JOURNAL OF VIROLOGY, Feb. 1977, p. 713-723 Copyright © 1977 American Society for Microbiology

Vol. 21, No. 2 Printed in U.S.A.

Replication Process of the Parvovirus H-1. VII. Electron Microscopy of Replicative-Form DNA Synthesis IRWIN I. SINGER AND SOLON L. RHODE III* Putnam Memorial Hospital Institute for Medical Research, Bennington, Vermont 05201

Received for publication 11 June 1976

The geometry of replicative form (RF) DNA synthesis of the H-1 parvovirus was studied with the electron microscope using formamide or aqueous variations of the Kleinschmidt spreading procedure. H-1 DNA was isolated from human or hamster cells infected with a temperature-sensitive mutant, tsl, which is deficient in progeny single-stranded DNA synthesis at the restrictive temperature (S. L. Rhode, 1976), thus minimizing possible confusion between RF and progeny DNA replicative intermediates (RIs). The purity of the isolated H-1 DNA, as determined by gel electrophoresis, ethidium bromide staining, autoradiography, and digestion with endo R EcoRI, was high. H-1 RF DNAs were linear doublestranded molecules, 1.53 ,m in length. H-1 RIs of RF DNA replication were double-stranded, Y-shaped molecules, with the same length as RF DNAs. The replication origin was localized no more than 0.15 genome lengths from one end of the RF DNA, with replication proceeding toward the other end at a uniform rate. Similar RF and RI molecules of dimer size were also observed. The length of H-1 single-stranded DNA extracted from purified virions was measured relative to that of OX174 and it had a very similar contour length, so that the molecular weight of H-1 single-stranded DNA would be at least 1.48 x 106 to 1.59 x 106 (Berkowitz and Day, 1974). The parvovirus H-1 contains a singlestranded (ss) DNA (22), and is capable of autonomous replication (12, 20, 21). During infection, a double-stranded (ds) replicative form (RF) DNA is synthesized and replicated semiconservatively at a nearly exponential rate (13). Progeny ssDNA is produced simultaneously, presumably by displacement from RF DNA engaging in asymmetric DNA synthesis (14), and encapsidated into virions. In this study we have examined H-1 replicative form DNA and its replicative intermediates (RI DNA) with the electron microscope to define the geometry of RF DNA replication. To minimize any confusion with progeny viral DNA synthesis, we analyzed the replicating DNA of a temperaturesensitive mutant of H-1, tsl, which is deficient in progeny ssDNA synthesis, but not in RF DNA replication (15). Previous studies using ethidium bromideCsCl density gradient centrifugation, velocity sedimentation, and gel electrophoresis produced no evidence for covalently closed circular H-1 RF DNA (13). Electron microscope visualization of H-1 RF DNA reveals it to be a linear linRIs molecule, 1.53 um in length. Analysis of shows that they are Y-shaped, with the origin of replication located no more than 0.15 genome lengths from one end, and that replication pro-

ationlofuH-iR. DAm reveal. itatosbe

ceeds from the end containing the origin to the opposite terminus at a uniform rate. The branched RI DNA appeared to be entirely ds. Dimer-length RF DNA molecules and RIs were also observed. The lengths of H-1 viral and RF DNA relative to those of pX174 were measured, and the molecular weight of H-1 ssDNA was determined to be at least 1.48 x 106 to 1.59 x 106, similar to the value obtained by gel electrophoresis (16). Additional data on the location of the origin of replication has been obtained by partial denaturation mapping, and will be presented in the following paper of this series (19). MATERIALS AND METHODS Virus and cells. Parasynchronous cultures of sec-

ondary hamster embryo fibroblasts or human NB cells were prepared and infected with tsl or wildtype (wt) H-1 as previously described (16). Escherichia coli H-502 and H-4714, and OX174 (wt) and am3 were kindly provided by R. L. Sinsheimer. Viral DNA preparation. H-1 virion DNA was extracted from purified virus (12) labeled with [3H]thymidine ([3H]TdR) by lysis in 0.2 N NaOH and in an alkaline sucrose (16). centrifugation viral DNA was prepared as gradient in (11), and OX174 XX174 RF DNA was produced as outlined by Johnson and Sinsheimer (8). H-1 RF DNA was labeled and prepared as previously using the Hirt extraction with Pronase digestion (16). The Hirt supernatants 713




