- Email: [email protected]
Intracellular and virion 35 S RNA species of murine sarcoma and leukemia viruses
VIROLOGY
69,
253265
Intracellular
(1974)
and
Virion
35
and
S RNA
Leukemia
NOBUO TSUCHIDA’ Institute
for
Molecular
Species
Sarcoma
Viruses
AND
MAURICE
Virology, Saint Louis University St. Louis, Missouri 63110 Accepted
of Murine
January
GREEN School
of Medicine,
23, 1974
RNA molecules from the following sources were treated with dimethyl sulfoxide to dissociate noncovalent aggregates and resolved by electrophoresis on polyacrylamide gels : (1) transformed MSV(MLV) -producing Balb/3T3 cells (MSV-39, clone 24), (2) transformed MSV(MLV)-producing rat cells (78Al), (3) MSV transformed, non virus-producing hamster cells (HT-I), (4) MLV-producing Balb/3T3 cells, (5) MLVproducing NIH/3T3 cells (6) 6&70 S RNA from MSV(MLV) virions, and (7) 60-70 S RNA from MLV virions. Virus-specific RNA was detected by hybridization of RNA in gel fractions with the 3H-DNA product of the MSV(MLV) RNA-directed DNA polymerase. Two well defined viral RNA species with sedimentation coefficients of 35 S and 20 S, but none of intermediate size, were detected in both MSV(MLV) producing cell lines. The non virus-producing HT-1 cell line contained a viral RNA species slightly smaller than 35 S, about 33 S, but no detectable 20 S virus-specific RNA. These results with 78Al and HT-1 cells agree with previous conclusions based on rate-zonal centrifugation in sucrose density gradients (Tsuchida et al., 1972). The two MLV-producing mouse cell lines contained a well defined 35 S RNA peak, a somewhat less pronounced 20 S RNA peak, and heterogeneous RNA species of smaller molecular weights. Although both 35 S and 20 S virus RNA species were detected in cells replicating murine oncornaviruses, labeled 60-70 S RNA isolated from MSV(MLV) virions (consisting mainly of MLV) and MLV virions, and dissociated with dimethyl sulfoxide or by heat, consisted of 35 S RNA and about l(r15o/o 7 S RNA plus 4-5 S RNA, but no detectable 20 S RNA. Hybridization-competition experiments using MSV(MLV) 60-70 S 3H-RNA (consisting mainly of MLV RNA) and saturating amounts of the unlabeled DNA product of the MSV(MLV) RNA-directed DNA polymerase showed that 35 S RNA from cells replicating MSV(MLV) shares virtually all of its nucleotide sequences with MSV(MLV) 70 S RNA; these data suggest that intracellular 35 S RNA species are the major precursors to virion 70 S RNA. In contrast, 33 S RNA from HT-1 cells shares only about 5070 of its sequences with MSV(MLV) 70 S RNA, indicating that only part of the sequences of MSV(MLV) 6&70 S RNA are integrated and/or transcribed in this non virus-producing MSV transformed cell. INTRODUCTION
The 60-70 S RNA genome (referred to here as 70 S RNA) of murine oncornaviruses dissociates under conditions that disrupt hydrogen bonds to 3040 S RNA and smaller heterogeneous species (Blair and Duesberg, 1968; Bader and Steck, 1969). When RNA l Present address: Flow Laboratories, Inc., 1701 Chapman Avenue, Rockville, Ma.ryland 20852. 253 Copyright All rights
@ 1974 by Academic Press, Inc. of reproduction in any form reserved.
