Early synthesis of virus-specific RNA and DNA in cells rapidly transformed with Rous sarcoma virus

VIROLOGY

56, 532-548 (1973)

Early Synthesis of Virus-Specific RNA and DNA in Cells Rapidly Transformed with Rous Sarcoma Virus ANTHONY L . SCHINCARIOL

AND

WOLFGANG K . JOKLIK

Department of Microbiology and Immunology, Duke University Medical Center, Durham, North Carolina 27710 Accepted September 4, 1973

Chick embryo fibroblasts (CEF) were infected with the Prague strain (subgroup C) of RSV (PrC-RSV) under conditions of rapid transformation and the synthesis of RNA and DNA capable of hybridizing with single-stranded DNA transcripts of 70 S PrC-RSV RNA (PrC-RSV DNA) was measured by hybridization kinetics analysis . PrC-RSV DNA was synthesized in the presence of high concentrations of actinomycin D and was freed from double-stranded DNA by chromatography ; it protected over 90% of 70 S PrC-RSV RNA from digestion by ribonuclease at DNA :RNA ratios greater than 9 . Virus-specific RNA sequences were measured with cellular RNA in vast excess, using the hybridization kinetics of the PrC-RSV DNA with 70 S PrC-RSV RNA ae the reference . Virus-specific DNA sequences were measured with cellular DNA in vast excess, using the kinetics of self-annealing of the unique sequences of cellular DNA as the reference . Uninfected gs CEF contained on the average one viral genome copy of DNA per cell and no detectable virus-specific RNA . DNA capable of hybridizing with the probe began to be synthesized within 3 hr after infection, and by 24 hr 2 viral genome copies had been synthesized . No additional virus-specific DNA was synthesized thereafter . During the first 6 hr after infection, CEF contained about 40 viral genome equivalents of RNA per cell, about 83,x, of which were in the cytoplasm . This represented most probably RNA in inoculum virus . Between 6 and 24 hr the number of viral gecome equivalents per cell increased to 100 . This newly synthesized RNA was equally distributed between the nucleus and polyribosomes . Between 24 and 96 hr the amount of virus-specific RNA in the nucleus and in polyribosomes increased by just under 2-fold, but in the remainder of the cytoplasm it rose by over 8-fold . By 96 hr cells contained about 340 genome equivalents of RNA per cell, which was about one-half of the number present in cells 14 days after infection . The size of the virus-specific RNA present in polyribosomes was predominantly 35 S, but some 10-30 S material was also present . INTRODUCTION

body of evidence that is consistent with this notion : the results of studies with inhibitors of DNA and RNA synthesis (Bader, 1964, 1965 ; Temin, 1963, 1964, 1967 ; Vigier and Golde, 1964) ; the demonstration that virus replication and cell transformation are prevented if the DNA that is synthesized is rendered nonfunctional (Boettiger and Tcnin, 1970) ; the fact that genetically different viruses require the synthesis of different DNAs (Duesberg and Vogt, 1969)

The successful replication of exogenous RNA tumor viruses is unique in its requirement for both DNA and RNA synthesis . According to the DNA provirus hypothesis (Temin, 1971), the virion-associated RNAdependent DNA polymerase transcribes the genetic information in viral RNA into DNA, which is then integrated into cellular DNA and in time serves as the template for the transcription of viral RNA . There is a large 532 Copyright © 1973 by Academic Press, Inc . All rights of reproduction in any form reserved .



EARLY SYNTHESIS OF RSV-SPECIFIC RNA AND DNA and that this DNA is not cell DNA (Temin, 1968 ; Murray and Temin, 1970) ; the discovery of the virus-associated DNA polymerase (Temin and Mizutani,1970 ; Baltimore, 1970) ; and finally the demonstration of virusspecific infectious DNA in cells infected with and transformed by RNA tumor viruses (Hill and Hillova, 1972) . Further, while the background of endogenous viral genetic material in chicken and mouse cells, variously reported as 1-4 (Baluda and Nayak, 1970 ; Rosenthal et al ., 1971 ; Neiman, 1972; Baluda, 1972 ; Harel et al ., 1972 ; Neiman, 1973) or 12-15 genome equivalents/cell (Varmus et al ., 1972 ; Gelb et al ., 1973), has prevented the clear and unequivocal demonstration that virus-specific DNA is synthesized following infection, such DNA has been shown to be synthesized when such background is absent, as, for example, in mammalian (mouse) or duck cells infected with avian RNA tumor viruses (Varmus et al., 1973a, b) . As for virus-specific RNA, it seems clear that infected and fully transformed cells contain a great deal (0 .3-1 % of total cell RNA, equivalent to some 2000-3000 genome equivalents per cell (Leorrg et al ., 1972 ; Bishop et al ., 1973)), while uninfected cells that express resident viral information to the extent of gs antigen synthesis (gs* cells) contain much smaller, but still readily detectable amounts (30-50 genome equivalents), and uninfected cells that do not express resident viral information (gs cells) contain none (Leong et al., 1972 ; Hayward and Hanafusa, 1973) . The object of the work reported here was to determine how soon after infection of gs chick embryo fibroblasts with PrC-RSV, virus-specific DNA and RNA species are synthesized, to quantitate them, and to characterize them with respect to size and location within the cell . For this purpose we used as a probe PrC-RSV-specific DNA transcribed in the presence of actinomycin D by the DNA polymerase in detergent-treated PrC-RSV particles . In agreement with Garapin et al . (1973), we find that such DNA is predominantly single-stranded and not highly reiterated, since it almost completely protects viral RNA from single-strand-specific nuclease attack when annealed to it in 10-fold excess . This DNA was used as a probe

533

for measuring virus-specific RNA by hybridizing it with a vast excess of cellular RNA, and as a probe for measuring virus-specific DNA by similarly hybridizing it with a vast excess of cellular DNA . In both cases, amounts of nucleic acid as low as one genome equivalent per cell could easily be detected and measured . While this work was prepared for publication, two reports which bear on it were published : Salzberg et al . (1973) demonstrated that viral RNA synthesis commences within 7 hr after infection of 3T6 cells with a murine sarcoma-leukemia virus complex, and Varmus et al . (1973b) showed that RSV-specific DNA was synthesized in duck cells within 4-12 hr after infection . MATERIALS AND METHOD

