VIROLOGY

70,

(1976)

65-79

Infectivity ASHLEY

T. HAASE,

BETTY

of Visna Virus DNA L. TRAYNOR,

AND

PETER

E. VENTURA

Infectious Disease Section, Veterans Administration Hospital, 4150 Clement Street, San Francisco, California 94121, and Departments of Medicine and Microbiology, University of California, San Francisco. California AND

DAVID NIAID,

National

Institutes

W. ALLING of Health,

Bethesda,

Maryland

20014

Accepted October 22, I975 Deoxyribonucleic acid isolated from cells infected with the RNA containing slow virus visna confers on uninfected cells the capacity to synthesize new virus. The cytopathic agent isolated in these experiments has the biological and biochemical properties of visna virus, and the active material is DNA by several criteria, the most important of which is the loss of infectivity with prior treatment of the DNA with deoxyribonuclease. The efficiency of infection with DNA is markedly enhanced by presenting DNA to the cell as a calcium DNA phosphate precipitate. Because of the lytic nature of visna virus infection, the infectivity of visna DNA can be assayed quantitatively by plaque formation. Our major findings with this infectivity assay include the following: (1) Duplex DNA is infectious while denatured DNA is not. (2) At least one complete copy of proviral DNA is integrated in the host genome. (3) The minimal infective size of duplex DNA corresponds to a transcript of one subunit of viral RNA. (4) However, the concentration dependence of infectivity is two hit over the size range of lo-30 million molecular weight. These findings suggest that the unique genetic information of the viral genome is distributed over more than one subunit and that the proviral DNA transcripts are unlinked in the cell. The usefulness of the DNA infectivity assay and the implications of the visna provirus for persistent viral infections are discussed. INTRODUCTION

In the present work we investigated the infectivity of DNA isolated from sheep choroid plexus (SCP)’ cells acutely infected with the RNA containing slow virus visna, a virus closely related to RNA tumor viruses in morphologic and biochemical properties (Haase, 1975). We did so for two reasons. First, prior investigation by nu-

Temin’s proposal (1971) that the life cycle of RNA tumor viruses involves the transfer of genetic information from the RNA of the virus to a DNA intermediate in the cell is convincingly supported by the demonstration of infectious DNA in cells transformed by avian tumor viruses (Hill and Hillova, 1972a,b). Cooper and Temin (1974) recently extended these findings by developing a quantitative assay for infectivity of DNA by end-point dilution. With this assay they determined that the minimal size of DNA required for infectivity is about 6 million and that a single molecule of DNA can initiate infection in the avian system.

I Abbreviations used: Cot, product of DNA concentration and time; CPE, cytopathic effect; ds DNA, double-stranded DNA; ss DNA, singlestranded DNA; EDTA, ethylene dinitrilo tetraacetate; G, gauge; HBS, Hepes buffered saline; MM, maintenance medium; MW, molecular weight; PBS, phosphate buffered saline; RSV, Rous sarcoma virus; SCP, sheep choroid plexus; SDS, sodium dodecyl sulfate; SSC, standard saline-citrate (0.15 M NaCl, 0.015 M Na citrate. 65

Copyright 0 1976 by Academic Press, Inc. All rights

of reproduction

in any form reserved.

66

HAASE

ET AL.

subpassaged at 1:4 split ratio, and used at the fifth subpassage. These procedures, media, and serum employed, and the source, propagation, and plaque assay of visna virus have been detailed previously (Haase and Levinson, 1973; and Haase and Baringer, 1974). Labeling and extraction of DNA. DNA was extracted from confluent monolayers of SCP cells. For infectious DNA, cultures representing 1 to 3 x lOR cells were inoculated with 3 ml of MM containing 3 PFU of virus/cell. After adsorption of virus for 2 hr, additional MM (15 ml) was added which contained 1 &i/ml of [3HldTr if DNA was to be labeled. After incubation for 24 hr, the medium was discarded, the cell sheet was washed with phosphate buffered saline (Dulbecco and Vogt, 1954), and the cells were removed with 0.05% trypsin/0.02% EDTA. After centrifugation, the pellet of cells was washed once more with PBS and resuspended at 5 to 10 x 10” cells/ml of 0.1 M NaCI, 0.05 M TrisHCl (pH 8.1), 0.01 M Na,EDTA. SDS was added to a final concentration of 0.4% and mixed by rolling into the cell suspension. When the lysate cleared, Pronase MATERIALS AND METHODS was added to 500 pglml and the lysate was incubated at 37” for 2-8 hr. The concentraReagents and chemicals. Phenol (Mallinkrodt) was distilled prior to use and tion of SDS was then increased to 1% and equilibrated with 0.02 M Tris-HCl (pH the mixture was deproteinized by gently rolling (40-60 times/min) with an equal 8.1), 0.01 M Na,-EDTA; DEAE-dextran, average MW 5 x lo”, purchased from volume of phenol at room temperature for Pharmacia, was dissolved in distilled wa- 15 min. The phases were separated by centrifugation and the upper aqueous phase ter at 10 mg/ml, and sterilized by filtration. Sodium dodecyl sulfate (SDS) was was removed with a pipet having an interrecrystallized from ethanol. Pronase was nal diameter of 3.7 mm. The extraction purchased from Calbiochem, dissolved at was repeated, and DNA was precipitated 10 mg/ml in 0.02 M Tris-HCI (pH 8.1), and from the aqueous phase by addition of 2 vol autodigested for 2 hr at 37”. RNAse and of cold ethanol. After storage at -20” for DNAse I were the electrophoretically pure more than 2 hr, DNA was recovered by washed once with 90% grade from Worthington. To inactivate re- centrifugation, sidual deoxyribonuclease, RNAse (2 mg/ ethanol, and allowed to dry. The DNA was (pH ml in acetate buffer 0.02 M, pH 5) was held then dissolved in 0.02 M Tris-HCl 7.4), 0.001 M Na,-EDTA in one-third the at 90” for 10 min. DNAse was alkylated original volume. This process normally (Zimmerman and Sandeen, 1966) to inactitakes several hours at 37” and can be hasvate traces of residual ribonuclease. [3H]Thymidine (20 Ci/mmole) was ob- tened by gently pipeting the gel-like aggregates of DNA through a wide mouthed tained from New England Nuclear. pipet (internal diameter, 5 mm). This unCells and uirus. Sheep choroid plexus questionably shears DNA, but molecular (SCP) cells were established by explantaweights (MW) in excess of 10” were nevertion from tissues of 6-month-old animals,

cleic acid hybridization techniques (Haase and Varmus, 1973) had indicated that visna virus replication proceeds through a DNA intermediate or provirus. Because of the potential implications of a provirus for understanding virus persistence in slow disease, it seemed important to unequivocally establish that the transfer of genetic information from virion RNA to DNA is complete by demonstrating that the DNA is sufficient to complete the replicative cycle. Secondly, it seemed reasonable to expect that advantage could be taken of the lytic nature of visna virus growth in SCP cells to assess infectivity by plaque formation. In this paper we show that DNA isolated from SCP cells infected with visna virus will elicit virus synthesis in uninfected cells and that the infectivity of DNA can be assessed by plaque assay. The efficiency of the method, when calcium is used to increase the uptake of DNA, and the quantitative aspects of the plaque assay provide the basis for studies of the relationship of the size and conformation of DNA to infectivity,