from the rat embryo cells (78Al) transformed by the Moloney strain of murine sarcoma virus (M-MSV) and producing both MSV and murine leukemia virus (MLV) were sedimented in sucrose density gradients (Tsuchida et al., 1972), two peaks of virusspecific RNA sedimenting at 35 S and 20 S were detected. In the present paper, we describe the further characterization by
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polyacrylamide gel electrophoresis and by hybridization-competition of the viral RNA speciesfound in transformed mouse and rat cells replicating MSV(MLV), in mouse cells replicating MLV, in non virus-producing MSV transformed hamster cells, and in the MSV(MLV) and MLV virus particles. MATERIALS
ANI)
METHOUS
Cells. The following cell lines were used in these studies: transformed rat embryo cells (78Al) (Green et al., 1970) producing M-MSV(MLV), transformed Balb/3T3 cells (MSV 39, clone 24) producing M-MSV(MLV), M-3lLV producing Balb/3T3 cells (the latter two cell lines were kindly provided by Dr. Stuart Aaronson), M-MLV producing NIH/3T3 cells (kindly provided by Dr. George Todaro), non virus-producing MSV transformed hamster cells (HT-1) (Tsuchida et al., 1972), and adenovirus 2 transformed rat cells (8617) (Fujinaga et aZ., 1969). Cells were grown in monolayer culture in Eagle’s minimal essential medium supplemented with 10% fetal calf serum. Preparation of cell RNA. Nearly confluent monolayers xere trypsinized [2.5 mg/ml of trypsin in phosphate-buffered saline (PBS) (Dulbecco and Vogt, 1954) without Mg2+ and Ca2+], collected by centrifugation at 700 g for 3 min, and washed twice with cold PBS. After suspending in 15 volumes of 0.05 M Na acetate buffer (pH 5.5) containing 0.1% diethylpyrocarbonate (Baycovine, Nafton, Inc., Chicago), cells were lysed with 1% Na dodecyl SO4 and nucleic acids were extracted by the hot phenol method (Parsons and Green, 1971). Nucleic acids were precipitat,ed with 2 volumes of ethanol, suspended in 2 ml of HSB buffer (0.5 M R’aCl, 0.05 M Tris .HCl (pH 7.4), 0.05 M MgC12) per 1 ml of original packed cells, and incubated with 25 &ml of pancreatic DNase (Sigma Chemical Company, RNase-free) for 5 min at 25”. After the addition of 4 volumes of AES buffer (0.05 M Na acetate, pH 5.5, 0.02 M EDTA, 0.5 % SDS), the solution was extracted with equal volumes of watersaturated phenol and chloroform-isoamyl alcohol (24 : 1) at 5-10” by vigorous agitation for 2 min. R,NA was precipitated from the aqueous phase with 2 volumes of ethanol at
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35 S RNA
259
- 20” overnight, dissolved in NTE buffer (0.01 M TrisTHCl (pH 7.4), 0.1 M NaCl, 0.001 M EDTA) and treated with 95% dimethyl sulfoxide (DMSO) to dissociate RNA aggregates that might interfere with the resolution of individual RNA species (Tsuchida et al., 1972). The concentration of RNA was determined by the orcinol reaction {Green, 1959). Virus-specific DNA. The viral 3H-DNA used to detect intracellular viral RNA was prepared by the endogenous reaction of JJ-MSV(MLV) virions as described (Green et al., 1970, 1971; Rokutanda et al., 1970). The reaction mixture contained 40 mM Tris.HCl (pH S.l), 5 mM dithiothreitol, 30 mM NaCl, 0.01% Nonidet P-40, 2.5 mM MgClz , 10-d M each of dATP, dCTP, and dGTP and 2 X lo+ M 3H-dTTP (lo-11 Ci/mmole, New England Nuclear Corp.). Unlabeled DNA was prepared using 1O-4M of each of the four deoxyribonucleotide triphosphates. Enzyme reactions were performed at 37” for 90 min and stopped by adding EDTA to 10 mM, NaCl to 100 mM, and SDS to 0.5 %. The mixture was extracted with phenol plus chloroform-isoamyl alcohol, with water-saturated ether, and treated with 0.05 volumes of 2 N NaOH at 80” for 30 min to destroy RNA. After neutralization with 0.05 volume of 3 M NaH2P04, the DNA product was dialyzed at 4” against three changes of 1000 volumes of 0.1 X SSC (SSC = 0.15 M NaCl-0.015 M Na3 citrate). The