RD-114 virus: Analysis of viral gene sequences in feline and human cells by DNA-DNA reassociation kinetics and RNA-DNA hybridization

VIROLOGY

66, 484-495 (1973)

RD-114 Virus: Human

Analysis Cells

of Viral

by DNA-DNA RNA-DNA

KEI

FUJINAGA,

Gene

Sequences

Reassociation

in Feline Kinetics

and

and

Hybridization

ANNE RANKIN, HIROKO YAMAZAKI, KENJI JANICE BRAGDON, AND MAURICE GREEN

SEKIKAWA,

Institute for Molecular Virology, Saint Louis University School of Medicine, 3681 Park Avenue, Saint Louis, Missouri 63110; and Department of Molecular Biology, Sl, Wl7, Chuo-ku, Sapporo Medical College Cancer Research Institute, Sapporo, Japan Accepted

August 27, 1973

RD-114 virus is a C-type virus found growing in a human rhabdomyosarcoma cell line (RD) after inoculation into a fetal cat. To investigate the host species of origin of RD-114 virus and its relationship to other oncornaviruses, we performed molecular hybridization and reassociation kinetic experiments using three radioactive probesthe double stranded (ds) and single stranded (ss) DNA products of the viral RNAdirected DNA polymerase and the viral 60-70 S [3H]RNA genome. DNA was synthesized by the endogenous DNA polymerase reaction of purified RD.114 virions disrupted with detergent and dsDNA was isolated by hydroxyapatite chromatography. The most efficiently transcribed dsDNA segment represented approximately 50yo or more of dsDNA transcripts and had a sequence complexity of about 0.8 X lo6 daltons. The kinetics of reassociation of this rapidly reassociating dsDNA segment, in the presence of cellular DNA was used to detect viral gene sequences in human and feline cells. The RD.114 cell line which chronically produces RD-114 virus contains 20-30 copies of this DNA sequence while normal feline tissues contain loo-200 copies. Less than one copy of DNA sequences homologous to this RD-114 viral dsDNA segment were detected in uninfected human, hamster, and rat cells. RD-114 viral [zH]ssDNA did not hybridize significantly with RNA of several strains of feline and murine leukemia and sarcoma viruses. RD-114 viral 60-70s [3H]RNA hybridized to the same extent and with similar kinetics to RD-114 cell and feline tissue DNA indicating that similar numbers of copies of most, if not all, of RD-114 viral genome, are present in normal feline cells as well as in RD.114 virus-producing human cells. No hybridization was detected between RD-114 viral 6&7OS RNA and the DNA of the parental human RD cell line, or the DNA of uninfected human, hamster or rat cells. These measurements indicate that RD-114 virus incorporates less than 10% human cell DNA sequences even after replicating in human cells for many generations. We conclude that the major portion of the RD.114 virus genome is of feline derivation, that it shares base sequences with a repetitive class of normal feline cell DNA, and that it is genetically distinct from known isolates of feline leukemia-sarcoma viruses. These properties suggest that RD-114 virus is an endogenous virus of normal feline cells. INTRODUCTION A large number of viruses with the chemical and physical properties of oncornaviruses (Melnick, 1973) have been isolated from

mammalian species, including primates, during the past few years. These isolates were shown to grow readily in cells of other mammalian species, often in human cells. 484

Copyright All rights

@ 1973 by Academic Press, of reproduction in any form

Inc. reserved.

RD-114

VIRUS

GENE

The ease with which oncornaviruses can cross species lines, at least in cultured cells, complicates the identification of the host species of origin. The problem becomes acute in view of the current search for RNA tumor viruses in humans. Most new virus isolates have been readily identified with regard to host species, e.g., mouse, hamster, rat, or primate, by the immunological properties of the two distinguishing proteins of the oncornavirus particle, the major internal protein containing the species-specific antigenie determinant (gs-1), and the RNAdirected DNA polymerase. Since the corresponding proteins of the putative human oncornavirus family have not been identified, the possible human derivation of a new oncornavirus is judged by negative criteria, i.e., the lack of relatedness to previously known viruses. The above considerations point to an unresolved problem-a human isolate not identified with regard to host species by these immunological criteria could be either a huma.n oncornavirus or a representative of a new family of mammalian oncornaviruses with an immunologically distinct gs antigen and DNA polymerase. This dilemma is illustrated by RD-114 virus, a type C oncornavirus t#hat was discovered in a human rhabdomyosarcoma cell line cultivated in a fetal cat (McAllister et al. 1971). Studies utilizing immunological reagents showed that the group-specific antigenic determinant and the Rn’A-directed DNA polymerase of RD-114 virus were distinct from those of previously characterized type C viruses (McAllister et al., 1972; Oroszlan et al., 1972; Scolnick et al., 1972; Long et al., 1973). These findings raised the possibility that RD-114 virus was of human origin, although as cautioned by McAllister et al. (1971), the occurrence of more than one family of feline oncornaviruses was possible. To answer questions concerning the origin of RD-114 virus and its relatedness to other oncornavirus genomes, we have utilized molecular hybridization and kinetic measurements of the rate of reassociation of nucleic acid molecules, procedures capable of detecting small numbers of viral DNA sequences in eukaryotic cells (Britten and Kohne, 1968; Gelb et al., 1971). Several