Growth of cells and virus . Primary cultures of gs C/O CEF' were prepared from 10-11day-old White Leghorn embryos (Spafas ; Dr . R . Luginbuhl, University of Connecticut, Storrs), as described by Vogt (1969) . The tissue culture medium for secondary cultures was Ham's F10, supplemented with beef embryo extract (0 .1 %), streptomycin (50 µg/ml), penicillin (50 units/nil), mycostatin (10 units/ml), and tryptosc phosphate broth (10%) (Smith and Bernstein, 1973) . Growth (GM) and maintenance (MM) medium contained 5 % and 1 % calf serum (Gibco), respectively . Dimethyl sulfoxide (1%) was added to media used for the production of virus (Smith and Bernstein, 1973) . The Prague strain (subgroup C) of Rous sarcoma virus (PrC-RSV) used in this study was recloned from a single focus (Smith and Bernstein, 1973) . It was concentrated from tissue culture medium by centrifugation (Spinco rotor SW 27, 25,000 rpm, 60 min, or rotor 19, 18,000 rpm, 90 min) after removal of cell debris (800g for 10 min) . Pellets were resuspended in TE buffer and purified by equilibrium and velocity cenI

The following abbreviations are used : CEF,

chick embryo fibroblasts ; TR buffer, 0 .005 M Tris- HCl, 0 .001 M EDTA, pH 8 .6 ; EDTA, ethylenediaminetetraacetic acid ; FFU, focus-forming units ; BSS, balanced salt solution ; TCA, trichloroacetic acid ; SDS, sodium dodecyl sulfate ; NTE, 0 .1 M sodium chloride, 0 .01 M Tris • No, 0 .001 M )ETA, pH 7 .1 .



534

SCHINCA1tJOL AN!) JOKLIK

trifugation in sucrose density gradients [1560 % (w/v) and 15-40 % (w/v) in TE buffer, respectively] (Smith and Bernstein, 1973) . Labeling virus with nP and $II-uridiiu . The medium on confluent CEF cultures producing virus was replaced with phosphate-free MM for 5 hr . Fresh MM containing 200 µCi/ml 62P-orthophosphate was then added ; 12 hr later it was replaced with unlabeled MM, and collection of supernatants for virus isolation was begun . The procedure for the preparation of virus labeled with 3H-uridine was the same except that complete MM containing 200 µCi/ml 3H-uridine (28 Ci/ mmole, Schwarz-Mann) was used . Assay for focus forming units . Primary CEF were trypsinizcd and seeded in 60-mm Falcon plastic Petri plates at the rate of 1 .2 X 10 6 cells (in 5 ml CM) containing 2 µg,/ml polybrene per plate . They were infected within 4 hr with 0 .1-ml samples of appropriate virus dilution . Twenty-four hours later, the monlayers were overlaid with .5 ml of GM containing 0 .9% agar (Difco) and kept at 37° until foci developed (in 7-14 days) . Rapid transformation of C,EF . In order to initiate infection as synchronously as possible, 4- to 6-day-old primary cultures were trypsinized and the cells, suspended in GM, seeded into Falcon plastic Petri plates (7 .5 X 10 6 cells in 25 ml/plate) . Within 4 hr the supernatant was removed and the cells were infected with 7 .5 ml of a virus suspension containing 3-4 X 10' FFU in GM supplemented with 2 µg/ml Polybrene . Virus suspensions were always supernatants (800 g for 10 min) freshly prepared from medium removed from confluent cultures of transformed cells . After 60 min for adsorption, the medium was removed and 25 ml of fresh GM lacking polybrene was added . Cultures were kept at 37° and fed daily with GM . Measurement of 2-deoxy-n-glucose uptake . The uptake of 2-deoxy-n-glucose by uninfected and infected cells was measured essentially as described by Martin et al . (1971) . Duplicate monolayers in 35-nun Petri plates were rinsed three times with warm Hanks' BSS lacking glucose ; Hanks' BSS without glucose but containing 0 .25 µCi of 'H (G)-2deoxy-n-glucose (10 Ci/mmole ; New England Nuclear Corporation) was then added ;

and after 10 nun at 37° the monolayers were rinsed twice with cold modified flanks' BSS . Cells were removed by adding 0 .5 ml of 0 .25 % trypsin for 1 min and scraping with a rubber policeman . One-half of the cells was used for measuring cell number with a Coulter cell counter, while the other half was used for the measurement of radioactivity . Preparation of nuclei and eytoplasmic fractions . Cells were trypsinized and suspended at 4 X 10° cells/ml in ice-cold extraction buffer (0 .1 M NaCl, 0 .02 M MgCh, 0 .1 M Tris, pH 8 .4) . After 15 min 0 .15 ml of a mixture of one part of 10% (w/w) sodium deoxycholate and two parts of 10% (w/w) Twecn 40 was added and the cells were broken by 10 strokes in a Dounce homogenizer (Penman, 1966) . The nuclei were deposited by centrifugation at 800g for 2 min, and washed once with the bufferdetergent mixture . In order to isolate and characterize cytoplasmic polyribosomes, the cytoplasmic extract was layered onto a 11 .5 ml 7 .5-45 `% (w/w) linear sucrose density gradient in extraction buffer and centrifuged for 70 min at 4° at 36,000 rpm in an SW 41 rotor . Gradients were fractionated with continuous monitoring of absorbance at 260 nm using a Gilford recording spectrophotometer . Fractions containing polyribosomes were pooled, treated with 0 .5% SDS, precipitated with ethanol and deproteinized with phenol at room temperature in NTE containing 0 .1 SDS . Isolation of viral and cellular RNA . The isolation of nucleic acid was carried out using autoclaved solutions and glassware rendered nuclease-free by heating for 4 hr at 140 ° . Unlabeled PrC-RSV 70 S RNA was extracted from purified virus resuspended in NTE buffer (1-2 mg viral protein/ml) . The suspension was made 0.5 % with respect to SDS and rapidly deproteinized at 4 ° using an equal volume of double-distilled phenol which contained 0 .1 % 8-hydroxyquinoline, 10 % (w/v) water and was saturated with NTE buffer . After three phenol extractions and one extraction with an equal volume of chloroform (containing 4 % isoamyl alcohol), the aqueous phase was made 0 .2 M with respect to NaCl and the RNA was precipitated by addition of 2 vol of 95 % ethanol and