INFECTIOUS

theless obtained routinely. RNAse was added at 100 pglml to the DNA solution to degrade contaminating RNA, to prevent irreversible aggregation, the enzyme was warmed to 37” and mixed rapidly with the DNA solution. The digestion by RNAse was at 37” for 3-12 hr. DNA was again deproteinized with phenol, and residual phenol and oligonucleotides were removed by successive dialysis in 1 x SSC and 0.1 x SSC. DNA solutions were stored at 4”. The results of this report were derived from 14 preparations of DNA, each representing a separate SCP cell line. Ten preparations were from infected cultures and four were from uninfected cells. Yields of DNA from 1 to 3 x lOacells varied from 2-4 mg. DNA prepared by the method described above had OD,,,/OD,,, ratios of 1.84-1.96 and, on melting, exhibited the hyperchromic shifts in excess of 25% expected of DNA. Protein, determined by the method of Lowry et al. (1951) with bovine serum albumin as a standard, was less than 1%. Specific activity of labeled DNA ranged from 15,00-25,00 cpm/pg. Salmon sperm DNA was obtained commercially (Calbiochem) and further purified by sequential digestion with Pronase and RNAse, phenol extraction, and dialysis as noted above. Adenovirus type 5 DNA was prepared from infected KB cell cultures labeled with [3HldTr as described by Tibbetts et al. (1973). Network DNA. A portion of several DNA preparations was used for network formation (Britten and Smith, 1969). DNA (450 pg in 2 ml) in polycarbonate tubes was denatured by heating to 100” for 3 min and reannealed to a Cot of 10 (68, 1 hr, 0.6 M NaCl). At this low Cot, repeated sequences scattered throughout the genome reassociate and cross-link strands of DNA into networks. These were recovered by brief sedimentation (40,000 rpm, 15 min, lo”, 65 rotor). The supernate was removed with a Pasteur pipet and the network DNA was resuspended in 1 ml of 0.1 x SSC. From 75-83% of DNA was recovered in networks. Rate zonal sedimentation of DNA. Estimates of MW were obtained by sedimentation of DNA preparations at 40,000 rpm

VISNA

DNA

67

(15”, Beckman SW41 rotor) through linear 5-20% sucrose gradients containing 0.1 M NaCl, 0.05 M Tris-HCl (pH 7.4), 0.001 M Na,-EDTA (neutral gradient) or 1 M NaCl, 0.6 N NaOH, 0.01 M Na,-EDTA (alkaline gradients). The average MW of native DNA was estimated from its sedimentation velocity in neutral gradients relative to a DNA of known MW (Studier, 1965; Levin and Hutchinson, 1973). 3H-labeled A-DNA, generously provided by Dr. D. Dussoix, was used as the reference DNA. The MW of single-stranded (ss) DNA was determined after denaturation by relative rate of sedimentation in neutral or alkaline gradients in siliconized cellulose nitrate tubes. DNA was denatured with NaOH (0.6 N, 15 min, 37”) and layered directly on alkaline gradients, or neutralized with 3 N HCl/l M Tris (neutral gradients). Samples were loaded with a l-ml plastic pipet with an internal diameter of 2.7 mm. Duplex DNA of defined size class. DNA preparations had duplex MWs of 50 to over 100 million after extraction, depending on rapidity of lysis of cells with SDS, and other factors which were not identified. More highly sheared DNA was obtained by repeated passage of 3H-labeled DNA through hypodermic needles (Pyeritz et al., 1972) or by sonication. DNA in 0.1 x SSC was sheared in 150-~1 portions by drawing the solution into a l-ml plastic syringe and then expelling it with steady pressure through a needle. Passage of the DNA 10 times through a l-in. hypodermic needle of 20 gauge resulted in fragments with an average MW of 25 million. DNA with an average MW of 10 million was obtained by ejecting the DNA five times through a 26gauge needle (0.5-in. long). Subsequent passage 10 times through a 30-gauge needle (0.5-in. long) further reduced the DNA to a MW of 5 million. Lower MW DNA was obtained by sonication. DNA in 500 ~1 of Hepes buffered saline (HBS) was sonicated with the medium probe of a Bronson sonicator at 50% output for 30 set at 2”. Sedimentation profiles of DNA obtained by these procedures, illustrated in Fig. 1, were essentially superimposable with each preparation. The figure also

68

HAASE

FRACTION

NUMBER

1. Sedimentation of sheared DNA. Infected 3H-labeled SCP DNA was sheared and centrifuged in parallel sucrose density gradients for 3 hr as described in Methods. The position of h reference DNA is indicated by an arrow. The direction of sedimentation is from right to left, DNA prior to shearing (&@I; DNA sheared with a 26-G needle (n---a); DNA sheared with a 20-G needle (U--U); sonicated DNA (A--A). Unsheared DNA (O--O) was also precipitated with calcium, added to SCP cells as in the infectivity assay, and then recovered through a wide-bore pipet. After release from the precipitate by addition of 100 m&f Na,EDTA, the DNA solution was diluted and centrifuged. FIG.

shows that DNA manipulations involved in adding DNA to cells does not further reduce the size of the DNA. The procedures may introduce ss regions at fragment termini (Pyeritz et al., 1972) but sheared preparations manifested the same low degree (~5%) of sensitivity to S, single-strand specific nuclease as DNA that had not been purposefully sheared. Solid phase S, n&ease and chromatography on hydroxylapatite. S, nuclease was prepared from crude amylase powder (Sigma) by precipitation with ammonium sulfate and chromatography on DEAE-cellulose (Sutton, 1971). Coupling of the enzyme to cyanogen bromide activated sepharose was performed by previous procedures (Haase and Pereira, 1972) except that the ratio of enzyme:sepharose was 1 mg:l ml of packed gel. Digestions were carried out at 50” for 2 hr with 200 ~1 of insolubilized S, enzyme to

ET AL.