SEQUENCES

485

types of radioactive probes containing viral gene sequences are available for such studies. The RNA-directed DNA polymerase of RNA tumor virions copies portions of the viral 60-705 RNA in vitro to form both single-stranded (ss) and double-stranded (ds) DNA segments. The dsDNA product of avian and murine RNA tumor virions consists of at least two populations of molecules, rapidly reassociating and slowly reassociating DNA (Varmus et al., 1972; Gelb et al., 1971). These dsDNA segments, containing sequences derived from a portion of the viral genome, have been used as radioactive probes to analyze the number of viral DNA copies in normal and transformed cells by reassociation kinetics. Such studies have shown that uninfected murine and avian cells share base sequences with their native oncornavirus genome. Another approach which uses viral 60-70s RNA as a probe has the additional advantage that, unlike the dsDNA fragments generated by the viral polymerase, viral RNA contains sequences from the entire viral genome. Thus quantitative studies on hybridization with viral RNA may provide information on the fraction of the viral genome represented in cell DNA; however the difficulty in preparing RNA of sufficiently high specihc activity so that conditions of DNA excess are achieved, and the uncertain kinetics of RNA-DNA hybridization limit precise estimates of the number of viral gene copies in cells (Neiman, 1972). Studies on the kinetics and extent of hybridization of labeled viral RNA with cell DNA in excess have shown that normal avian cells contain a small number of copies of most if not all of the genome of Rousassociated virus, RAV-0, the endogenous leukosis virus of chickens (Neiman, 1973). The base sequence homology thus far established between murine and avian cells and their species-specific oncornavirus suggest the evolutionary origin of this class of viruses from the host cell genome and provides a means to determine by molecular hybridization the host species of origin of new oncornaviruses. We report here some properties of the viral DNA sequences synthesized by the RNA-directed DNA polymerase of the RD-