EARLY SYNTHESIS OF N .S4-SPECIFIC RNA AND DNA

storage overnight at -20 °. The precipitate was collected by centrifugation, redissolved in NTE buffer containing 0 .1 % SDS and centrifuged into a 11 .5 ml 15-30% (w/w) linear sucrose (in NTE buffer) density gradient (SW 41 rotor, 36,000 rpm, 19 °, 210 min) . Fractions containing RNA were pooled, made 0.3 M with respect to NaCl, and the RNA was precipitated with ethanol . The precipitate was collected by centrifugation at 30,0008 for 60 min, and resuspended in NTE buffer . The concentration of RNA in the sample was determined from its absorbance at 260 ran, using the conversion factor 1 OD unit at 260 run = 40 µg of RNA/ml . When isolating 70 S RNA from small amounts of labeled virus a similar deproteinization procedure was employed but crude virus pellets were used, yeast transfer RNA (50 µg/ml) was added as a carrier, and the 70 S RNA was isolated in 4.8 ml 1530% (w/w) sucrose density gradients (SW 50 .J, 46,000 rum . 19°, 120 min) . Whole cell RNA was extracted using a low pH, high temperature technique that reduces DNA contamination to a minimum . Briefly, cells collected by try psinization were suspended in ice-cold extraction buffer (0 .5 M sodium acetate, 0.01 M FDTA, pH 5 .1) at a concentration of 0 .5-1 X 10' cells/ml. The cells were lysed by the. addition of SDS to a final concentration of 0.75 % and then rapidly deproteinized at 60° according to the procedures described by Scherrer (1969), using distilled phenol containing 0 .1% 8-hydroxyquinoline . The nucleic acids in the aqueous phase were precipitated with ethanol and then redissolved in NTE containing 0.5 % SDS . 1'ronase (grade B, Calbiochem, self-digested for 2 hr at 37°) was added (50 sg/ml), and the solution was incubated at 20° for 60-90 min . Pronase was removed by deproteinization. with phenol at room temperature, and the RNA was concentrated by ethanol precipitation and centrifugation . It was then treated with 10 µg,/ml of electrophoretically purified DNase (Worthington Biochemical Corp .) for 30 min at 0° in 0.01 M Tris, 0 .05 M NaCl, 0.001 M VIgClz, pH 7 .0 ; the DNase had been tested for absence of RNase by demonstrating its inability to digest labeled PrC-RSV RNA and poly(U) to TCA-soluble components .

535

EDTA was then added to 0 .002 M, the solution was deproteinized with phenol, the RNA was concentrated with ethanol as described above and it was redissolved in NTE . Cytoplasmic RNA was extracted from cytoplasmic extracts (obtained as described above) containing 0 .02 III EDTA and 1 0 SDS, precipitated with 2 vol of ethanol, redissolved in extraction buffer containing 0.5 % SDS and deproteinized as described above . Nuclear RNA was extracted from purified nuclei with phenol and chloroform as described by Penman (1966) . Preparation of cellular DNA . Unlabeled DNA from infected or uninfected cells was purified by modifications of the methods of Marmur (1961) and Berns acid Thomas (1965) . Cells suspended in 0 .1 M NaCl, 0.01 M Tris, 0 .01 M EDTA, pH 8 .4, were made 0 .5 % with respect to SDS and digested with 500 µg/ml Pronase for 2-3 hr at 37°. Sodium perchlorate was added to 1 M, followed by an equal volume of chloroform (containing 4c/"O isoamyl alcohol), and the mixture was rotated on a rotary shaker at room temperature for 30 min at 60 rpm . After standing for 10 min, the mixture was centrifuged at 800g for 5 min . The chloroform phase was removed and the chloroform extraction was repeated (rotating for 15 min at 60 rpm) until little or no interphase remained. Ethanol was then added to the aqueous phase, the nucleic acids were collected by centrifugation, dissolved in 0 .005 Al EDTA, pH 7 .0, and treated with 100 gg/ml pancreatic ribonuclease (boiled for 10 min) for 60 min at 37 °. Pronase was then added to a final concentration of 50 µg/ml, and the samples were incubated for 60 min at 37 °. The salt concentration was then increased to 0 .1 M NaCl, 0 .01 M Tris, 0 .005 M EDTA, pH 8 .4, and the solution was extracted three times with buffer-saturated phenol and once with chloroform . The deproteinized DNA was then sonicated and passed through a 1 X 90 cm column of Sephadex G-50 equilibrated with NTE . The DNA in the void volume was precipitated with ethanol and dissolved in a small volume of 0 .005 M EDTA . Uninfected cell DNA was labeled by

536

SCHINCARIOL AND JOLKIK

seeding cells in petri plates at 30% confluency and adding GM containing 0 .25 µG5/ ml 14 C-thymidine (37 mCi/mmole) or 10 µCi/ml 'H-thynidine (20 Ci/mmole) . The DNA was then extracted as above . Synthesis of virus-specific DNA probes . High specific activity radioactively labeled DNA complementary to PrC-RSV 70 S RNA (PrC-RSV DNA) was synthesized using the virion RNA-directed DNA polymerase . Purified PrC-RSV (200-300 Ag/ml) was added to an incubation mixture containing 0 .1 M Tris, pH 8 .3, 0 .01 M MgCI 5 , 0 .0001 M each of dATP, dCTP, and dGTP, 0 .005 M dithiothreitol, 100 pg/ml actinomycin D, 0 .01% (v/v) Triton X-100, and 50 µCi/nd (about 3 X 10-1 M) of 1 H-dTTP (20 Ci/mmole, Schwarz-Mann)? The reaction mixture was incubated for 8-10 hr under nitrogen at 37 ° . The mixture was then made 0 .01 M with respect to EDTA, 0 .1 M with respect to NaCl, and 0 .5% with respect to SDS, calf thymus DNA was added to a concentration of 50 pg/ml and Pronase to 500 µg/ml, and the mixture was incubated at 37 ° for 60 min. The DNA was deproteinized three times with buffer-saturated phenol and once with chloroform, and precipitated with 2 volumes of ethanol . The precipitate was dissolved in 0 .01 2 phosphate buffer, pH 6 .8, and single and double strands were separated by hydroxyapatite column chromatography (Bernardi, 1969) . The single strands (85-90 % of the total) eluted at 0 .16 M phosphate ; they were pooled and passed through Sephadex G-50 to remove phosphate, precipitated with ethanol, and dissolved in 0 .005 M EDTA . The solution was then made 0 .3 M with respect to NaOH and kept at 100 ° for 5 min to hydrolyze RNA ; it was neutralized with HCl after the addition of one-tenth volume of 1 M Tris, pH 7 .4, and chromatographed on a Sephadex ' If the concentration of Triton X-100 was raised above 0 .01%, the yield of DNA decreased markedly (and template RNA was degraded to acidsoluble components) . Actinomycin D, at a concentration of 100 (or 200) µg/ml, reduced the yield of DNA by 75% ; but the transcripts formed in its presence contained a far higher proportion of single-stranded species and far fewer reiterated sequences than those formed in its absence (Bishop et al ., 1973) .