1 ml of reaction mixture. The solid phase enzyme degrades ~2% native A-DNA but digests single-stranded (ss) DNA to residual material of ~5% of the total DNA. Assessment of DNA conformation by SI, buffers employed, and chromatography on hydroxylapatite following previous protocols (Haase et al., 1974a). Determination of DNA adsorption. Uptake of DNA by cells was measured as described by Graham and van der Eb (1973). In experiments with DEAE-dextran, procedures delineated by Hill and Hillova (1972b) were followed. Assay of infectivity of DNA with calcium. DNA infectivity assays were performed on monolayers of SCP cells in 6-cm plastic petri dishes (Falcon). Cells at the fifth subpassage were seeded at 5 x lo” cells/dish and were used at 72-96 hr, when the cultures had reached 90% of confluent cell densities. DNA was diluted in HBS, usually to 10 to 15 Fg/ml, and precipitated by addition of 2 M CaCIB to 125 mmoles. Mixing was affected by gently rolling three to five times, and all measurements and manipulations of DNA were done in plastic pipets of 2.7 mm i.d. These pipets can be conveniently made by cutting the constriction of a l-ml plastic pipet off at the 0.9 mark with a scalpel heated in an alcohol flame; the rough ends may be polished and flared by holding the end of the pipet briefly in the flame. DNA precipitates, formed in 30 min at room temperature, were allowed to adsorb to cells (0.5 ml/dish) for an additional 30 min. Then 5 ml of MM was added to each dish and the cultures were incubated for 4 hr at 37”. The DNA precipitate was removed by suction, and the cell sheets were washed twice with PBS and overlaid with plaque medium. At 14 days, the cell sheet was fixed with 10% formalin and plaques were enumerated after staining with 1% crystal violet/20% ethanol-water. RESULTS

Uptake of DNA. We have shown previously (Haase and Varmus, 1973) that visna virus specific nucleic acids hybridize to SCP cell DNA after infection. In view of the relatively large number of copies of

INFECTIOUS

proviral DNA demonstrated in these experiments (about 20/diploid genome) we were surprized to find that DNA from infected SCP cells was only rarely infectious, using techniques successfully employed by Hill and Hillova (1972a,b) with avian tumor viruses. It seemed likely that the difficulty was at least in part a consequence of the limited uptake of DNA by SCP cells treated with DEAE-dextran; as Fig. 2 demonstrates, only about 3% of the exogenous DNA is taken up at the saturation plateau. The report (Graham and van der Eb, 1973) of increased uptake and infectivity of adenovirus DNA precipitated by calcium phosphate therefore prompted us to investigate uptake of SCP DNA employing the calcium method. We also found that the DNA was taken up more efficiently, some five- to tenfold, compared with DEAE-dextran. Adsorption was evident by 1 hr and reached a plateau by 3-5 hr (Fig. 2); DNA of lower MW was taken up somewhat more readily, but by 4 hr only modest differences of 5-10% were obtained for DNA of low and high MW. The greatest uptake occurred when DNA was denatured and neutralized prior to addition to cells. At 4 hr, only 20-25% of the DNA is resistant to DNAse. Because this degree of adsorption is considerably less than the 70% uptake of adenovirus DNA by KB cells (Graham and van der Eb, 19731, we repeated their experiments with results similar to theirs (data not shown). By contrast, only 25-30% of adenovirus DNA was adsorbed by SCP cells suggesting that the capacity of cells to take up exogenous DNA varies considerably between species. Optimal pH and concentrations of calcium phosphate were found to be identical to that described by Graham and van der Eb (1973). Their conditions were adopted in subsequent experiments.

VISNA

69

DNA

! HOURS

FIG. 2. Uptake of DNA by SCP cells. 3H-labeled SCP DNA was diluted in HBS (to 10 pg/ml) precipitated with calcium, and added to confluent monolayers of SCP cells. At designated times, adsorption was determined as described in Methods. Each point represents the average of quadruplicate dishes for each of three DNA preparations. Unsheared DNA (0-O); DNA sheared with a 20-G (W---m) or a 26-G needle (A-A); sonicated DNA (A-A); denatured DNA (O-O); DNA uptake with DEAEdextran (-----).

unsheared infected cell DNA was precipitated with calcium and added to 30 petri dishes. When all of the cultures manifested maximum CPE, the medium was collected, pooled, and stored at -70”. That CPE was due to production of visna virus was shown by the following tests: (11Virus with the buoyant density of 1.16 g/cm3, electron micrographic appearance, and distinctive polypeptide pattern of visna virus was recovered by carrying the medium through the procedures for purification of RNA slow viruses (Haase and Baringer, 1974). (21 In neutralization tests, summarized in Table 1, normal sheep serum had no significant effect on the cytopathic agent recovered from cultures infected with DNA, but sheep serum with neutralizing activity against visna virus reduced the virus titer by approximately 90%. These biological and biochemical properties identify visna virus as the cytopathic agent recovered from the cultures exposed Infectivity of DNA, recovery, and identification of Virus. There was a parallel in- to DNA. Lack of endogenous virus. We previcrease in infectivity associated with enhanced uptake of DNA. We routinely ob- ously reported (Haase and Varmus, 1973) served stellate cells and polykaryocytes, that DNA from normal SCP cells does not cytopathic effects (CPE) typical of visna hybridize to visna virus RNA, or measuravirus, in SCP monolayers 7-10 days after a bly accelerate the reassociation of highly single exposure to DNA from infected labeled virus specific DNA synthesized in cells. To identify visna virus positively, vitro by detergent activated virions. Under

70

HAASE TABLE 1 NEUTRALIZATION OF VIRUS ISOLATED IN DNA INFECTIVITY EXPERIMENT” Titer

Virus alone Virus + normal sheep serum Virus + sheep antivisna serum

(log PFU/ml) 6.96 7.03 6.10

0 Medium (0.5 ml) from SCP cultures infected with DNA was collected at maximum CPE, mixed with an equal volume of serum and allowed to stand at room temperature for 1 hr. Residual virus titer was determined by plaque assay. The antivisna serum (from an infected animal) was generously provided by Dr. H. Thormar and had previously been shown to have a reciprocal dilution titer of 512 (Thormar and Helgadottir, 1966). Controls included an original isolate of visna virus (from Dr. Thormar) and an unrelated virus (vesicular stomatitis virus) which were assayed concurrently. The antivisna serum inhibited visna virus to a degree comparable to the virus isolate but did not neutralize vesicular stomatitis virus.