amount of unlabeled DNA was estimated by the diphenylamine reaction (Green, 1959). Polyacrylamide gel electrophoresis. Polyacrylamidc gel electrophoresis was performed asdescribed (Bishop et al., 1967; Parsonsand Green, 1971). Gels (9.5 X 0.5 cm) contained 2.5 c/d acrylamide, 0.12 % bisacrylamide, 40 mM Tris-acetate (pH 7.2), 30 mM Na acetate, 1 mM EDTA, 0.08% N,N,N’,N’tetramethylethylenediamine, 0.08 % ammonium persulfate, and 10 % glycerol. The electrophoresis buffer contained 40 mM Trisacetate (pH 7.2), 30 mM Na acetate, 1 mM EDTA, 0.2 % SDS, 0.5 % diethylpyrocarbonate, and 10 % glycerol. Samples in 0.1 X SSC containing 20% glycerol and 0.005 % bromophenol blue were electrophoresed for
260
TSUCHIDA
2 hr at 5 mA per tube. After electrophoresis, each gel was transferred to a rectangular quartz cell (9.5 cm long) and the absorbancy at 260 nm was scanned in a Beckman ACTA V spectrophotometer. The gel was then placed in an aluminum trough, frozen in an acetone-dry ice bath, and sliced into about 2 mm segments. To measure radioactivity, each slice was incubated with 0.3 ml of 30 % Hz02 at 55” for 15 hr. After addition of 10 ml of aquasol (New England Nuclear Corp.), radioactivity was measured in a liquid scintillation counter. To measure the amount of virus-specific RNA, each slice was incubated at 66” for 20 hr with viral 3H-DNA (2000 dpm) in 0.3 ml of 3 X SSC. After hybridization, the fluid was removed from gel particles with a Pasteur pipette and the amount of hybrid formed was determined by batch elution with hydroxyapatite (Tsuchida et al., 1972). Radioactive viral RNA. 78Al cells and M-MLV infected Balb/3T3 cells were labeled with 32P-phosphate (100 &i/ml) in phosphate-free Eagle’s medium, or with 100 $Zi/ml of 3H-uridine (41 Ci/mmole, New England Nuclear Corp.) by the method of Bader and Steck (1969). Pooled culture fluids were clarified by centrifugation at 2000 g for 10 min and virus was recovered by centrifugation at 25,000 rpm at 4” for 60 min in the Spinco SW 27 rotor. The pellet was suspended in NTE buffer, layered on a 15 to 60 % sucrose density gradient in NTE buffer, and centrifuged as described (Tsuchida et al., 1972). Fractions containing virus (p = 1.16) were pooled, diluted with 2.5 volumes of NTE buffer containing 0.05 % diethylpyrocarbonate and 1% SDS, and radioactive RNA was extracted with equal volumes of phenol and chloroform-isoamyl alcohol. RNA was precipitated with 2 volumes of ethanol in the presence of 30 Mg/ml of yeast tRNA (Sigma type IV), dissolved in NTE buffer, layered on a 15 to 30% sucrose density gradient containing 0.5 % SDS, and centrifuged at 36,000 rpm for 2-2.5 hr at 20”. The fractions sedimenting at about 70 S were pooled, precipitated with 2 volumes of ethanol, and dissolved in 0.1 X SSC. Portions of RNA were treated with 95 % DMSO or heated at 70” for 1.5 min. DMSOtreated RNA was precipitated with 2
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GREEN
volumes of ethanol in the presence of 30 pg/ml of yeast tRNA, and dissolved in 0.1 x ssc. Intracellular 35 S RNA. Intracellular virus-specific 35 S RNA was isolated by zonal centrifugation of RNA extracted from 78Al or HT-1 cells in a 15 to 30% sucrose density gradient in NTE buffer containing 0.5 % SDS as described previously (Tsuchida et al., 1972). Fractions in the 35 S region were pooled and RNA was precipitated with 2 volumes of ethanol at -20” overnight. RNA was dissolved in 0.1 X SSC, reprecipitated with ethanol, dissolved in 0.1 X SSC, and dialyzed against 1000 volumes of 0.1 X SSC at 5” overnight. The 35 S RNA isolated from 78Al cells was free of detectable 20 S viral RNA as shown by polyacrylamide gel electrophoresis and hybridization with virusspecific DNA. RNA in each preparation was quantitated by hybridization with the 3HDNA product. When 2000 dpm of M-MSV (MLV) 3H-DNA was annealed with virion 70 S RNA, 78Al cell RNA, or intracellular 78A135 S RNA, 75 % of the DNA hybridized with saturating levels of RNA, while only 45 % of DNA hybridized with unfractionated HT-1 cell RNA at saturation. One hybridization unit of virus-specific RNA is defined as the amount that gives half-maximum hybridization; 1 unit corresponded to 0.012 pg of virion 70 S RNA, 1.2 pg of 78Al cell RNA, and 11 pg of HT-1 cell RNA. RESULTS