G-50 column equilibrated with NTE . The labeled DNA from the excluded region of the column was precipitated with ethanol . The specific activity of PrC-RSV DNA prepared in this manner was between 3 and 10 X 10' epm/pmole (1-3 X 10' cpm/µg) . Nucleic acid hybridization and reassociation . RNA-DNA hybridization were performed at 68° in 0 .3 M NaCl, 0 .01 2 Tris, pH 7 .2, 0 .001 M EDTA, and 0 .1 Tc SDS . Unlabeled RNA was used in excess at concentrations up to 20 mg/ml, and labeled single-stranded PrC-RSV DNA was added to a final concentration of 0 .001 pg/ml . The reactions were carried out in volumes of 50 pl in sealed microcapillary tubes . DNA-DNA reassociation was carried out in the same manner except that sonicated cellular DNA denatured at 100 ° C for 3 min and then made 0 .6 M with respect to NaCl was used in excess at concentrations up to 10 mg/ml . The extent of hybridization/reassociation was assessed by measuring resistance to Aspergillus oryzae 81 single-strand specific nuclease which was purified from Takadiastase or a-amylase powder as described by Sutton (1971) up to the DEAF-cellulose chromatography step and was stored at -20°C with no detectable loss of activity . Reaction mixtures contained 0 .03 M sodium acetate buffer, pH 4 .5, 0 .01 M ZnSO,, 0 .3 M NaCl, 10 pg/ml denatured calf thymus DNA and sufficient S1 nuclease (determined empirically) to degrade this amount of DNA . The samples were incubated at 37 ° for 30 min and then precipitated with 5 4i° TCA following addition of 1 drop of carrier yeast tRNA (2 mg/ml) . Precipitates were collected on GF/C glass fiber filters, dried, and counted in 10 ml of Omnifluor-toluene . The results are expressed as the fraction of DNA hybridized at a given value of C„t or Co l, computed as described by Britten and Kobne (1968) and corrected to standard conditions of salt concentrations (Britten and Smith, 1970) . The hybridization of PrC-RSV 70 S RNA labeled with '1 P to PrC-RSV DNA was measured by determining the resistance of the labeled RNA to pancreatic ribonuclease digestion (50 µg/ml) after diluting 10-fold

EARLY SYNTHESIS OF RSV-SPECIFIC RNA AND DNA

537

Fro . 1 . Phase contrast photomicrographs of uninfected (A-D) and infected (E-H) ebick embryo fibroblasts at 24 (A, E), 48 (f3, F), 72 (C, (1) and 96 (D, H) hr after infection with 5 FFU PrC-RSV per cell . X 162 .

538

SCIIINCAItIOL AND JOKLIK

into 0 .3 M NaCl, 0 .01 M Tris, 0 .001 M EDTA, pH 7 .4 . RESULTS The Time Course of Transformation of CEF with PrC-RSV Morphological changes . Infection of secondary cultures of CEF with PrC-RSV at a multiplicity of 4-o FFU/ccll shortly after reseeding results in their rapid and synchronous morphological transformation (Hanafusa, 1969) . Morphological alteration

was detectable microscopically in 10 15' ; of the cells within 24 hr after infection (Fig . 1) : uninfected cells retained a flat and stretched-out appearance, while transformed cells, either spindle-shaped or round, appeared more refractile and randomly oriented (see also Hanafusa, 1969) . After 40 hr, 4050 % of cells appeared altered ; after 72 hr, 80-90 %r, and by 96 hr, virtually all cells had transformed morphologically (Fig . 1) . Transport changes . The uptake of 'H-2deoxv-D-glucose was measured to provide a second index of cell transformation . As shown in Fig . 2A, the rate of uptake of this substance by uninfected cells remained constant during the 120 hr following mock infection, whereas that of infected cells increased markedly between 24 and 72 hr, and by 96 hr exceeded that of uninfected cells by a factor of about 5 . Cell growth . rate changes . The growth rate of infected and uninfected cells was measured by counting the number of cells in parallel cultures . Cells began to grow slightly faster

DNA synthesized in presence of Act D DNA synthesized in absence of Ac? D

TIME AFTER INFECTION (HR5) FIG. 2 . Measurement of the rate of (A) uptake of 'H-2deoxy-u-glucose, (B) cell multiplication, and (C) virus production and release, under conditions of rapid transformation . Secondary cultures of CEF were seeded into 35-nun petri plates at 5 to 7 X 10' cells/plate and infected within 2 hr . All points arc the averages of duplicate determinatione .

FIG . 3 . Measurement of the extent of the ability of PrC-1',SV DNA to hybridize with PrC-RSV virion RNA, DNA labeled with 'H was prepared as described in Materials and Methods (but with a specific activity of only 100 epm/pmole), and hybridized to A"P-labeled 70 S PrC-RSV RNA (100 epm/pniole) at various DNA :1tNA ratios for 48 hr in 0 .01 M Tris, pH 7 .4, 0 .3 M NaCl, 0.001 h EDTA containing 0 .1o%, SDS at 68° ; reaction volume 25 µl . After incubation, samples were diluted 10-fold in NTE, the RNA was digested with 50 µg/ml pancreatic RNase A and 10 µg/ml RNase T, for 40 min at 37°, and the samples were precipitated with TCA . The fraction of 'RNA resistant to digestion is plotted as a function of the DNA :RNA ratio .

EARLY SYNTHESIS OF RSV-SPECIFIC RNA ANT) DNA

than control cells as soon as they were infected (Fig . 2B) . Time course of virus production . Figure 2C indicates that progeny virus was first detected at 18 hr after infection ; it was then produced increasingly rapidly until 72-96 hr after infection, when a stable rate of virus production (about 2 FFU/cell/24 hr) was achieved. Characteristics of the Viral DNA Probe (PrCRSV DNA)

The following characteristics of the DNA probes were examined : 1 . Size and strandedness . The DNA sedimented with 4-S S in neutral sucrose density gradients . It was over 98 % single-stranded . 2. Extent of viral 70 S RNA represented . To measure the extent to which PrC-RSV DNA represented the sequences of viral RNA, 3H-PR-RSV DNA was hybridized with 32P-PR-RSV RNA at various DNA : RNA ratios . Eighty-eight percent of the RNA was protected from S l nuclease digestion at a DNA : RNA ratio of 7, 92 % at a ratio of 10 and 97 % at a ratio of 25 (Fig . 3) . The population of DNA transcripts synthesized in the presence of actinomycin D therefore represented essentially the entire RNA genome. 3. Kinetics of hybridization with 70 S RNA . Hybridization of labeled DNA probes to unlabeled viral and cellular RNA was per-

539

formed under conditions of RNA excess . Under these conditions, DNA-RNA hybridization is a first-order reaction in which the rate of the reaction is directly proportional to the genetic complexity of the RNA (Bishop, 1969 ; Birnstiel ct al ., 1972) . The hybridization data are presented on semilogarithmic graphs, plotting the fraction of DNA hybridized as a function of Crt [the product of initial RNA concentration X time of incubation (Birnstiel et al ., 1972)] . The fraction of RNA complementary to DNA transcripts in a given mixture can then be estimated by comparing the C,tr/2 value calculated for it with the C,t11 2 value obtained using only RNA complementary to the DNA probe. The sensitivity of this procedure was tested by hybridizing a standard amount of DNA to a range of 70 S PrC-RSV RNA concentrations . At RNA concentrations ranging from 0 .1 to 10 µg/ml, corresponding to RNA : DNA ratios of 100-10,000, 95-100% of the DNA hybridized, and all points fell on the same curve, the C„t,i 2 value of which was 2 X 10-2 mole-sec/1 (Fig . 4) . This curve closely followed the theoretical curve for hybridizing DNA to complementary RNA in excess . Since RNA concentrations as high as 20 rng/ml can be used, 1 part of complementary RNA in 200,000 can readily he measured ; this corresponds to about 1 virus genome equivalent per cell (on the basis of

C,t (mole-sec/liter)

FIG . 4 . Hybridization of viral and cellular RNA with 3H-labeled PrC-RSV DNA . Concentration of 70 S PrC-RSV RNA, 10 erg/ml (0), 1 µg/ml (0) and 0 .1 µg/ml (0) ; of transformed CEF RNA (isolated 14 days after infection with PrC-RSV), 210µg/ml ; of °H-PrC-RSV DNA, 0 .001µg/ml . For hybridization conditions and assay for Sl-nuclease resistant DNA, see Materials and Methods . The broken curve represents the theoretical curve expected for DNA-RNA hybridization with RNA in excess .