containing viral DNA were extracted with phenol stubs and their attached grids. The samples were after RNase treatment and before fractionation in shadowed by melting a 2-cm segment of platinumthe preparative neutral sucrose gradients. Benzoyl- palladium (80:20) wire in a vacuum of 2 x 10-5 mm of ated DEAE-cellulose (BDC) chromatography was Hg; a visible droplet of molten metal was maincarried out as described (16). tained on the tungsten electrode for the 1-min Gel electrophoresis. Agarose gel electrophoresis shadowing period while the table rotated at 120 rpm. in cylindrical gels has been described (16). Vertical The grids were shadowed at a 50 angle 5 cm from the gel electrophoresis was performed on an EC470 elec- metal source, and were then easily removed from trophoresis cell (E-C Apparatus Corp., Philadel- their stubs with fine forceps. Micrographs were phia, Pa.) with a gel (0.3 by 12 by 16 cm) of 1% made with a JEM-7 electron microscope (J.E.O.L. agarose. The gel and electrode buffers were buffer E Co., Inc., Medford, Mass.) equipped with liquid(40 mM Tris[pH 7.2]-20 mM Na acetate-1 mM nitrogen-filled cold traps at the diffusion pump and EDTA) as detailed (5). Electrophoresis was carried on top of the objective lens pole piece (immediately out with a constant voltage of 100 V at 16°C until the beneath the specimen), and a 30-,tm gold foil objecbromophenol blue marker was near the bottom of tive aperture. The lens currents and high voltage the gel. The gels were stained in E buffer with 5 ,ug (80 kV) were turned on at least 1 h before studying of ethidium bromide per ml for 1 h and photographed grids at a magnification of 14,200, calibrated freunder illumination with long-UV light. Autoradi- quently with a grating replica (E. Fullam, Inc., ographs were made of the gel after vacuum drying Schenectady, N.Y.). The intermediate lens current with Kodak no-screen X-ray film NS2T, exposed (which controls magnification on this instrument) for 24 h at 23°C. was never changed once a micrograph of the calibraElectron microscopy. Viral DNA, which had tion replica was made; the output magnification been banded to equilibrium in Cs2SO4, was dialyzed remained constant throughout the course of this against 0.1 M Tris(pH 8.5)-O.01 M EDTA, brought to work. Care was also taken to use a low electron0.2 M in Na acetate, precipitated with ethanol, and beam current (not exceeding 10 AA) and a reduced dissolved in 50 to 100 gl of the latter buffer for amount of condensor illumination to minimize damelectron microscopic study. Most of the DNA was age to the specimens. The electron micrographs prepared with the formamide technique. The spread- were enlarged 10 times by projection so that the ing solutior. contained 10ll of water, 10gl of an 0.5- DNA molecules could be accurately traced; their mg/ml solution of cytochrome c (Sigma, type III, in contour lengths were measured with a Dietzgen 0.5 M Tris-hydrochloride[pH 8.5]-0.05 M EDTA), planimeter, and expressed as the mean plus or mi5 gl of DNA sample in 0.1 M Tris-hydrochlo- nus the 99% confidence interval (CI). ride(pH 8.5)-0.01 M EDTA, and 25 ,ul of formamide (Matheson Scientific, Inc.). Immediately after thorRESULTS ough mixing, the entire 50 gl of DNA solution was

spread onto a freshly prepared hypophase of 20% formamide in 10 mM Tris-hydrochloride(pH 8.5)-i mM EDTA, in a Teflon-coated trough as described by Davis et al. (4). DNA was also spread by the aqueous method as outlined by the latter authors. Parlodion-coated 300-mesh copper grids were touched to the hypophase surface 1 min after DNA spreading; no talc was used. The grids were attached to stubs to avoid damaging their parlodion films when handling them with forceps, sampling the DNA, or during staining, dehydration, and shadowing. To do this, microscope slides were dipped into a 1% solution of parlodion in amyl acetate (wt/vol) and allowed to dry vertically. The parlodion films were stripped onto a distilled water surface, and portions having a silver-gold interference color were picked up with a wire loop, air dried, and placed onto the surface of a grid resting on a stub; the overhanging edges of the parlodion film firmly held the grid on the stub. This method minimizes damage to the parlodion film, which increases length variation of the adherent DNA molecules; contamination of the lower surface of the grid by the various solutions is also prevented. The grids were stained immediately after DNA sampling with freshly prepared 50 HM uranyl acetate-50 ,M HCl in 90% ethanol (pH 3.9) for 30 s, followed by a 10-s rinse in 90% ethanol and dehydration with 2-methylbutane for 10 s. Shadowing was accomplished with a Denton vacuum evaporator and a rotary table, which accommodated the