Resolution of Viral RNA Species in MSV Transformed Rat, Mouse, and Hamster Cells by Polyacrylamide Gel Electrophoresis Two size classes of virus-specific RNA were previously detected in 78Al cells by zonal centrifugation in sucrose density gradients (Tsuchida et al., 1972). To further characterize viral RNA, we utilized the higher resolution of polyacrylamide gel electrophoresis. Total cellular RNA isolated from M-MSV transformed rat cells (78Al) and mouse cells (MSV-39, clone 24) replicating M-MSV(MLV) was treated with dimethyl sulfoxide (DMSO), and electrophoresed for 2 hr on polyacrylamide gels. To identify virus-specific RNA, each gel fraction was
INTRACELLULAR 3$&F-
M.MSV(MLV) rat (78Al)
AND l.5
- 1.0
- 0.5 4s ‘--L.&O M-MSV(MLV) balbBT3 (MSV 39, clone 24)
VIRION
35 8 RNA
261
To search for additional viral RNA species, 35 S and 20 S RNA were further resolved by electrophoresis for 7.5 hr. As shown in Fig. 2, only 35 S and 20 S RNA species were detected. Resolution of Viral RNA Species in MLVProducing Mouse Cells by Polyacrylamide Gel Electrophoresis The RNA extracted from two mouse cell lines, Balb/3T3 and NIH/3T3, infected by the Moloney strain of MLV(M-MLV) was analyzed by polyacrylamide gel electrophoresis. Each cell line contained both 35 S and 20 S viral RNA species (Fig. 3). In addition, heterogeneous RNA species with smaller molecular weights were detected (Fig. 3). These small species were not detected in appreciabIe amounts in MSV~iM!I~~Producing mouse and rat cells (Figs.
Distance moved (cm)
1. Resolution of virus-specific RNA from M-MSV transformed cells by polyacrylamide gel electrophoresis. Cell RNA was treated with DMSO and electrophoresed on polyacrylamide gels for 2 hr at 5 mA per tube as described in Materials and Methods. After electrophoresis, the absorbancy at 260 nm was scanned and gel fractions were prepared and hybridized with 3H-DNA product as described in Materials and Methods. (A) M-MSV(MLV) rat (78Al) RNA. (B) M-MSV (MLV) Balb/3T3 (MSV 39, clone 24) RNA. (C) M-MSV hamster (HT-1) RNA. FIG,
Subunit Structure of 70 S RNA Molecules Present within MSV(MLV) and MLV Virions To study the relationship of intracellular viral RNA species to the viral 70 S RNA genome, we examined the subunit structure of 70 S RNA extracted from virus particles. Radioactive 70 S RNA was isolated from purified virions labeled as described by Bader and Steck (1969). Virus-producing cells were incubated with 3H-uridine or 82Pphosphate for 1 hr, and the medium was replenished every hour for 3 or 4 hr. Virus was isolated from the medium of each l-hour harvest, and viral 70 S RNA was extracted and purified by zonal centrifugation
annealed with a constant amount of a viral 3H-DNA prepared by the endogenous DNA polymerase of MSV(MLV). Two distinct virus-specific RNA species were clearly resolved (Fig. 1A and 1B). The sedimentation coefficient of the slower and the faster migrating RNAs were 35 S and 20 S, respectively, calculated from the 28 S and 18 S ribosomal RNA markers in sucrose density gradient centrifugation. However, 20 S RNA speciesmigrates in polyacrylamide gel more slowly than expected from its sedimentation coefficient, suggesting that secondary structure affects its mobility. Non Distance moved (cm) virus-producing iVlSV transformed HT-1 cells contain a single species migrating FIG. 2. Resolution of virus-specific RNA species slightly faster than 35 S RNA, estimated at of 78Al cells after polyacrylamide gel electrophoresis for 7.5 hr. For details see legend to Fig. 1. 33 S (Fig. l(J).