SCHINCARIOL AND JOKLIK

5 40

jig RNA/CEF (Leslie, 1955) and pg RNA per viral genome) . Since the curve for 0 .1 pg/mI in Fig . 4 coincided with those for the higher RNA concentrations, complementary RNA at concentrations far lower than 0 .1 pg/ml, and therefore 4 X 10- e

1 .7 X 10 -11

FIG . 5. Thermal denaturation profiles of DNARNA hybrids . 3H-PrC-RSV DNA was annealed to either 70 S PrC-RSV RNA (5 pg/ml) or cellular RNA extracted from transformed CEF at 14 days after infection (250 pg/ml) for a time sufficient to hybridize 95-100% of the DNA . Aliquots of 5 pl were then sealed in 50-Al capillary tubes, heated for 5 min at the indicated temperature and cooled rapidly in an ice-water bath . Each sample was assayed for the amount of DNA that remained resistant to Sl-nuclease . Filled circles, viral RNA ; open circles, cellular RNA .

corresponding to much less than 1 genome equivalent per cell, could clearly still be detected . Hybridization of PrC-RSV DNA to the RNA of CEF 14 days after infection with PrC-RSV, when they were fully transformed and producing virus, yielded a C,tt ; 2 of 7 .5 (Fig . 4) . This indicated that about 0 .27 % of total cell RNA was virus specific ; this corresponded to about 625 viral genome equivalents per cell . The fact that the hybridization curve with cellular RNA was similar in shape to that with 70 S PrC-RSV RNA indicated that, as expected, the transformed cells contained all the sequences present in PrC-RSV RNA. Thermal denaturation profiles of DNARNA hybrids. The precision of hybridization of the DNA probe to both viral and cellular RNA was assessed by determining the denaturation profiles of the hybrids, using resistance to S1 nuclease as the criterion of ordered secondary structure (Fig. 5) . The sharp profiles and relatively high Tms (S5 °) indicated a high degree of base-pairing between the DNA probe and the RNA from both virus and cells . The Synthesis of Virus-Specific RNA following Infection Kinetics . The uninfected gs- CEF used in these experiments contained no detectable

0 o

.I

N 02 a & 03m i Oq05-

060 z 070 u 08 a 0910 0001

001

0.1

I I

10

00

1000

10,000

C,3 (mole -sec/Ide,) FIG . 6 . Hybridization profiles for PrC-RSV DNA and RNA of normal and PrC-RSV-infected CEF . For experimental details see text and Materials and Methods . Specific activity of 3 H-PrC-RSV DNA : 104 epm/pmole . Concentration of 3 H-PrC-RSV DNA : 0.001 pg/ml ; of cellular RNA : 20 mg/ml . O, 70 S PrCRSV RNA ; 0, RNA from uninfected gs CEF ; 0, RNA from uninfected gs* CEF . The remaining curves represent RNA isolated from gs CEF at various times after infection under rapid transformation conditions . 0, 1 hr ; •, 3 hr ; 0, 6 hr ; A, 10 hr ; 0, 24 hr ; A, 48 hr ; Q, 72 hr ; and •, 96 hr after infection .



EARLY SYNTHESIS OF RSV-SPECIFIC RNA AND DNA amounts of RNA capable of hybridizing with PrC-RSV DNA (Fig . 6 ; Table 1) . By contrast, a line of gs+ cells kindly supplied by Dr . Thomas Graf yielded about 15 viral genome equivalents per cell of RNA that hybridized with about 70% of the DNA probe (Fig. 6, Table 1) . RNA was then isolated at various times from gs- CEF infected at a multiplicity of 4-5 FFU,/cell with PrC-RSV, and each RNA sample was hybridized to 311-labeled PrCRSV DNA with a specific activity of 10 4 epm/pmole . RNA isolated at 1, 3, and 6 hr after infection contained sequences representing the entire viral genome and yielded a Crt1 ;, value of 100 moles-sec/l which corresponded to about 50 viral genome equivalents per cell (Fig . 6, Table 1) . This RNA represented most probably the gcnomes of

TABLE 1 AMOUNT OF VIRUS-SPECIFIED RNA PRESENT IN CEF AT VARIOUS TIMES AFTER INFECTION WITH

PrC-RSV^

Source of RNA

G6„,^ (molesec,/liter)

Virusspecific RNA° (% of total RNA)

70 S PrC-RSV Virion RNA Uninfected CEF

2 X 10 -2

100

>10, 300

0 0 .0067

0 16

100 100 100 78 60 28 17 14

0 .020 0 .020 0 .020 0 .026 0 .033 0 .071 0 .118 0 .143

47 47 47 61 78 167 278 336

gs gs• Infected ge CEF (hr after infection) 1 Hr 3 Hr e Ifr 10 Hr 21 Hr 48 Hr 72 Hr 96 He

Viral genome equivalents/ cello

This table refers to the experiment described in Fig . 6 . 1, Ct„2 values from Fig . 6 .

541

parental virus particles ; since the cells were infected with 200-500 virus particles each (4-5 FFU with a particle : FFU ratio 50-100, Dr . R . Smith, personal communication), this amount of RNA would correspond to 1025 % of that in input virus . Between 6 and 10 hr after infection the amount of hybridizable RNA began to increase at an approximately linear rate . At 96 hr after infection, the latest sample examined, each cell contained on the average 330 viral genome equivalents, that is, just over half of the number present in cells which had been transformed for 2 wk (see above) .

Distribution of virus-specific RNA between nuclei and cytoplasm . Experiments were carried out to determine the intracellular location of virus-specific RNA in cells as infection progressed, and in particular, to determine whether it was restricted to the nucleus at early times . The experiment described in Fig . 6 was therefore repeated, but each cell sample was fractionated into nuclei and cytoplasm prior to hybridization analysis . The hybridization curves were similar to those for whole-cell RNA, and from the Cr t1 ;, values derived from them the virusspecific RNA contents of the nuclear and t lasmic fractions at various times after infection were calculated . The results are shown in Fig . 7 . During the first 48 hr the amount of virus-specific RATA increased

I I I 24

48

72

96

TIME AFTER INFECTION (HRS)

C.Aty for PrC-RSV RNA 100 C,t„z for cellular RNA X 1 ^ Assuming 4 X 10-6 Pg RNA per cell (Leslie, 1955) and 1 .7 X 10-11 µg RNA per viral genome equivalent .