Purification of H-1 RF and RI DNA from infected cells. H-1 DNA was extracted from tsl-infected cultures of parasynchronous human NB cells or secondary hamster embryo fibroblasts by the method of Hirt as previously by the met supertas wre subdiled detailed (16). The Hirt supernatants were subjected to velocity sedimentation in a preparative sucrose gradient, and fractions were pooled as illustrated in Fig. 1. Pool A contains primarily monomer RF DNA, and pool B contains monomer RF DNA, dimer RF DNA, and RI molecules, which sediment more rapidly than monomer RF DNA (16). Mock-infected cultures cted cultures were simer similarly extrA extracted, and the yield of radiolabeled DNA in these regions of the sucrose gradient was less than 2% of that from H-1infected cultures. The homogeneity of the radiolabeled DNA to competitive hybridization (13), or to cleavage by bacterial restriction endonucleases (16) indicated that the H-1 DNAs were at least 90% radiochemically pure. However it e, S possible that mock-infected cultures are inadequate controls for purity. For example, the cytopathic effects of H-i infection might induce degradation of cellular DNA, although this was not observed when cells with prelabeled DNA were infected (13), or contamination


VOL. 21, 1977


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with long-UV light (Fig. 2a). As shown before (16), the predominant bands are the monomer RF DNA in the mixture of A and B and in A alone, dimer RF in B alone, and the partially cleaved dimer RF, EcoRI-A, dimer B, and B fragments in the digest of A-plus-B mixture. These stained DNA bands were shown to correspond to the radiolabeled DNA by an autoradiograph of the gel (Fig. 2b). Thus, after the sucrose gradient step, the preparations consist largely of monomer and dimer H-1 RF DNAs, as judged by electrophoretic mobility and specilficity of cleavage with EcoRI. It should be noted that the monomer and dimer RF bands, as well / as those of the EcoRI-A and -B fragments, appear as doublets in the ethidium bromidestained gel, but not in the autoradiogram. This difference in resolution is probably due to our deliberate overexposure of the autoradiogram to visualize the light bands in the EcoRI digest. A B The occurrence of two distinct EcoRI-B fragments has already been documented (16); the of doublet monomer and di20 25 possible existence 15 A






FIG. 1. Preparative sucrosegradient of PH]BUdR containing H-1 RF DNA. Parasynchronous cultures of hamster embryo cells infected with tsl H-1 at a multiplicity of infection of 5 to 10 PFU/cell were incubated at 39.5°C. The cultures were treated with FUdR (10.5 m.g/ml) 14 to 14.5 h p.i., and labeled with [3H]BUdR as in Results. Viral DNA extracted by the Hirt method was redissolved in 50 mM Tris(pH 7.5)-i mM EDTA and treated with pan-

mg/ml) for 30 min at 37°C. RNase was removed by phenol extraction and the DNA was precipitated with 0.15 M NaCl and 2.5 volumes of ethanol at -20°C for 16 h. The DNA was redissolved in the gradient buffer and sedimented in a 5 to 20% sucrose gradient for 18 h at 24,000 rpm, 4°C, in an SW27 rotor as done previously (16). Fractions of i ml were collected through the bottom of the tube, and 20pi aliquots used to determine the positions of radioactivity. Regions were pooled as illustrated, precipitated with ethanol, and redissolved in 20 mM Tris(pH 8.0)-i mM EDTA-0.15% Sarkosyl before The direction of isopycnic centrifugation in sedimentation is from right to left. creatic RNase (50


with degraded unlabeled cellular DNA may be in excess of the viral DNA. Such contamination was ruled out by comparing radiolabeled DNA to total DNA by agarose gel electrophoresis. H1 tsl viral DNA labeled for 12 to 16h postinfection (p.i.) with 32p was prepared through the sucrose gradient step. Equal portions of pool A and pool B (Fig. 1) were analyzed separately and combined both with and without digestion with endo R EcoRI in a slab gel (3 mm by 12 cm by 16 cm) of 1% agarose. The gel was stained with ethidium bromide, and the fluorescent DNA was photographed under illumination

mer RFs and EcoRI-A fragments is currently under investigation. Also, the material remaining at the origin of the lanes containing DNA from fraction B of the sucrose gradient is presumed to be entangled molecules not greater than dimer size, since electron microscopy never revealed longer DNAs in fraction B, and this entrapment is not consistently observed. It is possible that this material is associated with but we believe that this is unlikely, sice the DNA was digested with Pronase, and extracted with phenol. The DNA prepared for electron microscope analysis was density labeled with [3H]bromodeoxyuridine([3H]BUdR). Specifically, H-1 tslinfected NB cultures at 39.5°C were incubated with medium containing 5'-fluorodeoxyuridine


