White, and L. McKerlie. 1972. Growth characteristics of Kilham rat virus and its effect on cellular macromolecular synthesis. J. Virol.

10:57:3-577. 15. Siegl, G. 1973. Physicochemical characteristics of the DNA of parvovirus LuIII. Arch. Gesamte Virusforsch. 43:334-344. 16. Siegl, G., and M. Gautschi. 1973. The multiplication of parvovirus LuIII in a synchronized culture system. I. Optimum conditions for virus replication. Arch. Gesamte Virusforsch. 40:105-118. 17. Siegl, G., and M. Gautschi. 1973. The multiplication of parvovirus LuIII in a synchronized culture system. II. Biochemical characteristics of virus replication Arch.

Gesamte Virusforsch. 40:119-127. 18. Sinsheimer, R. L. 1959. A single-stranded deoxyribonucleic acid from bacteriophage XX 174. J. Mol. Biol. 1:43-53. 19. Tattersall; P., L. V. Crawford, and A. J. Shatkin. 1973. Replication of the parvovirus MVM. II. Isolation and characterization of intermediates in the replication of the viral deoxyribonucleic acid. J. Virol. 12:1446-1456. 20. Tennant, R. W., and R. E. Hand, Jr. 1970. Requirement of cellular synthesis for Kilham rat virus replication Virology 42:1054-1063. 21. Vogt, V. M. 1973. Purification and further properties of single-strand-specific nuclease from Aspergillus orYzae. Eur. J. Biochem. 33:192-200.