the conditions employed, we would have been able to detect as little as one-half copy of virus specific DNA per diploid genome. In this section we show that transfection is not a result of activation of a visna-like agent from ostensibly normal SCP cells. Uninfected SCP cultures do not spontaneously exhibit CPE or undergo morphological transformations, and medium from such cultures is not infectious. Since a defective virus would not be detected in this way, we examined cells and media for virus by other methods, again with negative results (Haase and Baringer, unpublished work). Media, concentrated by banding in sucrose gradients or pelleting, and cells, are free of virus by electron microscopy and lack RNA-dependent DNA polymerase activity assayed under conditions where virus infected cultures are positive. Furthermore, medium from uninfected SCP cells labelled with 13Hluridine does not give rise to material banding at the virus density in sucrose gradients. Exposure of SCP cells to DNA from uninfected SCP cells does not activate virus by the foregoing criteria, whether the DNA is applied alone, in DEAE-dextran, or as a calcium phosphate precipitate. This result was obtained with DNA extracted

ET AL

from four lines of uninfected SCP cells derived from different animals. Moreover, DNA that should be indifferent with respect to virus genetic information, such as salmon sperm DNA, is not infectious. These experiments argue against the presence of an endogenous visna-like agent in SCP cells and its activation by DNA. Plaque Assay of DNA Infectivity. The consistency of infection by DNA with the calcium method, and the fact that cytopathic effect occurs pari passu with production of visna virus, suggested that it should be possible to quantitate infectivity by plaque assay. This objective was realized as shown in Fig. 3. Within 2 weeks of exposure to DNA, plaques characteristic of visna virus developed and could be enumerated. These plaques have a central clear area with a fuzzy border and range in diameter from 2-4 mm. The macroscopic features of the plaque reflect the mode of formation. Virus production results in the formation of multinucleated cells. With succeeding growth cycles this effect progresses outward and cells at the periphery are recruited into the syncytium. By 2 weeks the cells at the center have undergone degeneration and give rise to the relatively clear center of the plaque. Polykaryocytes at the periphery take up less stain than surrounding normal cells and give rise to the fuzzy lighter staining border. These features of the mature plaque are illustrated in the middle and lower portions of Fig. 3. Nature of the active material. Our basic observations have been presented as though infection were known to result from DNA. To substantiate this assumption we compared the properties of the active material in the DNA preparation with visna virus. Although virus probably would not have survived the DNA extraction procedure, we verified this by showing that the infectivity of visna virus is sharply reduced by exposure of virus to neutralizing antiserum or to trypsin (Table 21, but the infectivity of DNA is not affected by these treatments. More importantly, infectivity is abolished if the DNA is subjected to digestion with deoxyribonuclease prior to addition to cells (Table 31,

INFECTIOUS

VISNA

DNA

71

but this enzyme does not destroy the infectivity of intact virus. The role of secondary structure. Having established the infectivity of DNA and conditions for quantitative assay, we next investigated the relationship of secondary structure of DNA to infectivity. Infectious DNA is double stranded by two criteria: DNA used in transfection elutes entirely as duplex material from hydroxylapatite; and digestion by S, single-strand specific nuclease degrades less than 2% of native DNA to acid solubility. Infectivity is lost when DNA is denatured by treatment with base or by melting (Table 4). This result, reproducible with six preparations of DNA with the calcium method, and with two preparations of DNA with DEAE-dextran, leaves little doubt that ss DNA is ordinarily not infectious. Svoboda (19731, Levy et al. (1974), and Cooper and Temin (19741, also found that alkali denaturation of DNA from Rous sar(RSV) transformed cells coma virus largely destroys the infectivity of DNA, but others have reported (Hillova et al., 1974) that alkali does not abolish infectivity. In an attempt to resolve these apparent discrepancies, we investigated factors that might be responsible for the failure of ss DNA to transmit infection. The first and obvious problem, poor uptake of denatured DNA, has been dealt with already; in uptake experiments, denatured DNA is adsorbed to SCP cells with greater efficiency than duplex DNA. A second trivial explanation could be idiosyncratic toxicity of ss DNA in SCP cells. This is not the case, since after 4 hr of exposure to ss DNA or ds DNA the same proportion (80-90%) of trypsinzed cells are viable (judged by exclusion of trypsin blue). A third consideration is the size of ss DNA. One can imagine that an extensive

FIG. 3. Top: Plaques from DNA. Magnification, 2x. Infected cell DNA was sheared with a 26-G

needle, adjusted to 10 pg/ml with HBS, precipitated with calcium, and added to SCP cultures in petri dishes. Plaques were allowed to develop for 2 weeks under 0.3% agarose. The cell sheet was fixed, stained, and photographed. Middle: Plaque morphology. Clear central area of plaque consisting of lysed cells. Magnification, 30x. Bottom: Inset showing polykaryocytes at plaque periphery (indicated by arrows). Magnification, 250x.

72

HAASE TABLE

2

THE EFFECT OF NEUTRALIZING SERUM AND PRONASE ON THE INFECTIVITY OF VISNA VIRUS AND DNA” Composition

of test mixutre

Virus Virus + pronase Virus + antiserum DNA DNA + pronase DNA + virus + pronase DNA + virus + antiserum

Average number of plaques per dish 32 1

6 13

16

ET AL

nary step to determine whether a complete copy of visna proviral DNA is covalently integrated into host DNA. The loss of infectivity with denaturation was unfortunate, since it precluded experiments with alkaline gradients to see if infective DNA were inseparable from host DNA. The problem of linkage can nevertheless be pursued from another point of view which provides additional insight into the role of secondary structure in infectivity.

15 TABLE

20

(I Incubation mixtures of 100 ~1 were prepared in 0.1~ SSC containing 30-40 PFU of visna virus, 50 Fg of sheared DNA (26 Gl, or both, in the reconstruction controls. Pronase (sterilized by filtration) was added to mixtures so labeled; these were incubated at 37” for 30 min and diluted with HBS, and infectivity was determined by conventional plaque or transfection assay. In the neutralization test, normal sheep serum or sheep serum with neutralizing activity was heated at 70” for 10 min to destroy nuclease activity (Kaiser and Hogness, 19601, sterilized by filtration, and added to designated mixtures of DNA and virus at a final concentration of 10%. At the end of 1 hr at room temperature, mixtures were diluted and assayed as noted above. Infectivity was determined on four dishes.

number of breaks in ss DNA would reduce the chance that a cell would receive a complete copy of a provirus. There is no doubt that the extensive purification procedures employed to isolate DNA and exposure to nucleases in the cytoplasm after lysis with SDS provide ample opportunities for introduction of single-strand scissions. Indeed, sedimentation analysis of several DNA preparations indicated that our DNA preparations contain on the average two to five breaks per chain. However, the average MW of ss DNA, which was infectious in the native state, was over 13 million. These ss pieces are long enough to accommodate a complete copy of the RNA genome with a MW of about 10 million. Therefore, it seems improbable that the loss of infectivity with denaturation of DNA can be attributed solely to fragmentation of proviral DNA. Network DNA. The investigation of infectivity of ss DNA was in part a prelimi-