486

FUJISAGA

BY’ ill,

and Grac(b, 1967), Fischer rat embryo (IT1853), RD-114 culls, and a cat thgmus cell line producing Rickard FSV(FLV) (Salzbcrg and Grcpn, 1972), were grown in Eagle’s minimal essential medium supplemented wi-ith 10 % calf serum, trypsinized, and washed wiDh phosphat’e-buffered saline. DNA was isolated by a modification of the procedure of Berns and Thomas, 1965; Petterson and Sambrook, 1973). Cells were lysed in lo-20 volumes of buffer A (0.1 36 EDTA, 0.01 M Tris.HCl (pH 8.1), 0.01 M KaCl) with 0.5 % SDS and digested overnight with 100 pg/ml of Pronase at. 37”. After extraction with phenol plus chloroforn-isoamyl alcohol (24: I), 2 volumes of ethanol were added to the aqueous phase. The precipitate was dissolved in buffer A and treated with RNase A (50 pg/ml, 2 hr at 37”), digested with Pronase (100 pg/ml, 2 hr at 37”), and extracted with equal volumes of phenol and chloroform-isoamyl alcohol. After precipitation with ethanol, t’he DNA was dissolved in 0.1 X SSC (SSC = MATERIALS AND METHODS 0.15 A1 SaC1-0.015 M Na3 citrate) and Virus and viral RNA. RD-114 virus was dialyzed against 0.1 X SSC. DNA was grown in RD-114 cells, provided initially by isolated from feline tissues obtained from cats after homogenization in Dr. Robert McAllister, and purified as healthy buffer A by the procedure described above. described (Green et al., 1970). Additional preparations of RD-114 virus were kindly Alkali treatment (0.2 N YaOH, 37”, 2 hr) was performed to remove contamination supplied by Pfizer Laboratories, Maywood, with RNA when necessary. Sew Jersey, under the auspices of the Virus Preparation of RD-114 viral [3H]dsDNA. Cancer Program of the National Cancer Institute. Viral 60-70s RNA was isolated by Viral [3H]Dr\‘A was synthesized by the zonal centrifugation of detergent-lysed viendogenous DNA polymerase reaction of rions (Tsuchida et al., 1972). Viral 60-70s disrupted RD-114 virions as described for [3H]RNA was prepared from [3H]uridineother viruses (Green et al., 1970; Fujinaga labeled RD-114 virus as follows. To each of et uZ., 1970; Fujinaga and Green, 1971) exfive monolayers (Pfizer bottles) of RD-114 cept that 0.1 mM dTTP (4.52 Ci/mmole, cells containing 200 ml of Eagle’s minimal unless otherwise specified) was used with a essential medium supplemented with 10% longer incubation period (18 hr, 37”) to calf serum was added 10 mCi of [3H]uridine permit accumulation of dsDNA. The re(45 Ci/mmole), 2 mCi of [3H]adenosine action was terminated by addit.ion of EDTA (18.3 Ci/mmole), and 2 mCi of [3H]cytidine to a final concentration of 10 mM. The DNA (26 Ci/mmole). After incubation for 47 hr, product was purified by the SDS-phenol the culture supernatants were used to isolate method (Rokutanda et al., 1970), followed by labeled RD-114 virus. Because of the small precipit’ation with 2 volumes of ethanol in amounts of RD-114 viral 60-705 [3H]RNA the presence of carrier E. coli tRNA (150 that was isolated, its specific act#ivity could I*g/ml), and treatment with RNase A (100 not be determined. pg/ml) in 0.007 M phosphate buffer, pH 6.8 Isolation of cell DNA. Human KB, P3- (PB) for 60 min at 37”. Viral dsDNA was HRIK, a human lymphoblastic cell line isolated on hydroxyapatite by elutions with derived from a Burkitt lymphoma (Hinuma 0.4 M PB after removal of ssDNA with 0.14 114 virion, and the use of three molecular probes, dsDNA, ssDNA, and viral 60-70s RNA, to study the relationship between RD114 viral genomc and several cell and viral genomes. While we did not detect any homology between RD-114 virus and known feline oncornaviruses, wc found that a major portion, if not the entire RD-114 viral genome, is represented with similar frequency in normal feline tissue and in virusproducing, human RD-114 cells. Moreover about 4 % of the RD-114 viral genome is represented with a frequency of 100-200 in normal feline cells. RD-114 viral sequences were not detected in RD cells, the parent of RD-114 virus producing cells, or in uninfected human, rat, or hamster cells. These findings suggest that RD-114 virus is an endogenous feline oncornavirus. The recent isolation from feline cells of oncornaviruses closely related to RD-114 virus supports this view (Fischinger et al., 1973; Livingston and Todaro, 1973; Sarma et al., 1973).

RD-114

VIRUS

GENE

M PB containing 0.4% SDS. After the addition of E. coZi tRNA (150 pg/ml) as carrier, DNA was precipitated with 2 volumes of ethanol and dissolved in 0.01 X SSC and dialyzed aginst 0.01 X SSC. Preparation of [3H]ssDNA. Viral [3H]ssDNA was synthesized by purified A&IV polymerase (PCII) in the presence of actinomycin D (200 pg/ml) using RD-114 viral 60-70s RNA (10 rg/ml) as a template with an incubation period of 2 hr at 37” as described previously (Grandgenett et al., 1973), and purified by the SDS-phenol method (Rokutanda et al., 1970). Reassociation of viral &DNA. [3H]Labeled RD-114 viral dsDNA plus cell DNA in 0.12 M PB or 0.01 X SSC were sonicated for 10 min at full power in the Raytheon DF101 sonic oscillator and denatured at 100” for 10 min. As analyzed by zonal centrifugation in sucrose density gradients and analytical centrifugation, adenovirus DNA, is broken reproducibly to pieces 800-900 nucleotides in length. After adjusting to 0.40 M PB, the solution was incubated at 67”. Aliquots were removed at various times and resolved into ssDNA and dsDNA fractions by batch elution on hydroxyapatite (Tsuchida et al., 1972). The radioactivity of each fraction was measured by precipitation with 0.6 M trichloroacetic acid and counting in a liquid scintillation counter. RESULTS

Kinetics of Reassociation of RD-114 dsDNA

SEQUENCES

487 Reassaciatian

I

I

lo-5

lo"

of RD-114 dsDNA

I

1

10-3

10-T

lo"

Cat(mole-sec,IIter)