FIG . 7 . Number of viral genome equivalents of RNA in the nuclei and cytoplasm of ge CEF at various times after infection with PrC-RSV . The numbers plotted here were calculated from C .tvs values as described in Table 1 .



SCHINCARTUL AND JOKLTK

5 42

equally in both nucleus and cytoplasm ; from then on that in the nucleus remained virtually constant, while that in the cytoplasm began to increase rapidly . The amount of virus-specific RNA associated with polyribosmnes . In order to deterTABLE 2 AMOUNT OF VIRLS-SPECIFIC RNA ASSOCIATED WITH THE POLYRIBOSOMDS OF CELLS INFECTED WITH PrC-RSV^

Time after infection (hr)

Viral genome equivalents/cell

C1, 12

(mole-sec/liter)

1 3 6 10 24

450 450 450 ISO 110 90

48

72 96

10 10 10 26 43 52 59 71

80

66

For experimental details sec text and legend to Fig . S . The number of viral genome equivalents/ cell was calculated from the C.t, ;, values as described in Table 1 .

mine whether virus-specific RNA was associated with polyribosomes, cytoplasmic extracts were prepared from cells at various times after infection and centrifuged in sucrose density gradients so as to separate out the polyribosomes . A typical profile, obtained from cells 24 hr after infection, is shown in Fig . 8A . The RNA in polyribosomes corresponding to region 11 was pooled, isolated and assayed for virus-specific sequences by hybridization with the same probe as was used in the previously described experiments . The amount of virus-specific . RNA associated with ribosomes increased sharply between 6 and 24 hr after infection and more slowly thereafter (Table 2) . At 96 hr after infection about 20%n of the total cellular virus-specific RNA was associated with polyribosotnes . Size of virus-specific RNA associated with polyribosomes . In order to determine the size of the potyribosome-associated virus-specific RNA, a cytoplasmic extract was prepared from cells 24 hr after infection and centrifuged as illustrated in Fig . 8 . The polyribosome-containing fractions (region II) were 20

A

B

gas 505

E N W

Z a

o.

0

~I

0

I

II I 5

I 10

I

I

I

15

20

25

FRACTION

a

F^I

5

1

1I

10

IS

20

I

25

FRACTION

Polyribosome profiles from CEF infected with PrC-RSV for 24 hr . For experimental details see text and Materials and Methods . (A) Polyribosomes from 5 X 107 cells ; (B) same as (A), but the extract was made 0 .02 M with respect to EDTA prior to centrifugation so as to dissociate polyribosomes . FIG . 8 .

EARLY SYNTHESIS OF RSV-SPECIFIC TINA AND DNA

pooled, the RNA was extracted with SDS and phenol, and fractionated in a sucrose density gradient . Each fraction was then hybridized with 'H-labeled DNA probe . The density gradient profile revealed in this manner does not necessarily reflect with full fidelity the actual distribution of virusspecific RNA sequences (for a detailed analysis, sec Fan and Baltimore, 1973) ; however, it indicates their overall size distribution, and species that it fails to reveal arc very unlikely to be actually present in more than small amounts . As shown in Fig . 9, the predominant hybridizable RNA species that was present sedimented at 35 S ; in addition, virus-specific RNA also sedimented heterogeneously between 10 and 30 S . No RNA species with sedimentation coefficients greater than 35 S were capable of hybridizing with the probe .

0 N

70

60

70S f

35S 4

C

m 50 r = 40 a 0 30 z

w ¢ w a

20 tEDTA

10

00

5

10 15 20

25

FRACTION

The size distribution of polyribosomeassociated virus-specific RNA . Fifty million CEF were used for each density gradient . The fractions designated region 11 in Fig . 8 panels A and B were pooled in each case, made 0 .17 with respect to SDS, and precipitated with 2 volumes of ethanol . The RNA was dissolved in NTE containing 0 .5% SDS, extracted with phenol and then centrifuged in 15-30% (w/v) sucrose in NTE + 0 .1% SDS density gradients (SW 50 .1 rotor, 100 min, 45,000 rpm, 20° ) . After fractionation, 5 sl of 2 .5 M NaCl were added to 50 µl aliquots of each fraction, followed by 5 pl of 'H-labeled PrC-RSV DNA (0 .001 µg, 500 epm) in 0 .3 AI NaCl, 0 .01 M Tris, pH 7 .4, 0 .001 M EDTA . The samples were sealed in capillaries and annealed for 24 hr at 68 ° . Each sample was then diluted 10-fold as described in Materials and Methods and assayed for DNA resistant to Sl-nuclease . Fin. 9 .

543

In order to determine whether the hybridizable RNA was associated with polyribosomes in the specific manner of mRNA, cytoplasmic extracts were prepared from parallel cultures and treated with 0 .02 Al EDTA prior to centrifugation in sucrose density gradients . Such treatment causes disaggregation of polyribosomes and release of ribosomal subunits as shown in Fig . 8B . RNA from region II of that density gradient was also tested for the presence of hybridizable RNA in the manner described above . As shown in Fig . 9, treatment with EDTA reduced the amount of hybridizable RNA to one-third . The virus specific RNA in polyribosomes therefore appears to be present as mRNA . Measurement of the Amount of Virus-Specific DNA in Normal and Infected Cells The reiteration frequency of cell DNA sequences can be estimated by measuring the rate at which their RNA transcripts hybridize with a vast, excess of DNA (for example, Nlelli et al ., 1971 ; Bishop, 1972 ; Gelderman et al ., 1971) . We used the singlestranded DNA transcripts of 70 S PrC-RSV RNA in the same manner in order to measure the number of viral gcnomc copies in the DNA of uninfected and infected CEF . This technique has the advantage that DNADNA, rather than RNA-DNA hybridization is measured . Estimation of the number of viral genome DNA copies per cell requires a reference point ; this is provided by the reassociation kinetics of nonreiterated host sequences which by definition arc present as single copies per genome . The reassociation kinetics of bulk cellular DNA was therefore measured using 1"C-labeled CEF DNA . At the same time, 8H-labeled PrC-RSV DNA was included in the hybridization mixture so as to determine. whether it hybridized more slowly or more rapidly than nonreiterated cellular DNA sequences . As shown in Fig . 10, cellular DNA reassociated in a biphasic manner : about 30 %, presumably representing the highly reiterated sequences, reassociated very rapidly with a C 0 1t12 much less than 10, while the remainder, taken to represent the sequences present as single copies, reassociated very slowly with a Cotta value of