3

THE EFFECT OF DEOXYRIBONUCLEASE ON TRANSFECTION” DNAse concentration mixture (fig/ml)

in

Average number of plaques per dish 0 0 0

10 1 lo- ’ 10-Z

6

104

11 15 15

10-J 0

fl Mixtures of 100 pl of sheared DNA were made 10 mM in MgCl,. Alkylated DNAse was added to concentrations specified above. After incubation at 37” for 30 min, the mixture was diluted with HBS and the residual infectivity was determined by transfection assay on four dishes. The alkylated DNAse did not reduce infectivity of intact virus and did not degrade trace amounts of S’P-labeled virion RNA, as measured by electrophoretic profiles of RNA or conversion to acid solubility. TABLE

4

STRUCTURE AND INFECTIVITY OF DNAR Treatment

Average number of plaques per dish

None

56 100"

16 14 0

NaOH

0

” Infectivity of DNA was determined by plaque assay on quadruplicate dishes before and after denaturation. DNA (50 Fg in 100 ~1) was heated to designated temperatures in 0.1x SSC for 10 min. After briefly chilling in an ice bath, the transfection assay was carried out. Alkali denaturation was carried out with similar amounts and volumes of DNA. The DNA solution was made 0.3 N in NaOH held at 37” for 15 min, neutralized with Tris-HCl, diluted in HBS, and assayed for infectivity.

INFECTIOUS

Repeated sequences are widely scattered throughout the genome of mammalian cells (Britten et al., 1969). When DNA is melted into its constituent strands and allowed to reanneal for brief periods, association of repeated sequences links separate strands of DNA into a network that can be isolated by brief centrifugation. Varmus et al. (1973) have shown that this procedure does not trap significant amounts of exogenous DNA. Hence, the occurrence of virusspecific sequences in network DNA can be taken reliably to indicate covalent linkage to the host genome. We have employed this technique in nucleic acid hybridization work to show that visna virus-specific sequences are stably associated with SCP DNA after infection (Haase and Varmus, 1973). The description of network formation is intended to convey the impression of a structure comprised of relatively long ss chains of DNA linked and interwoven by shorter segments of duplex DNA, representing the interspersed repetitive sequences. Table 5 summarizes the results of the structural analysis of networks, which indicate that this description is reasonably accurate. As would be expected from the foregoing discussion, the short regions of duplex DNA are sufficient to cause a substantial portion of the network to elute from hydroxylapatite as ds DNA. However, the more stringent S, assay indicates that only lo-15% of the network DNA represents duplex DNA. These double stranded regions of repetitive DNA are approximately 200-300 nucleotides in length, the single-stranded chains about 1800 nucleotides. Although we have not investigated the situation rigorously, the estimates of the size of interspersed repetitive sequences by our methods agree quite well with other workers (Davidson, 1973). The outcome of infectivity assays are also given in Table 5. Network DNA is infectious, albeit at a much reduced level. Since isolated double-stranded regions were not infectious, we infer that ss DNA can be infectious if complemented by some degree of secondary structure which functions in an unknown manner in the process of transfection. We presume that under

VISNA

73

DNA TABLE CHARACTERISTICS

5

OF NETWORK

Property Fraction ds on hydroxylapatite Fraction ds by S, nuclease Estimated length of ds DNA Estimated length of ss DNA Infectivity of ds DNA Infectivity of network Infectivity of ds portion of network DNA ___--__-___

DNA -Result

---

0.40 0.11 220 nucleotides 1800 nucleotides 0.34 pglplaque 15 pg/plaque Not infectious (5 &dish) ___--- -.

” The secondary structure of networks from three DNA preparations was determined by batch chromatography on hydroxylapatite and by digestion with ss specific S, nuclease (cf. Methods). The fraction listed as ds has been normalized by reference to ss DNA and ds “H-labeled h-DNA standards assayed concurrently (Haase, et. al., 1974a). Estimates of size were obtained as follows: A reaction mixture of 1 ml containing 100 pg of “H-labeled network DNA in S, buffer was digested with 200 ~1 of S,-sepharose; subsequently S, enzyme was removed by centrifugation and the digest was applied to a sucrose gradient. The size of S, resistant cores was estimated by relative sedimentation (SW41 rotor, 7 hr. 40K, 15”). ss chain length was estimated by denaturation of network DNA and sedimentation in neutral sucrose gradients. The marker DNA for ss estimates was hbacteriophage DNA; for the ds marker, sonicated sH-labeled SCP DNA which sedimented at 10 S was used. Recovery from gradient was nearly 100%. Infectivity of intact networks was assessed by transfection assay employing 5-10 pg of network/dish. Twelve dishes were employed in the assay and the results, expressed in FG DNA/plaque, are compared with infectivity of native DNA (see text). Infectivity of ds regions was assayed at comparable concentrations after digestion by S, nuclease. In a typical experiment, a reaction mixture of 1 ml containing 400 pg of network was incubated with solid phase S, enzyme as noted above. At the end of the reaction the enzyme was removed by centrifugation and presumptive ds DNA and digestion products were precipitated with 2 vol of cold ethanol. The precipitate was collected by centrifugation, dissolved in 0.01 M phosphate buffer pH 6.8, and adsorbed to 1 ml of hydroxylapatite. The ds DNA eluting in 0.4 M phosphate buffer was exhaustively dialyzed against 0.1 x SSC to remove phosphate and oligonucleotides. The dialysate was concentrated by precipitation with ethanol, recovered by centrifugation, redissolved in 0.1x SSC, and assayed for infectivity. Recovery (computed from specific activity of network DNA) in the S, digestion and hydroxylapatite steps was 6080%. Final recoveries were 30-40s