FIG. 1. Reassociation of RD-114 viral [aHIdsDNA with different specific activities. Viral [3H]DNA synthesized by the virion DNA polymerase was isolated and purified as described in the text. After sonication and denaturation, reassociation of denatured DNA (500-3000 cpm per 200 ~1) was performed at 67” in 0.40 M phosphate buffer (pH 6.8). The fraction of reassociated DNA fragments was measured by hydroxyapatite chromatography as described in Materials and Methods. The [3H]dTTP precursor was 0.50 mM and 0.981 Ci/mmole (O), 0.50 mM and 0.199 Ci/mmole (A), 0.10 mM and 4.52 Ci/mmole (a), and 0.10 mM and 0.977 Ci/mmole (A). The concentration of [HDNA in the reassociation reaction was calculated from the specific activity of [3H]dTTP, assuming equimolar amount of the four deoxyribonucleotides.

Viral

Four preparations of viral DNA were synthesized using two different concentrations of [sH]dTTP (0.1 and 0.5 mM) of four different specific activities. Since the amount of viral DNA synthesized was too small to measure by absorbance or diphenylamine analysis, the concentration of viral DNA was calculated from the specific activity of precursor dTTP, assuming an equimolar content of the four deoxyribonucleotides. The time course of reassociation of the four DNA products were almost identical (Fig. I), illustrating the reproducibility of the kinds and amounts of viral DNA sequences that were synthesized in vitro. To characterize the population of dsDNA

fragments, the kinetics of reassociation were further analyzed. The rate of reassociation of denatured dsDNA fragments, containing equimolar amounts of each DNA sequence, follows second-order kinetics (B&ten and Kohne, 1968) and may be expressed as follows : dC,,/dt

= -h-c:,

(1)

then? c,,/c,

= l/(1

+ kCd)

(2) where C,, is the concentration of ssDNA in moles per liter at time t, Co is the initial concentration of ssDNA, and k is the reassociation rate constant. Equation (2) is used for the standard Cot plot (see Fig. 1). A

488

FUJINAGA

simple rearrangement (3) : Cd&s

ET AL.

of Eq. (2) gives Eq.

0.5

0.4 =

kcot

admo 7 I 3zP~ DNA f urMx4ed adeno 2 DNA

(3)

where cds is the concentration of dsDNA. A plot of Gds/Gss against cot provides a simple and precise estimate of the C&2, i.e., concentration X time required for 50% reassociation of ssDNA; k is the slope of the line and l/lc is Cotljz. A linear relationship is expected for unique, i.e., nonrepetitive DNA sequences, as illustrated by the reassociation of sheared, denatured adenovirus 2 DNA in Fig. 2. In contrast, the reassociation of RD-114 viral L3H]DNA in Fig. 2 shows complex kinetics, reflecting unequal amounts of different base sequences in the population of RD-114 viral dsDNA segments. The complex reassociation kinetics observed with RD-114 viral dsDNA can be reconstructed by reassociating adenovirus DNA in the presence of a second adenovirus DNA molecule containing partially homologous DNA sequences. As shown in Fig. 3, the reassociation of adenovirus 7 [32P]DNA gives

”

0.3 -

U

1.0

2.0

3.0

cot xl0+h?-sec/lIter)

FIG. 3. Reassociation of adenovirus 7 [32P]DNA in the presence of unlabeled homologous and heterologous viral DNA. Adenovirus 7 [32P]DNA plus unlabeled adenovirus DNA was sonicated and denatured. Reassociation of denatured viral DNA fragments (1.0 ml) was performed at 67” in 0.40 M PB. The fraction of reassociated adeno 7 [32P]DNA fragments was determined by hydroxyapatite chromatography. (0) Adenovirus 7 [32P]DNA (11.6 rig/ml, 735 cpm/ml). (0) Adenovirus 7 [32P]DNA (11.6 rig/ml, 735 cpm/ml) + adenovirus 7 DNA (6.28 rig/ml) (A) Adenovirus 7 LS2P]DNA (11.6 rig/ml, 735 cpm/ml) + adenovirus 2 DNA (97.5 rig/ml).

the linear plot expected for nonrepetitive DNA. The addition of unlabeled adenovirus 7 DNA does not affect the linear kinetics, but t,he addition of unlabeled heterologous adenovirus 2 DNA gives complex reassociation kinetics of labeled adenovirus 7 DNA. Since adenovirus 7 and 2 genomes share a portion of their sequences (Lacy and Green, 1965), the addition of adenovirus 2 DNA results in unequal abundancies in the population of adenovirus 7 specific DNA sequences. Cot xlO*(mole-set/liter)

2. Reassociation kinetics of RD-114 viral dsDNA and adenovirus type 2 DNA. (0) RD-114 viral dsDNA (500 cpm/l50 ~1) was reassociated after sonication and denaturation as described in the text. The concentration of [3H]DNA product was estimated as described in the legend to Fig. 1. The fraction of reassociated DNA fragments was determined by hydroxyapatite chromatography. (0) Adenovirus 2 [3H]DNA (1.60 X lo6 cpm/pg) was sonicated, and denatured by heating at 100’ for 10 min. Denatured viral DNA (35.0 rig/ml, 200 rl) was then reassociated in 0.40 M PB, at 67”. The fraction of reassociated DNA fragments was measured as described above. FIG.