(FUdR) and BUdR (10-5 M) 14 to 16 h p.i. and then FUdR with [3H]BUdR (2 gCi/ml, a X 106 M) 16 to 18 h p.i. Similarly, ts-l

infected hamster embryo cells at 39.5°C were incubated with FUdR and BUdR 14 to 14.5

h p.i., and then FUdR and [3H]BUdR at 14.5 to 16 h p.i. After Hirt extraction and sucrose gradient centrifugation, the A and B pools (from Fig. 1) were banded to equilibrium in Cs2SO4 gradients (Fig. 3A and B). All DNA greater than hybrid density (arrow indicates a density of 1.440 g/cm3) was pooled, and dialyzed against 10 mM Tris(pH 8.5)-i mM EDTA for study with the electron microscope. In this way, the preparation was enriched for molecules that had replicated one or more times in the presence of BUdR, and control experiments indicated that 98% of contaminating light DNA was removed. The composition of the final dense H-1 DNA


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FIG. 2. Vertical slab-gel electrophoresis of H-i DNA after centrifugation in neutral sucrose. Parasynchronous NB cultures were infected with tsl H-i at 39.50C and labeled with 32P0 4 from 12 to 1 6 h p. i. (1 6). The viral DNA was extracted, treated with RNase, and subjected to preparative centrifugation in a neutral sucrose gradient. Fractions were pooled as in Fig. 1, and the DNA was dissolved in 1 00 p.l of 1 0 mM Tris(pH 7.5) -b mM NaCl-0 .1 mM EDTA. A liq uots ofl10 MI of pools A and B were combined and adjusted to 50 mM NaCl, 1 0 mM MgCl,, and 1 mM dithiothreitol, and digested with 100 U of EcoRI for 1 h at 370C. The reaction was

stopped by addition of 20 tii of 2.5% sodium dodecyl sulfate-50% glycerol-1 0 mM EDTA. Slab-gel electrophoresis was carried out as described in Materials and Methods. The gel contained, from left to right: pool B (20 pi), pool A (20 pI), EcoRI-digested A + B (10 /.d of each) mixture, and A + B (20 PIl each) mixture. Twenty microliters of each sample contained the yield of DNA from about 2 X 107 NB cells. The gel was stained with ethidium bromide, and the DNA was visualized with long-UV light (a), and by autoradiography (b). It has been shown (1 6) that the specific H-i DNA species observed are (in descending order): dimer RF (DI RF), RF with attached EcoRI-B fragment (RF + RIB, partial digest of DI RF), RF, EcoRI-A fragment (RI A), dimer EcoRI-B fragment (DI RI B), and EcoRI-B fragment (RI B). Relative contour lengths of H-i and OX174 preparation was examined by agarose gel electrophoresis (Fig. 4A) and found to be almost viral ssDNAs. Preliminary electron microentirely monomer with small amounts of dimer scopic examination of H-i ss viral DNA showed RF DNA. The 13H]TdR-labeled virion DNA that it is linear, and has a contour length simiused in this study was also analyzed by gel lar to that of circular pX174 viral DNA. Since electrophoresis as shown in Fig. 4B. The elec- we wanted to determine the molecular weight tropherogram of the latter preparation is domi- of H-i ssDNA relative to 4)X174 DNA by mixnated by a homogeneous peak, with a smaller ing these preparations and measuring the ratio portion of faster-migrating species assumed to of their lengths on the same grid, it was necesbe fragmented molecules. It should be noted sary to ensure that significant breakage of that the virion ssDNA migrates faster than the OX174 circles was not occurring so that broken ds RF under these conditions and that there is (linear) 4X174 ssDNA would not be confused no evidence of virion DNA in the RF DNA with H-i ssDNA. We therefore examined preparation (Fig. 4A). A very small peak of 4X174 ssDNA, and found that the extent of uncertain significance at the electrophoretic po- circle breakage was 9%. These viral DNAs were sition of RF DNA is noted in the virion DNA then mixed such that the concentration of H-i electropherogram. This could arise from an- DNA was twice that of the 4X174; the maxinealing of V strands to traces (0.25%) of C mum amount of linear 4X174 DNA contamistrands, but this has not yet been proven. nating the H-i viral DNA pool was therefore