SCHINCARIOL AND JOKLIK

b44 0

Colt ,hy mn s DNA

e1

Uninfec •e d cells and I hr after infection

02

N

n

C3

m 04 r r CS n OF p

~+a

O7~

Oe~ 09



101L

I I 100 1000

0

10 .000

C ot (male-secAifer7

. Reassociation kinetics of CEF DNA and hybridization of I'rC-RSV DNA to uninfected and Fio . 10 infected cellular DNA . "+C-Labeled gs CEF DNA (2000 cpuy'µg) and 'H-PrC-RSV DNA (0 .001 µg/ml, 500 cpm/pmole) were incubated at 68° in.0 .6?l1 NaCl with unlabeled cellular DNA (10 mg/ml) . The extent of reassooiation and hybridization was determined by 81-nuclease digestion, and plotted as a function of C ot (product of initial DNA concentration X time) . •, Normal cellular DNA reassociation ; 0, hybridization of PrC-RSV DNA to normal cellular DNA ; hybridization of PrC-RSV DNA to DNA ex48 (V), 72 (0), and 96 (*) hr, and tracted from cells infected for 1 (O), 3 (0), 0 (A), 10 (A), 24 with calf thymus DNA (J) . TABLE 3 DNA CAPAe1.E Dr ANNFAI .fva wren PrC-RSV DNA IN NORMAL AND INFEOTDD g3 CEF°

AMOUNT OF

Source of DNA

Calf thymus DNA Uninfected gs CEF gR COW infected with PrC-RSV (hr after infection) I Hr 3 Hr 6 Hr 10 Hr 21 Hr 48 Hr

C,t r , 3 (,mole-

sec/liter)

Viral genome copies/ cell

> 10000

<0 .1

1000

1

low

1 1 .2 1 .6 2 2 .8 3 3 3

S40 640 500 360 3'l)

bridized completely to uninfected gs CEF DNA with a C 0t 1 1s value of about 1000, which indicated the presence of a single copy of the viral genome per cell (Fig . 10) . However the probe hybridized more rapidly with the DNA from infected cells (Fig . 10, Table 3) . There was no increase in the reassociation kinetics within 1 hr of infection, but by 3 hr it bad increased significantly, by 10 hr it corresponded to 2 viral genome copies per cell, and at 24, 48, 72, and 96 hr the reassociation kinetics indicated the presence of 3 viral genome copies per cell. i) I SCUSSION

The conditions of infection used in this study were sufficient to cause rapid transformation of chick embryo fibroblasts by PrC-RSV . Transformation was first visible 72 Hr 3 .30 microscopically in a portion of the infected 96 Hr 330 cells at. 24 hr and by 72-96 hr all cells were morphologically altered . These morphologiI The GI I /2 values were taken from Fig . 10 . cal changes were paralleled by an increased The number of viral genome copies per cell was calculated by comparison with the C„ h/ ; value for rate of transport of 2-deoxy-D-glucose, which the DNA of uninfected cells on the assumption has been shown to be one of the earliest that it represented the reassociation kinetics of manifestations of transformation (Martin unique sequence DNA . et at ., 1971 ; Iawai and Hanafusa, 1971) . Viral replication followed a similar time about 1000 . These values are in accord with course . Release of progeny virus into the those in the literature (for example, Britten culture medium was first detected at I8 hr and Kohne, 1968 ; Nciman, 1972) . after infection ; the rate of virus production The 311-labeled PrC-RSV DNA probe by- then increased and reached a steady value by



EARLY SYNTHESIS OF RSV-SPECIFIC RNA AND DNA 48-72 hr . This rapid transformation and replication was achieved by infecting cells in the presence of Polybrene to enhance the efficiency of infection and by infecting cells under conditions assuring the active replication of host cells . The latter condition was obtained by infecting secondary cells shortly after subculture, when they were partially synchronized and about to enter the S phase (Hanafusa, 1969) . Using this system, the synthesis of virusspecific RNA and DNA was measured by determining the kinetics of hybridization of highly labeled PrC-RSV DNA to a vast execss of cellular RNA or DNA . The DNA probe, synthesized in the presence of actinomycin D with purified virions as the source of template and RNA-dependent DNA polyrrierase, and freed from small amounts of double-stranded DNA by chromatography, closely resembled that described by Garapin et al . (1973) in reannealing with 70 S PrC1 RSV RNA with a C,t11, of 2 X 1 molesec/ liter and in protecting 70 S PrC-RSV RNA almost completely when hybridized to it in 10-fold excess . Using this probe we found that (1) gs CEF contained no hybridizable RNA ; (2) a line of gs+ cells contained 16 genome equivalents of viral RNA, which however only represented some 70 of the sequences of PrC-RSV ; and (3) that CEF fully transformed by PrC-RSV contained about 620 of RNA genome viral equivalents per cell . All these values are in agreement with those . reported by Bishop et al. (1973) and Hayward and Hanafusa (1973) . Using this DNA as probe we measured, as a function of time after infection, the total amount of virus-specific RNA per cell as well as the amount in the nucleus and in polyribosomes . The results of several experiments are summarized in Table 4 . Up to 6 hr, cells contained about 40 genome equivalents of RNA, over 80 % of which was located in the cytoplasm . This RNA most probably represented parental viral genomes ; it corresponded to 10-25% of the RNA in the inoculum . This initial phase was followed by a second, extending from 6 to 24 hr, during which the total amount of RNA increased by about 150 %, but all the newly formed RNA was located either in the nucleus or in polyribosomes, With the increase in the former

TABLE

545

4

AMOUNT OF NUCLEIC ACID CAPABLE OF HYARIDIZ1NG WITH PrC-RSV INFECTED WITH

Time after infection (hr)

DNA IN gs PTC-RSVa

CEF

Viral genome equivalents DNA

RNA

Nucleus

Cytoplasm

Total

Poly- Reriho- mainseries der 1 1 7 10 3 1 .2 10 6 1 .6 7 to 26 10 2 25 24 2 .8 32 43 48 3 49 52 72 3 52 59 3 63 71 96 gs CEF, uninfected gs` CEF, uninfected gs- CEF, infected for 14 da y s

25 25 25 25 25 58 169 206

42 42 42

76 100 159 280 340 0 16 620

a The figures presented here are derived from the experiments described in Tables 1-3 and Figs . . 6, 7, and 10 . slightly preceding that in the latter . This appeared to be a period during which viral RNA was transcribed in the nucleus and functioned as a messenger in the cytoplasm . Then followed a third phase (24-96 hr) during which the amount of RNA in the nucleus and in polyribosomes increased by less than 2-fold in 72 hr while the amount of RNA in the cytoplasm increased over 8-fold to an average of 340 genome equivalents per cell ; this is over half of that found in fully transformed cells 14 days after infection . This third phase coincided with the phase of rapid development of virus production (see Fig . 2) and therefore corresponded to the phase when the cell acquired the means to synthesize and release progeny virus at a steadystate level . In this steady state about 20 % of the viral RNA is in the nucleus, 207c is in polyribosomes, and about 607e probably represents RNA in various subviral forms, budding virus, and possibly even readsorbed virus . The amount of virus-specific DNA per cell was measured by determining the rate at which the same DNA probe hybridized to a vast excess of cell DNA . The probe annealed to the DNA of infected gs CEF at the same