The Viral Base Sequences Most Emiently Copied by the RD-114 DNA Polymerase RD-114 viral dsDNA is heterogeneous with regard to the relative concentration of different base sequences, but the major portion representing approximately 50 % or more of the DNA product (see Figs. 2 and 4) is relatively homogeneous and reassociates. with second-order kinetics, as shown by the initial reassociation of the four RD-114 viral DNA products in Fig. 4. The genetic complexity of this fraction was estimated from its rate of reassociation. The reassociation of

RD-114

r

VIRUS

GENE

Initial reassociationof RD-114 dsDNA 1

, 5

0

j

10

Cotx lO’(mole-sditer) FIG. 4. Initial kinetics of reassociation of RD114 viral [3H]dsDNA. Purified RD-114 viral dsDNA (5003000 cpm per 200 ~1) was reassociated after sonication and denaturation, and the fraction of reassociated DNA fragments was measured as described in the legend to Fig. 1. Concentrations and specific activities of precursor dTTP were (I) 0.50 mM and 0.981 Ci/mmole (0), (II) 0.10 mM and 4.52 Ci/mmole (a), (III) 0.50 mM and 0.199 Ci/mmole (A), and (IV) 0.10 mM and 0.977 Ci/mmole (v).

adenovirus DNA of molecular weight 23 X lo6 (Green et al., 1967) under the same conditions provided a molecular weight calibration. As shown in Table 1, the four DNA products have sequence complexities of about 0.8 X lo6 and thus represent approximately 4% of the viral 60-705 RNA genome of assumed molecular weight 10’ (Green, 1970). RD-114 viral dsDNA was characterized further by annealing to viral 60-705 RNA in excess as shown in Table 2. Almost half of dsDNA hybridized with 70s RNA, as expected for the hybridization of one viral DNA strand to complementary viral RNA. From this we may conclude that RD-114 viral dsDNA is copied entirely from the viral 60-70s RNA genome, and does not contain appreciable amounts of cell DNA sequences. No significant hybridization occurred with RNA from feline leukemia virus (FLV) grown in RD cells (Table 2). Reassociation of RD-114 viral dsDNA in the Presence of Cell DNA The reassociation of viral dsDNA fragments in the presence of cell DNA was used as a sensitive means to detect viral DNA sequences in feline and human cells. Unlabeled cell DNA mixed with RD-114 viral

TABLE ESTIMATION

OF SEQUENCE

COMPLEXITY

Viral dsDNA

RD-114 (I) RD-114 (II) RD-114 (III) RD-114 (IV) RD-114 (IV) Adenovirus type 2 DNA

OF RAPIDLY

489

SEQUENCES

1 REASSOCIATED

[3H]dTTP precursor Concentration (m&f)

Specificity activity (Ci/mmole)

0.5 0.1 0.5 0.5 0.1

0.981 4.52 0.199 0.199 0.977

FRACTION

Sequence complexityb

Cdl/P

5.2 4.1 4.3 4.3 4.7 1.28

X x x x x x

OF D-114 VIRAL

lo+ 10-d 10-d 10-d 10-d 10-Z

0.94 0.74 0.77 0.77 0.85 23

x x x x x X

106 106 106 106 106 lo6

dsDNA

Percent viral RNA transcribed”

4.7 3.7 3.9 3.9 4.3 -

a Cotllz values for rapidly reassociating fractions were estimated from the slopes, assuming that 50% of RD-114 viral dsDNA was rapidly reassociating dsDNA, as most of our experiments indicated. No correction was made for the small differences in length between adenovirus DNA fragments (SOt-900 nucleotides) and the RD-114 viral DNA product (6OC-800 nucleotides). b Sequence complexity of rapidly reassociated fraction of RD-114 dsDNA was estimated from C&l/z values using adenovirus 2 DNA of molecular weight 23 X lo6 (Green et al., 1967) to calibrate the reIationship between molecular weight and Cotl/n. c Based on molecular weight for viral 60-70s RNA of 1.0 X 10’.