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(Tsuchida et al., 1972). As shown in Fig. 4, viral 70 S RNA was converted to relatively homogeneous 35 S RNA when denatured with DMSO or with heat (70” for 1.5 min) (Fig. 4A-D). The mobility of 35 S RNA obtained by dissociation of viral 70 S RNA coincided with that of intracellular virusspecific 35 S RNA, as shown by electrophoresis on parallel gels. No differences in electrophoretic patterns were observed between dissociated 70 S RNA from MSV (MLV) and MLV (Fig. 4A-D) ; this is as expected, since MLV is present in excess in MSV(MLV) preparations (see Discussion). Most important, no peak of viral RNA corresponding to intracellular viral 20 S RNA (less than 10 %) was detected in dissociated 70 S RNA from MSV(MLV) and mv virions in four separate experiments. In addition to 35 S RNA, small amounts of 7 S and 4-5 S RNA, representing about 10-15X of the total radioactivity, were reproducibly detected. Relationship between Intracellular and Viral ‘70 X RNA
35s
2%
GREEN M-MSV(MLV)
70s =P RNA OMSOtreated
2
M-MLV 70s 3H RNA
2 d
I
DMSO treated
I
2.0 400 1.0
200 I 0 5 400 ,r
0
35 S RNA
Intracellular viral 35 S RNA may be precursor to virion 70 S RNA. Since 70 S RNA could consist of 3 or 4 subunits of 35 S 30 A
AND
M-MLV balb 3T3
I I
O,*
i 0.4
M-MLV NIH 3T3
1.0
0
2.
4~
6
a
D~slance moved (cm)
FIG. 4. Analysis of 32P-RNA from dissociated virion 70 S RNA molecules by polyacrylamide gel electrophoresis. Radioactive 70 S RNA was prepared, treated with 95% DMSO or heated at 70” for 1.5 min, and electrophoresed on polyacrylamide gels for 2 hr at 5 mA per tube, as described in Materials and Methods. M-MSV(MLV) 70 S RNA after treatment with (A) DMSO, or (B) heated; and M-MLV 70 S RNA after treatment with (C) DMSO, or (D) heated.
0.8
RNA, intracellular 35 S RNA may contain only a portion of the sequences of 70 S RNA. It was therefore of interest to determine the relatedness between 70 S RNA and intracellular 35 S RNA. One approach would be to compare the extent of hybridization of viral RNA with the viral DNA product, but FIG. 3. Resolutionof virus-specificRNA species such measurementsmay be ambiguous since in M-MLV infected cells by polyacrylamide gel viral DNA does not consist of equimolar electrophoresis. RNA was isolated from (A) transcripts of the viral genome. To overcome M-MLV infected Balb/3T3 cells and (B) M-MLV this difficulty, we have determined the extent infected NIH/3T3 cells, electrophoresed, and of competition of intracellular 35 S RNA virus-specific RNA identified as described in the legend to Fig. 1. with viral 3H-RNA for hybridization with - 0.6
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virus-specific DNA under conditions where excess viral DNA saturates 70 S RNA. A similar procedure was described recently by Garapin et al. (1973). It was first necessary to prepare a viral DNA product which contained sequences derived from most of the viral genome, and second to establish conditions for saturating most of viral 70 S RNA with viral DNA. As shown in Fig. 5, when 3H-labeled 70 S RNA (0.012 pg) or 3Hlabeled 35 S RNA (0.015 pg) isolated from denatured 70 S RNA by zonal centrifugation was annealed with increasing amounts of viral DNA, more than 80 % of RNA became hybrid at a ratio of DNA to RNA of 10: 1, as shown by resistance to RNase digestion. Thus most if not all of the viral genome is transcribed to DNA under these reaction conditions. No significant difference in saturation levels was observed with 35 S or 70 S RNA indicating that most sequences of 70 S RNA are present in 35 S RNA subunit(s). The DNA-RNA saturation conditions designated by the arrow in Fig. 5 were used in the hybridization-competition experi-
IO’
100 DNA/RNA
FIG. 5. Hybridization of labeled virion 70 S and 35 S subunits withsaturating amounts of unlabeled M-MSV(MLV) DNA. Virion70 S 3H-RNA (0.12 pg) and 35 S $H-RNA subunits (0.15 pg) were annealed with increasing amounts of unlabeled viral DNA in 0.1 ml of 3 X SSC for 36 hr at 66’. The reaction was stopped by the addition of 1 ml of 1 X SSC. RNase-resistant hybrids were measured by treatment with RNase A (50 pg/ml, 20 min, 37” precipitated with trichloroacetic acid in the presence of 100 rg of carrier calf thymus DNA, and counted. RNase-resistant radioactivity (5-7y0 of the input) of the RNA annealed in the absence of DNA was subtracted. Virion 70 S 3H-RNA (a-0) and 35 S RNA subunits (O-O) derived from DMSOtreated RNA by zonal centrifugation.