74

HAASE

our conditions of denaturation, this degree of secondary structure is lost, an assumption that may not hold in the experiments of Hill and Hillova (1972a; cf. Discussion). Thus, there are two points to be emphasized from the structural analysis: First, infection by network DNA establishes that at least one complete copy of the visna genome is integrated in host DNA after infection. Second, the ss DNA is of such low infectivity with current methods as to restrict further analysis of transfection to duplex DNA. Distribution of unique nucleotide sequences in RNA C-type genomes. The RNA genome of visna virus, like that of RNA tumor viruses, is a segmented structure comprised of two to four subunits, each with a MW of about 3 million (Haase et al., 1974a; Friedmann et al., 1974). The content of unique genetic information may be equivalent to just one (polyploid genome) or to all of the subunits (haploid genome), as first suggested by Vogt (1973). Attempts to directly determine the complexity of the RNAs of C-type viruses have lead to conflicting conclusions. Data obtained in studies of the kinetics of hybridization suggest, with one exception (Baluda et al., 19741, that the genomes of avian tumor viruses (Taylor et al., 19741, murine tumor viruses (Fan and Paskind, 19741, and visna virus (Haase et al., 1974a) carry largely unique sequences on each subunit. On the other hand, when the complexity of RNA from avian sarcoma viruses is assessed by analysis of the oligonucleotides generated by limited digest with T, RNAse, the chemical complexity is equivalent to that expected of a polyploid genome (Billeter et al., 1974; Beemon et al., 1974; Quade et al., 1974). These results have recently been generalized to other Ctype viruses with an estimated chemical complexity equivalent to a MW of 3.6 to 4.0 x 10” for the visna genome (Beemon et al., in preparation). The content and distribution of unique nucleotides in virion RNA obviously will be reflected in proviral DNA. It is important to consider this in the design and interpretation of experiments to be described below, which are directed at defining the relationship of infectivity to the

ET AL.

size and concentration of DNA. Table 6 summarizes the simplest models. If the subunits are identical (polyploid genome) one or all of the subunits may be transcribed and integrated at the same site (model IA) or at different sites (model IB) without affecting the predictions. It should be possible to shear duplex DNA to about 6 million average MW (allowing for adjacent host sequences) without loss of infectivity, and one DNA molecule of this or greater size should be able to initiate the infectious process. Below 6 million, infectivity should become multihit. Alternatively, some unique sequences might be arrayed on a second or third subunit, and the proviral DNA replicas might either be clustered or scattered (model IA, IB). If the unique sequences are integrated in tandem, (model IIA), DNA larger than 20 million might display one hit kinetics; from 20 million to about 6 million infectivity should display two or three hit kinetics; and below 6 million the kinetics would be greater than three, the precise number determined by the number of fragments produced by shearing. By contrast, unlinked proviral replicas (model IIB) would display two to three hit kinetics over the same size range. These considerations provide the rationale for experiments which examine the dependence of infectivity on size and concentration of DNA. Size dependence and specific infectivity. The relationship between the average MW of duplex DNA and specific infectivity is presented in Fig. 4. In accord with the observations of Levy et al. (1974), DNA of very high MW functions much less efficiently in transfection than DNA in the MW range of lo-30 million. Maximum infectivity occurs with DNA of about 10 million MW with a specific infectivity of 0.1 to 0.3 pg/plaque. This is comparable to that reported for avian tumor viruses (Cooper and Temin, 1974). Infectivity falls sharply below 10 million and is undetectable in sonicated DNA. The data points fall on a straight line in this region, and give an estimate of 7 million for the average MW at which 50% of maximal infectivity has been lost. Concentration dependence. The rela-

(B) Unlinked

Identical subunits complexity equivalent 3 million (A) Linked

host nuclear

DNA sequences;

y

w...

A

;+A -

I? ,-...

. ..--tii...

... .

. ..Y

. ..%I

..

duplex

...

7 w’...

*

...j-w

f . ..I-

IN

A

,q 1 L,...

Y;...

h

PROVIRAL

DNA total unique nucleotides

B. c XI... T ~ -c . . . . .Tz ,-.‘a, I . ..-kt.,+ ... ... B 1 . .. -

%‘I...

+

6

SEQUENCES

TABLE

Representation

UNIQUE

proviral

. ..Y

. ..-

OF

= A;

DNA=

2-3 hit

1 hit

1 hit

1 hit

20 million

DNA $wnr

2-3 hit

2-3 hit

1 hit

1 hit

DNA between 6 and 20 million

Kinetics

by shearing

B and C additional

quences. ( t ) shear points; the maximum distance between two shear points indicates DNA of average MW >20 million obtained needle, DNA of MW >6 to 10 million indicated by minimum distance between two points. See text for explanation of models.

a (,.,--l

(B) Unlinked

(II) Unique sequences on more than one subunit complexity >3 million (A) Linked

(I)

Designation

Model

DISTRIBUTION

with

sea 20-G

unique

>3 hit

>3 hit

Multihit

Multihit

DNA smaller than 6 million

:

2

76

HAASE

ET AL

lated to cooperative interaction between subunits having different genetic information, but is related to some other aspect of the infectious process, such as uptake or processing of the DNA. For example, Graham and van der Eb (1973) found that the infectivity of the adenovirus DNA was not linearly related to the amount of DNA added, but rather to the third power of DNA concentration. In this case, the role of more than one molecule in infection FIG. 4. Size of DNA and infectivity. Infected SCP DNA was prepared and sheared to the average MW shown on the abscissa, as described in Methods. Infectivity, determined at concentrations of 4.5 and 6.3 pg/dish, is expressed as the average number of plaques (at each concentration/fig of DNA.

tionship between the amount of DNA added and the plaques which result is displayed in Fig. 5. The plaque count increases in a distinctly nonlinear manner up to 6-7 ygldish. Above this level the cells are apparently saturated, as no further increase in plaque count occurs, and fewer plaques are found when more than 7.5 pg of DNA is added per dish, attributable to increasing cell toxicity (data not shown). When the data in Fig. 5 are replotted on a logarithmic scale (Fig. 6) a linear relationship obtains between plaque counts and DNA added up to the saturation plateau. The number of plaques (p) is therefore’ related to some power (K) of the DNA (D) added; or p = c(D)” where c is a proportionality constant (Graham and van der Eb, 1973) and K corresponds to the hit number; that is, the number of DNA molecules which participate in the infectious regression process. Using a weighted method to estimate hit number (Alling, 19711, K was found to be 1.88 + 0.18 (SD) for DNA with an average MW of 10 million, andK = 2.0 2 0.10 (SD) for DNA with average MW of 25 million. The results evidently conform to those expected for unlinked proviral DNA molecules, carrying the unique nucleotide sequences of virion RNA on different subunits (model IIB). It might be argued, however, that the requirement for two molecules of DNA in infection is not re-

ug DNA DISH

FIG. 5. Concentration

dependence of infectivity. DNA sheared to an average MW of 10 (O-0) or 25 million (m-W) was adjusted to a concentration to give the number of wgidish noted on the abscissa, precipitated with calcium and added to quadruplicate dishes. The data points represent average number of plaques/dish which resulted at each concentration A linear dose response is indicated by the dotted line, using the number of plaques obtained at 6.3 +g of DNA/dish as reference.