FUJINAGA

490 TABLE HYBRIDIZATION OF RD-114 VARIOUS VIRAL RNA

2 VIRAL dsDNA WITH PREPARATIONS=

E2’ AL. 0.5 A 0.4

Source of RNA (5 pg/ml)

Reassociation time (hr)

[3H]DNA in RNADNA hybrid (%)

RD-114 viral 60-70s RNA (preparation I) RD-114 viral 60-70s RNA (preparation II) H-MSV(MLV) viral 60-70s RNA Gardner FLV (grown in RD cells) viral RNA Escherichia coli tRNA

1.0 2.0 1.0 2.0 1 .O

49.7 45.3 42.5 45.7

2.0 1.0

10.1 1.7

2.0

3.8

1.0 2.0 1.0 2.0

1.Q 5.0 1.0 3.4

No RNA

-

RD.114 cell DNA

7.6

a RD-114 viral[aH]ds DNA (565 cpm/l50 ~1) was annealed with 5 rg/ml of RNA in 0.40 M PB at 67’ for 1 or 2 hr, and the extent of hybridization wae measured by hydroxyapatite chromatography. H-MSV(MLV) was grown in a transformed mouse cell line (Green el al., 1970) and Gardner FLV in human RD cells (McAllister et al., 1973). Viral RNA was extracted from purified virions as described (Tsuchida et al., 1972).

[3H]dsDNA was sonicated, denatured, and annealed as described in Materials and Methods. The rate of reassociation of the rapidly reassociating fraction was measured by hydroxyapatite chromatography. As shown in Fig. 5A, viral [3H]DNA reassociated about three times more rapidly in the presence of RD-114 cell DNA. In three different experiments with cell to viral dsDNA ratios of 1.7 to 2.3 X 105, the increase in reassociation rate was 2.5-3.5 (Table 3). From this we calculate that 20-30 DNA copies of the sequences of rapidly reassociating dsDNA are present in RD-114 cells. The reassociation of dsDNA was accelerated much more rapidly in the presence of DNA from normal cat kidney tissue and from a cat thymus cell line producing the feline sarcoma-leukemia virus (FSV(FLV)) (Fig. 5B and Table 3). From the rate of reassociation, we calculate that there are loo-200 copies of this portion of the viral genome in feline tissues and in the cultured feline cell line (Table 3). No appreciable acceleration of the rate of reassociation occurred in t,ho

FSV(FLV)

Infected

DNA “I

”

.

I.”

^

^”

L.”

^A

3.”

Cot x103 (mole-w/liter) FIG. 5. Initial reassociation reaction of viral [aH]dsDNA in the presence of different cell DNAs. (A) RD-114 viral dsDNA (414 cpm/2OO ~1) was reassociated in the presence of RD-114 cell DNA (a), rat embryo cell DNA (A), BHK cell DNA (O), and calf thymus DNA (0), at a concentration of 171 pg/ml, as described in the text. (B) RD-114 viral dsDNA (491 cpm/l50 ~1) w&s reassociated in the presence of cat kidney DNA (A), FSV(FLV) infected cat thymus cell DNA (v), RD.114 cell DNA (a), and calf thymus DNA (O), at a concentration of 289 pg/ml, as described in the text.

presence of DNA from normal human spleen, the human P3-HRIK cell line, hamster or rat cells (Table 3). Hybridization of RD-114 viral 70s RNA with DNA from Human and Feline Cells To estimate the fraction of the viral genome present in feline cells, we compared the extent of hybridization of RD-114 viral 60-70s [3H]RNA with DNA of feline cells and of RD-114 cells. The latter cell line contains the entire viral genome since it replicates RD-114 virus. The extent of hybridization was monitored by treatment with RNase A (Gelderman et al., 1971; Melli et al., 1971; Nieman, 1972). Data from three

RD-114 TABLE

VIRUS

GENE

3

RD-114 @I-70s 13Hl RNA annealed with DNA from:

THF, NUMBER OF COPIES OF RD-114 VIRAL dsDNA IN MAMMALIAN CELLS Expt. No.