516

SCHINCARIOI, AND .IOKIIK

rate as their nonreiterated DNA sequences reannealed, indicating that . the uninfected cells contained one genome copy per cell . This amount increased very soon after infection ; by 3 hr it had increased by 2V ; (a highly significant increase since it represented a reduction in the C u tl r, value from 1000 to 840, both values being based on at least 10 experimental points), by 6 hr by 60`%, and by 24 hr it had almost tripled ; that is, by- that time two viral genome equivalents of DNA had been synthesized on the average in each cell (Table 4) . Very little virus-specific DNA was synthesized after 24 hr (Table 4) . The finding that virusspecific DNA is only synthesized at very early stages of infection agrees with the results of biochemical and biological studies (see Introduction) . These results may be related to sonic recent reports dealing with both avian and mammalian RNA tumor viruses . (a) Salzberg et al . (1973), using a 3T6 ;H-12SV (NILV) system that transformed about as rapidly as that used by us found that the transcription of virus-specific RNA could first be detected at 7 hr, that is, at about the same time as in our system, and that it reached maximal levels at 24 In, much earlier than in our system ; however they did not use cellular RNA in vast excess . (b) Parsons et al . (1973), measuring the ability of pulse-labeled RNA from cells fully transformed with SR-RSV to hybridize to excess AMV DNA transcripts, found that virusspecific RNA was first synthesized in the nucleus and then appeared in the cytoplasm and finally in mature viruses . This progression is in accord with our findings during the, initial phases of infection . (c) The size of intracellular virus-specific RNA has been determined repeatedly (Tsuchida et al ., 1972 ; Leong et al ., 1972 ; Fan and Baltimore, 1973) . Whereas in the undenatured state the size of such RNA is extremely heterogeneous, ranging from 90S to 10S (Leong et al ., 1972 ; Fan and Baltimore, 1973), it is predominantly 35 and 20 S when denatured (Tsuchida et al ., 1972), and in polyrihosomes it is mostly 35 S (Fan and Baltimore, 1973) . Our findings indicate that the predominant virus-specific RNA species in polyribosomes

sedimented with 3.5 S, bulb that smaller material was also present. . Our findings that uninfected gs CEF contain one viral genome DNA copy, that this amount begins to increase immediately after infection, before any virus-specific RNA is formed, and that within 24 hr the equivalent of two genome copies of virus-specific DNA is synthesized are in accord with several published reports . Three methods have been used to detect such virus-specific DNA : (1) annealing highly labeled viral 70 S RNA to a vast excess of host DNA (Baluda and Nayak, 1970 ; Rosenthal et al ., 1971 ; Baluda, 1972 ; Harel et al ., 1972 ; Neiman, 1973) ; (2) measuring the effect of cell DNA on the reassociation kinetics of double-stranded virus-specific DNA sequences (Varmus et al ., 1972, 1973a, h) ; and (3) measuring the hybridization kinetics of single-stranded virusspecific DNA sequences in the presence of vast DNA excess (Ruprecht et al ., 1973), the method also used by us . Most of these reports agree that gs+ as well as gs CEF contain a very low number (less than 4) viral DNA genome copies [except Varmus et al . (1972), who found 12-15 copies] ; and that after infection the formation of virus-specific DNA is detectable within 4 hr and essentially complete by 16 hr . Varmus el al . (1973b) also found that virus-specific DNA was integrated into the host gcnome during the interval from 16 to 64 hr ; we made no attempt to determine when the two newly formed DNA copies that we detected by 24 hr after infection were integrated .

ACKNOWLEDGMENTS We would like to thank Drs . Thomas Oral and Ralph Smith for stimulating discussions . This work was supported by Research Grant No . AT08909 and Health Sciences Advancement Award No . 5 S04 RR 00148 from the United States Public Health Service . ,A .L .S . was a National Cancer Institute of Canada Research Fellow . REFERENCES The role of deoxyribonucleic acid in the synthesis of Rous sarcoma virus . Virology 22, 462-468 . BADER, J . P . (1966) . Metabolic requirements for infection by Rous sarcoma virus . 1 . The transient requirement for DNA synthesis_ Virology 29, 444-451 . BADER, J . P . (1964) .

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Lion suitable for use in DNA reassociation experiments . Biochirn . Biophys . Acta 240, 522-531 . TEMIN, TI . M . (1963) . The effectt of actinomycin D on growth of Rous sarcoma virus inn vitro . Virology 20, 577-582 . TEMIN, H . M . (1964) . The participation of DNA in Rous sarcoma virus production . Virology 23, 480-494 . TIMIN, H . M . (1967) . Studies on carcinogenesis by avian sarcoma viruses . V . Requirement for new DNA synthesis and for cell division . J . Cell Physiol . 69, 53-61 . TEMIN, H . M . (1968) . Carcinogenesis by avian sarcoma viruses . Cancer Res . 28, 1835--1838 . TUMIN, H . M . (1971) . Mechanism of cell transformation by RNA tumor viruses . Annu . Rev. Microhiol . 25, 609-6648. TEMIN, H . M ., and MIZUTANI, S . (1970) . TINAdependent DNA polymerase in virions of Rous sarcoma virus . Nature (London) 226, 1211-1213 . TsUCHIDA, N ., ROnIN, M . S ., and GREEN, M . (1972) . Viral RNA subunits in cells transformed by RNA tumor viruses . Science 176, 1418-1420 . VARMUS, IT . E ., WFISs, R . A., FRIES, It . R ., LEVINsoN, W,, and BISHoP, J . M . (1972) . Detection of avian tumor virus-specific nucleotide sequences in avian cell DNAs . Pros. . Nat . Acad. Sci . U .S . 69, 20-24 . VARMUS, If . E ., BISHOP, J . M ., and VOGT, P . K . (1973a) . Appearance of virus-specific DNA in mammalian cells following transformation by Rous sarcoma virus . J . Mot . Biol . 74, 613-626 . VARMUS, H . E ., BISHOP, J . M ., and VOGT, P . K . (1973b) . Synthesis and integration of Rous sarcoma virus-specific DNA in permissive and non-permissive hosts . Proc . 1973 ICN-UCLA Symp . Virus Res . (in press) . VIGIER, P., and GOLDA, A . (1964) . Effects of actinomycin D and of mitomycin C on the development of Rous sarcoma virus . Virology 23, 511519 .