w

4 6810 ~g DNA;DISH

FIG. 6. Concentration dependence The data in Fig. 5 have been replotted mic scale.

of infectivity. on a logarith-

INFECTIOUS VISNA DNA could be assumed by another kind of DNA (salmon sperm). Under these conditions p = c(D) (D + S) K-’ where S is the amount of salmon sperm DNA added as carrier (Graham and van der Eb, 1973a). Accordingly addition of carrier DNA to infectious DNA should enhance infectivity. We, therefore, determined the infectivity of increasing amounts of infectious DNA to which salmon sperm had been added to keep the total DNA concentration constant (6.3 kg/dish). In contrast to the results obtained in the adenovirus system, we found that the addition of carrier DNA did not increase infectivity. Indeed, the concentration dependence was nearly identical to that observed with infected SCP DNA to which no carrier had been added. To determine if there was any specificity to the carrier DNA, we repeated the experiment with SCP DNA from uninfected cells from the same cell line and passage from which the infected SCP DNA had been isolated. Again, identical or lower infectivity was observed when carrier DNA was added. Although we cannot exclude a mechanism which requires the participation of specific host cell DNA sequences, the outcome of these experiments is consistent with the notion that two viral DNA molecules are needed for infection. DISCUSSION The provirus hypothesis and slow viruses. The experiments reported in this paper provide incontrovertible evidence that the replication of the RNA containing slow virus visna involves a DNA intermediate, and are in complete agreement with the tenets of the provirus hypothesis formulated by Temin (1971). After synchronous infection of SCP cells by visna virus, all of the genetic information in virion RNA is transferred to a DNA provirus in the cell, and this event occurs during eclipse, prior to the onset of synthesis of progeny RNA or virus. This event involves addition of new genetic information, since SCP cells lack virus specific sequences and transfection does not activate an endogenous virus related to visna. The transfer of information is complete, since DNA from

77

the infected cell is able to complete the replicative cycle when it is added to uninfected cells. Finally, at least one complete set of proviral DNA sequences is covalently linked to the host genome because network DNA is infectious. We have speculated elsewhere (Haase, 1975) as to the role of the visna provirus in slow infections. Visna virus causes an inflammatory and demyelinative disease of the central nervous system of sheep in which t,he natural history of disease may span a period of as long as 8 years (Thormar and Palsson, 1967). During this time, virus production and attendant tissue destruction proceed at a slow tempo in the face of a clear-cut immunological and inflammatory response to the virus by the host animal. We believe that the provirus provides an attractive explanation for persistence of visna virus on this novel time scale, by allowing some virus to always remain inside the cell where it is inaccessible to the defensive measures instituted by its host. Comments on the assay. The overall efflciency of transfection with visna DNA is about 10” molecules of viral DNA/plaque, computed from the number of copies of proviral DNA cell estimated by DNADNA reassociation kinetics (Haase and Varmus, 1973). This is comparable to the infectivity of the DNAs of SV40 and herpes simplex virus, and about loo-fold greater than adenovirus DNA (Graham et al., 1973). The sensitivity of the method and its quantitative aspects make it a particularly valuable adjunct to nucleic acid hydribization techniques, since the transfection assay measures the amount of complete duplex proviral DNA, while the hybridization methods are limited because of the variable representation of the viral genome in the probes employed. Therefore, the transfection assay should be useful in compiling a detailed account of the kinetics of synthesis and distribution of proviral DNA in cells infected with visna virus. Secondary structure, size, and concentration dependence. Transfection is ordinarily obtained with ds DNA but not with ss DNA. The infectivity of network DNA indicates that the requirement for duplex

78

HAASE

structure is not absolute and can be satisfied by a relatively modest degree of organized structure in DNA. To reconcile this conclusion with the report of successful transfection in chicken cells by DNA isolated from alkaline gradients (Hill and Hillova, 1972a) we postulate that their preparations contained residual ds DNA. This explanation is supported by the welldocumented observation of aggregated structures which persist in denatured DNA (Simpson et al., 1973). The minimal infective size of visna DNA is about 6 million, in agreement with findings in the avian tumor virus system (Hill and Hillova, 1974a; Cooper and Temin, 19741, and corresponding to the expected MW of a ds DNA copy of one subunit of virion RNA. However, in contrast to the avian system, the concentration dependence of infectivity is not one but two hit. The simplest model which can accommodate these data, and the recent lower estimates of the complexity of the visna genome 3.6 to 4 x 10” MW (Beemon et al., in preparation), requires that the unique genetic information of the virus be distributed over at least two of the RNA subunits in the virion. In the cell, these subunits are transcribed into unlinked proviral replicas, and therefore, the information for successful infection must be reconstituted by a cooperative interaction between subunits. Although two hit kinetics might also result from interactions at the level of RNAs of two closely related virus populations, this is unlikely to be the case, since plaque formation with visna virus increases as a linear function of virus concentration (Harter, 1969). ACKNOWLEDGMENTS We thank Drs. J. Bishop and H. E. Varmus for helpful discussions, and Ms. Angie Papastefan for preparation of the manuscript. This work is supported by grants from the NIH (NS11782) and from the American Cancer Society (VC-120A), and is project MRIS 3367 within the Veterans Administration. REFERENCES ALLING, D. W. (1971). Estimation of hit number. Biometrics 27, 605-613. BALUDA, M., SHOYAB, M., MARKHAM, P., EVANS, R.,