1

2

Cell DNA wmu

Viral RDcri&ed 1 14/13H] rate tlsm$ factor

Calf thymus(171) 1.00 RD-114 cells 1.00 (171) Rat embryo (171) 1.00 BHK (171) 1.00 Calf thymus (171) 0.762 RD-114 cells 0.762

491

SEQUENCES

a&r

feline

0

E. coli

2% copies per cell

1.00 3.10

31

1.04 1.02 1.00


3.47

28

18.4 19.5 1.09

207

2.45

20

11.9 12.9 1.00 1.12

148 162
1.00 1.01 4.18

<1 111

(171) 3

Cat kidney (171) 0.762 Cat thymus (171) 0.762 Calf thymus (289) 1.57 RD-114 cells 1.57

195 -

(289) 4

5

Cat kidney (289) 1.57 Cat thymus (289) 1.57 Calf thymus (289) 0.953 Normal human 0.953 spleen (289) 1.06 E. coli (289) P3-HRIK (289) 1.06 1.06 Cat kidney (72) + E. coli (217)

a The number of viral DNA copies in the cell was calculated from the increased rate factor, using values of 2.0 X lOi daltons for the molecular weight of haploid cell DNA, 0.8 X lo6 daltons for the sequence complexity of rapidly reassociating RD-114 dsDNA segments, and 5Oyn content of rapidly reassociating dsDNA in total RD-114 viral dsDNA segments.

different experiments with two different labeled 60-70s RNA preparations are plotted in Fig. 6. An average of 8 % of viral RNA was resistant to RNase A before and after annealing with DNA from E. coli, calf thymus, normal human spleen, or RD cells. This resistance represents in part the presence of poly(A) sequences of 60-70s RNA (Lai and Duesberg, 1972; Green and Cartas, 1972; Gillespie and Marshall, 1972; Ross et al., 1972). When viral 60-705 [3H]RNA was annealed with DNA of cat liver, lung and kidney, or RD-114 cells, about 60 % of RNA was rendered RNase resistant (Fig. 6). The lack of complete hybridization may be explained by competition of the more rapid

O41’ 0

I 100

I

10’

102

I 103

I

10”

Cot (mole-see/liter)

FIG. 6. Hybridization of RD-114 viral 60-70s RNA with cell DNA. Heated RD-114 viral 6&7OS [3HlRNA (lOO’C, 2 min, 2OCMOO cpm/lOO ~1) was annealed with cell DNA in 0.40 M PB at 67”. Concentrations of cell DNA ranged from 1.27 mg/ml to 8.84 mg/ml in different experiments. Aliquots of the reaction mixture were removed at different times and resistance to RNase digestion (RNase A, 50 rg/ml in 2 X SSC, 37’ for 30 min) was measured as described by Neiman (1972).

DNA-DNA hybridization with DNA-RNA hybridization (Gelderman et al., 1971; Melli et al., 1971; Straus and Banner, 1972) since DNA may not be in sufficient excess in some experiments. The similar rate and extent of reassociation suggests that similar numbers of copies of most of the RD-114 viral genome are present in feline tissue and in human RD-114 cells, although as shown above, much larger numbers of DNA segments homologous to the rapidly reassociating RD114 viral dsDNA are present in feline tissue. Lack

of Hybridization of RD-114 Viral ssDNA to RNA of FLV, FSV(FLV), an,d MSV(MLV) Virions

Single-stranded RD-114 viral DNA was synthesized by the action of the purified DNA polymerase of avian myeloblastosis

492

FUJINAGA

virus on viral 60-70s RNA in the presence of actinomycin D, conditions that generally result in the transcription of most of the viral genome (unpublished data). Viral [3H]ssDNA was annealed with various viral RNA preparations (data not shown). No significant hybridization was found with RNA from FLV and FSV(FLV) grown in feline cells, from FLV grown in human RD cells and from M-MSV(MLV) grown in murine cells [78Al cell line (Green et al., 1970)]. DISCUSSION

Molecular hybridization and reassociation kinetic measurements were used in this study to analyze the relationship between the genome of RD-114 virus and the DNAs of uninfected human cells, human RD-114 cells producing RD-114 virus, the parent RD cell line, uninfected feline, rat, and hamster cells, and the RNA genomes of FLV, FSV(FLV) and MSV(MLV). Three radioactive probes containing different fractions of the viral genome were used: the dsDNA fraction most frequently transcribed by the RD-114 viral DNA polymerase with a genetic complexity of about 0.8 X 106, the RD-114 viral ssDNA product which presumably contains sequences from a major fraction of viral genome, and RD-114 viral 60-70s RNA which represents the entire viral genome. From the rate of reassociation of RD-114 viral dsDNA, we estimate that 100-200 copies of DNA sequences representing approximately 4 % of the RD-114 viral genome are present in normal feline cells, whereas only 20-30 copies are present in virusproducing RD-114 cells. Thus a portion of the viral genome is homologous to a class of repetitive feline cell DNA sequences. These repetitive sequences, shared with the presumed endogenous feline oncornavirus, RD114 virus, could possibly play a role in gene regulation (Britten and Davidson, 1969). From the similar extent (at least 60%), and rate of hybridization of viral 60-705 RNA with RD-114 cell DNA and feline cell DNA it would appear that sequences derived from most, if not all of the RD-114 viral genome, are present in the DNA of normal feline cells, and that similar numbers of viral gene copies are present in feline and RD-114 cells.