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263
35 S RNA
aI 0 1 0
,
ID 50
1
20 Hybrfdiralm
I
30 units
100 150 ~8 8617 cell RNA
200
FIG. 6. Hybridization-competition between intracellular viral 35 S RNA from virus-producing and viral 33 S RNA from non virus-producing transformed cells with virion 70 S RNA. M-MSV(MLV) 70 S 3H-RNA (0.012 rg) was annealed with 0.17 rg of unlabeled M-MSV(MLV) DNA product in the presence of increasing amounts of unlabeled RNA; virion 70 S RNA (@-a), 78Al cell RNA (em), 78Al intracellular 35 S RNA (A-A), HT-1 intracellular 33 S RNA (u-n), and 8617 cell RNA (O-O). 3H-radioactivity resistant to RNase digestion was measured as described in the legend to Fig. 5. The radioactivity of the sample without competing RNA was normalized to 100%.
ments of Fig. 6. Viral 70 S 3H-RNA (0.012 pg) and the MSV(MLV) DNA product (0.17 pg) were annealed in the presence of increasing amounts of unlabeled (1) MSV (MLV) 70 S RNA, (2) 78Al whole cell RNA, (3) 35 S RNA from 78Al cells, (4) 35 S RNA from HT-1 cells, or (5) RNA from adenovirus 2 transformed rat cells (8617). The amounts of virus-specific RNA present in these preparations were quantitated by hybridization with viral 3H-DNA and are expressed in “hybridization units”, as described in the legend to Fig. 6. Figure 6 shows that intraceilular 35 S RNA from 78Al cells competed with virion 70 S 3HRNA to the same extent (90-95 %) and with similar kinetics as unlabeled 70 S RNA and unfractionated 78Al cell RNA. Control 8617 cell RNA did not compete significantly. These results indicate that hybridization between virus-specific RNA sequences and the DNA product is highly specific and that intracellular 35 S RNA from 78Al cells contains sequences derived from most if not all of the 35 S RNA subunits of virion 70 S RNA.
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Of considerable interest, the 33 S RNA from non-virus-producing MSV transformed HT-1 cells competed with only about 50 % of 70 S 3H-RNA (Fig. 6). Thus the 33 S RNA subunit of HT-1 cells contains only a portion of viral RNA sequences in MSV (MLV) 70 S RNA and of the virus-specific RNA sequences of the 35 S RNA subunit(s) present in 78Al cells.