ET AL. and DROHAN, N. (1974). Cold Spring Harbor Symp. Quant. Biol. 39, 869-874. BEEMON, K., DUESBERG, P., and VOGT, P. (1974). Evidence for crossing-over between avian tumor viruses based on analysis of viral RNAs. Proc. Nat. Acad. Sci. USA 71, 4254-4258. BILLETER, M. A., PARSONS, J. T., and COFFIN, J. M. (1974). The nucleotide sequence complexity of avian tumor virus RNA. Proc. Nat. Acad. Sci. USA 71, 3560-3564. BRITTEN, R. J., and SMITH, J. (1969). Carnegie Inst. Yearbook, 376-378. COOPER, G. M., and TEMIN, H. M. (1974). Infectious Rous sarcoma virus and reticuloendotheliosis virus DNAs. J. Vi&. 14, 1132-1141. DAVIDSON, E. H., HOUGH, B. R., AMENSON, C. S., and BRITTEN, R. J. (1973). General interspersion of repetitive with nonrepetitive sequence elements of the DNA of Xenopus. J. Mol. Biol. 77, l23. DULBECCO, R., and VOGT, M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99, 167-182. FAN, H., and PASKIND, M. (1974). Masurement of the sequence complexity of cloned Moloney murine leukemia virus 60 to 70s RNA: Evidence for a haploid genome. J. Viral. 14, 421-429. FRIEDMANN, A., COWARD, J. E., HARTER, D. H., LIPSET, J. S., and MORGAN,C. (1974). Electronmicoscopic studies of visna virus ribonucleic acid. J. Gen. Viral. 25, 93-104. GRAHAM, F., and VAN DER EB, A. (1973). A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 52, 456-467. GRAHAM, F., VELDHUISEN, G., and WILKIE, N. M. (1973). Infectious herpes-virus DNA. Nature New Biol. 245, 256-266. HAASE, A. T. (1975). The slow infection caused by visna virus. Curr. Topics Microbial. lmmun. 72, 101-156. HAASE, A. T., and BARINGER, J. R. (1974). The structural polypeptides of RNA slow viruses. Virology 57, 238-250. HAASE, A. T., GARAPIN, A. L., FARAS, A. J., TAYLOR, J. M., and BISHOP, J. M. (1974b). A comparison of the high molecular weight RNAs of visna virus and Rous sarcoma virus. Virology 57, 259-270. HAASE, A. T., GARAPIN, A. L., FARAS, A. J., VARMUS, H. E., and BISHOP, J. M. (1974b). Characterization of the nucleic acid product of the visna virus RNA dependent DNA polymerase. Virology 57, 251-258. HAASE, A. T., and LEVINSON, W. (1973). Inhibition of RNA slow viruses by thiosemicarbazones. Bioch. Biophys. Res. Commun. 51, 875-880. HAASE, A. T., and PEREIRA, H. G. (1972). The purification of adenovirus neutralizing antibody: Adenovirus type 5 Hexon immunoadsorbent. J. Zmmun. 108, 633-636.

I....._

,.ml--.”

lNI’LC;‘I’lUUS

HAASE, A. T., and VARMUS, H. E. (1973). Demonstration of a DNA provirus in the lytic growth of visna virus. Nature New Biol. 245, 237-239. HARTER, D. H. (1969). Observations on the plaque assay of visna virus. J. Gen. Viral. IGB) 5, 157160. HILLOVA, J., GOUBIN, G., COULAUD, D., and HILL, M. (1974). Nuclear localization and covalent linkage of infective virus DNA to chromosomal DNA of non-producer Rous sarcoma cells. J. Gen. Viral. 23, 237-245. HILL, M., and HILLOVA, J. (1972a). Virus recovery in chicken cells treated with Rous sarcoma cell DNA. Nature New Biol. 237, 30-35. HILL, M., and HILLOVA, J. (1972b). Recovery of the temperature-sensitive mutant of Rous sarcoma virus from chicken cells exposed to DNA extracted from hamster cells transformed by the mutant. Virology 49, 309-313. HILL, M., and HILLOVA, J. (1974). RNA and DNA forms of the genetic material of C-type viruses and the integrated state of the DNA form in the cellular chromosomes. Biochem. Biophys. Acta 355, 748. KAISER, A. D., and HOGNESS, D. S. (1960). The transformation ofEscherichia coli with deoxyribonucleic acid isolated from bacteriophage lambdadg. J. Mol. Biol. 2, 392-415. KAO, P. C., REGAN, J. D., and VOLKIN, E. (1973). Fate of homologous and heterologous DNAs after incorporation into human skin tibroblasts. Biothem. Biophys. Acta 324, 1-13. LEVIN, D., and HUTCHINSON, F. (1973). Neutral sucrose sedimentation of very large DNA from Bacillus subtilis. J. Mol. Biol. 75, 455-478. P. M., and VARMUS, H. E. LEVY, J. A., KAZAN, (1974). The importance of DNA size for successful transfection of chicken embryo fibroblasts. Virology 61, 297-302. LOWRY, 0. H., R~SEBROUGH, J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265275. PYERITZ, R. E., SCHLEGEL, R. A., and THOMAS, C. A. JR. (1972). Hydrodynamic shear breakage of DNA may produce single-chained terminals. Biochem. Biophys. Acta 272, 504-509.

VISNA

DNA

79

K., SMITH, R. E., and NICHOLS, J. J. (1974). Evidence for common nucleotide sequences in the RNA subunits comprising Rous sarcoma virus 70s RNA. Virology 61, 287-291. SIMPSON, J. R., NAGLE, W. A., BICK, M. D., and BELLI, J. A. (1973). Molecular nature of mammalian cell DNA in alkaline sucrose gradients. Proc. Nat. Acad. Sci. USA 70, 3660-3664. STUDIER, F. W. (1965). Sedimentation studies of the size and shape of DNA. J. Mol. Biol. 11,373-390. SUTTON, W. D. (1971). A crude nuclease preparation suitable for use in DNA reassociation experiments. Biochem. Biophys. Acta 240, 522-531. SVOBODA, J., HLOZANECK, I., MACH, O., MICHLOVA, A., REMAN, J., and URBANKOVA, M. (1973). Transfection of chicken fibroblasts with single exposure to DNA from virogenic mammalian cells. J. Gen. Virol. 21, 47-55. TAYLOR, J. M., VARMUS, H. E., FARAS, A. J., LEVIK SON, W. E., and BISHOP, J. M. (1974). Evidence for non-repetitive subunits in the genome of Rous sarcoma virus. J. Mol. Biol. 84, 217-221. TEMIN, H. M. (1971). Mechanism of cell transformation of RNA tumor viruses. Ann. Rev. Microbial. 25, 609-648. THORMAR, H., and HELGADOTTIR, H. (1966). A comparison of visna and maedi viruses II. Res. Vet. Sci. 6, 456-465. THOMAR, H., and PALSSON, P. A. (1967). Visna and maedi - two slow infections of sheep and their etiological agents. Persp. Virol. 5, 291-308. TIBBETTS, C., JOHANSSON, K., and PHILIPSON, L. (1973). Hydroxylapatite chromatography and formamide denaturation of adenovirus DNA. J. Virol. 12, 218-225. VARMUS, H. E., VOGT, P. K., and BISHOP, J. M. (1973). Integration of DNA specific for Rous sarcoma virus after infection of permissive and nonpermissive hosts. Proc. Nat. Acad. Sci. USA 70, 3067-3071. VOGT, P. K. (1973). The genome of avian RNA tumor viruses. In “Possible Episomes in Eukaryotes,” Int. Proc. 4th Lepetit. Collog. (Silvestri, L. G., ed.). North-Holland, Amsterdam. ZIMMERMAN, S. B., and SANDEEN, G. (1966). The ribonuclease activity of crystallized pancreatic deoxyribonuclease. Anal. Biochem. 14, 269-273.

QUADE,