VOL. 21, 1977






l mum molecular weight of H-1 viral ssDNA is D1.48 x 106 to 1.59 x 106, based on the 4X174 ssDNA molecular-weight determination of 1.59 l1.50 x 106 (1). In addition, we did not observe circu/11.45 larization (evidence of terminal self-complementarity) after incubation of H-1 viral DNA 1.40 under annealing conditions (50% formamide-50 I5 mM Tris-5 mM EDTA for 1.5 h at 23°C), fol{ \ 1.3 lowed by immediate spreading from 50% form- amide onto 20% formamide; the electrophoretic JC\ 4 profile also was unchanged after this treat' ' s ~ t8 ment. , Geometry of H-1 RF and RI dsDNA's. Regions of the Cs2SO4 gradients containing puta1.50 tive RI and RF (Fig. 3A and B), which exhibited 1\.45 [3H]BUdR-substituted H-1 tsl DNA of greater than hybrid density, were chosen for electron 1.40 \ - l.40 microscope study, since it would be unlikely for 1-.35 the host cell to produce any DNA of this density due to the semiconservative nature of cellular DNA synthesis, and the short labeling times employed. The fully substituted DNA from pool s , < s B (Fig. 3B) contained ds linear RF DNA of monomer and dimer lengths (Fig. 5B), and Y20 10 15 45




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Tube Number

FIG. 3. Isopycnic centrifugation of H-1 tsl DNA pools A and B from Fig. 1. The viral DNA pools prepared by velocity sedimentation in the neutral sucrose gradient (Fig. 1) were sedimented to equilibrium in gradients of Cs2SO4 as previously described (16). (A) Pool A; (B) pool B. Centrifugation conditions were 48 h at 35,000 rpm in a type 40 fixed-angle rotor at 10°C. Fractions of 0.2 ml were collected through the bottom of the tube, and 10-p. aliquots were dried on 25-mm filter paper disks for assay of radioactivity. DNA greater than hybrid density (arrows) was pooled, dialyzed against 0.1 M Tris (pH 8.5)-10 mMEDTA, adjusted to 0.3 MNaCl, precipitated with ethanol, and redissolved in 50 to 100 pl of 0.1 M Tris(pH 8.5)-10 mM EDTA for electron microscopy. The direction of sedimentation is from right to left.

3% (DNA spreading solution contained 50% formamide; hypophase contained 20% formamide). Figure 5A is a representative micrograph of this mixture, and histograms of the measured H-1 and 4X174 viral DNAs (Fig. 6) exhibit the same maximum peaks (at 1.0 ,Lm) and very similar contour length variation. Both H-1 and 4X174 viral ssDNA's had a mean length of 1.0 am under these formamide-spreading conditions. We also spread a mixture of H-1 and 4X174 ssDNA's from a solution containing 30% formamide onto a hypophase with 10% formamide. Under these codition, coditions, thee mean length g of 143 + 99 99 H-I ssDNA was 0.96 ± 00.03 z (n = 143 CI) and that of X174 DNA was 1.03 ± 0.02 ymI1 (n = 123). Since gel electrophoresis revealed some fragmented molecules (Fig. 4B), the mini-

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Fraction + FIG. 4. Analytical gel electrophoresis of [3H]BUdR-containing H-1 RF DNA and [3H]TdRlabeled H-1 ss viral DNA. H-1 RF DNA, prepared as in Fig. 3 (mixture ofpools A and B), and [3H]TdRlabeled virion DNA were analyzed in a cylindrical

gel (0.6 by 15 cm) of 1.4% agarose (16). Electrophoretic conditions were 30 Vfor 17.5 h at23C. (A) H-1 RF DNA; (B) H-1 virion DNA.


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Replication process of the parvovirus H-1. VII. Electron microscopy of replicative-form DNA synthesis.

JOURNAL OF VIROLOGY, Feb. 1977, p. 713-723 Copyright © 1977 American Society for Microbiology Vol. 21, No. 2 Printed in U.S.A. Replication Process o...
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