E2’ AL.

Similar conclusions were reached by Neiman (personal communication, Nature in press). It is difficult to estimate the number of viral gene copies from these data since the rate constant for RNA-DNA hybridization is not known. The dsDNA and/or 60-70 SRNA probes did not detect RD-114 viral sequences in the DNA of human RD cells, human spleen tissue, a human lymphoblastic cell line derived from a Burkitt lymphoma, and uninfected hamster and rat cells. The sensitivity of these measurements are such that about one copy of the sequences in dsDNA and approximately 10 % of the viral genome would have been detected. Experiments leading to similar qualitative conclusions were reported recently. RD-114 viral DNA was shown to hybridize to feline cell RNA (Okabe et al., 1973) and with feline DNA (Ruprecht et al., 1973), but not with human RNA or DNA. The lack of hybrid formation between RD-114 viral ssDNA and FLV and FSV(FLV) RNA suggests that there is little if any homology between feline leukemiasarcoma viruses and RD-114 virus, although quantitative conclusions are limited because RD-114 viral ssDNA may not represent the entire genome. Similar findings were reported recently by Haapala and Fischinger (1973) with total virion RNA, and by Dr. J. Michael Bishop (personal communication) using viral DNA probes representing a major portion of the viral genome. These results are consistent with immunological data on the lack of relatedness between the gs antigen and DNA polymerase of these virions (McAllister et al., 1972; Oroszlan et al., 1972; Scolnick et al., 1972; Long et al., 1973). The sharing of base sequences between avian and murine oncornaviruses and the host cell genome has been the subject of several recent studies (Baluda, 1972; Rosenthal et al., 1971; Varmus et cd., 1972; Yoshikawa-Fukada and Ebert, 1971; Gelb et al., 1971; Neiman, 1972). The more recent quantitative studies by Neiman (1973) with radioactive viral 60-70s RNA as probe show that most of the viral genome of an endogenous avian oncornavirus is present in normal chick embryo cells. Our data showing that a major portion, and possibly all, of the

RD-114

VIRUS

GENE

sequences of the RD-114 viral genome is present in the genome of normal feline cells whereas no detectable RD-114 viral sequences are present in human RD eel s suggests that RD-114 may be an endogenous feline oncornavirus. The extent to which known feline leukemia and sarcoma viruses share sequences with their host cell has not been established. Thus there appear to be at least two families of feline oncornaviruses, one represented by RD-114 virus and the second by previously isolated feline leukemia and sarcoma viruses. The recent isolation from feline cells of type C particles with properties similar if not identical to RD-114 virus support, this view (Fischinger et al., 1973; Livingston and Todaro, 1973; Sarma et al., 1973). It is of interest that RD-114 virus growing for a number of generations in human cells shares no detectable sequences with human cell DNA. According to current views (Green, 1972) the RD-114 viral genome is probably integrated in the human cell genome of RD-114 cells and is transcribed to form the RNA of progeny virus. Cells transformed by DNA tumor viruses, adenovirus (Tseui et al., 1972) and simian virus 40 (Wall and Darnell, 1971) transcribe polycistronic RNA molecules containing viral RNA and reiterated cell DNA sequences. If cell RNA sequences are transcribed covalently bound to RD-114 viral RNA, they must be removed prior to maturation of the viral RNA genome. In this regard, the nature of nuclear virus specific RNA would be of interest. ACKNOWLEDGMENTS We thank Michael Pursley and Maria Cartas for excellent technical assistance in some experiments, Dr. Duane Grandgenett for supplying the purified DNA polymerase of avian myeloblastosis virus, and Dr. Paul Neiman for advice on hybridization of viral RNA in DNA excess. This work was supported by research contract PH43-67-692 within the Virus Cancer Program of the National Cancer Institute and partly by funds from the Ministry of Education of Japan. REFERENCES BALUDA, M. A. (1972). Widespread presence, in chickens, of DNA complementary to the RNA genome of avian leukosis viruses. PTOC. Nat. Acad. Sci. U.S. 69,576-580.

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RD-114 ing viral sequences

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and highly reiterated cellular in adenovirus-transformed

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