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20 S RNA that is not contaminated with small amounts of degradation products from 35 S RNA. Although it is possible that 20 S RNA serves as precursor to 35 S RNA, since 35 S and 20 S RNA were calculated to have molecular weights of 3.0 X lo6 and 1.4 X lo6 daltons (Bishop el al., 1967), the assembly of large RNA molecules from smaller precursor molecules has not been reported. Both 35 S and 20 S viral RNA transDISCUSSION cripts could serve as mRNA for protein synthesis. We have localized viral mRNA in Both 35 S and 20 S viral RNA species 78Al cells in free and membrane-bound polywere detected in five MSV(MLV) and MLV ribosomes that contain nascent viral polyvirus-producing cells thus far examined. peptides (Vecchio et al., 1973). Our recent These RNA species are strictly intracellular experiments have detected native (i.e., not since harvesting cells with trypsin removes dissociated with dimethyl sulfoxide) 35 S virus particles from the cell surface (Levy RNA on both classes of polyribosomes; and Rowe, 1971; W. P. Rowe, personal communication). The 35 S RNA species are native 20 S RNA appears to be present only probably the major precursors to viral 70 S in membrane-bound polyribosomes (Shanmugan et al. 1974). The possibility is not exRNA since (1) hybridization-competition shows that intracellular and virion 35 S cluded as yet that 20 S RNA simply is a cleavage product of 35 S RNA. RNA species contain the same base The 33 S RNA species in non virussequences, and (2) 70 S RNA of MSV(MLV) producing HT-1 cells contains only approxiand MLV dissociates mainly to 35 S RNA species with the same sedimentation co- mately 50 % of the base sequences of MSV(MLV) 70 S RNA and of the 35 S RNA efficient (unpublished data) and electroof virus-producing 78Al cells (Fig. 6). The phoretic mobility as intracellular 35 S RNA. interpretation of this finding is complicated We have not detected significant 20 S RNA by the lack of information on (1) the nature in denatured 70 S RNA from the Moloney strain of MSV(MLV) or of MLV growing in of the MSV genome, (2) the extent of homology between the MSV and MLV four different cell lines by polyacrylamide gel electrophoresis that readily detects 20 S genomes, (3) whether the MLV genome is present in the DNA of HT-1 cehs, and (4) RNA in the RNA from infected ceils. This the relative content of MLV and MSV in result agrees with the early finding of Bader MLV appears to be present in and Steck (1969) that denaturation of the MSV(MLV). 70 S RNA of Rauscher-MLV gives rise to a large excessin MSV(MLV) stocks, as judged homogeneous 35 S RNA species. The sig- by the approximately lo-100-fold higher XC titer for MLV (Rowe et al., 1970) as nificance of the small amount of 18 S RNA compared to focus-forming units (generally reported in denatured 70 S RNA of MSV 10e105 FFU/ml, Rankin and Green, un(MLV) (McCain et al., 1973), in the same strain as used in our studies, is not clear; published data). That most of the 70 S RNA extracted from MSV(MLV) is MLVpossibly this represents RNA specific for the MSV genome which could be present in specific is, further indicated by the same ethciency of hybridization, 70-8070, of the smaller amounts in our MSV(MLV) prepaMSV(MLV) 3H-DNA product with MSV rations. (MLV) and MLV 70 S RNA (Tsuchida and The relationship between intracellular viral 20 S RNA and 35 S RNA is Green, unpublished data). The hybridization not known. Comparable hybridizationcompetition experiments presented here, competition studies with 20 S RNA are therefore, measure mainly MLV-specific sehampered by the difficulty in isolating by quences.Thus the intracellular 33 S RNA in zonal centrifugation sufficient quantities of HT-1 cells, which shares50 % of its sequences
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with MSV(MLV) 70 S RNA, could represent the expression of (1) only the defective MSV genome which may share sequences with MLV, or (2) both MSV- and MLV-specific gene sequences. Kirsten-MSV and KirstenMLV have been shown to differ extensively in nucleotide sequence (Stephenson and Aaronson, 1971). However, Ki-MSV and Maloney MSV are of different origin. KiMSV, derived by passage in rats, contains DNA sequences homologous to the endogenous rat virus (Scolmck et al., 1973), and is distinct from Moloney MSV, derived by passagein mice, which contains no rat viral DNA sequences, but does share sequences with MLV (Scolnick et al., 1973; H. Okabe, R. V. Gilden, and M. Hatanaka, personal communication). ACKNOWLEDGMENTS We thank Dr. Kei Fujinaga for advice and helpful criticism of the manuscript and Michael Pursley and Catherine Devine for technical assistance in part of these studies. This work was supported by research contract PH43-67.692 within the Virus Cancer Program of the National Cancer Institute. MG is a Research Career Awardee of the National Institutes of Health (5K6-AI4739). REFERENCES BADER, J. P., and STECK, T. L. (1969). Analysis of the ribonucleic acid of murine leukemia virus. J. Viral. 4, 454459. BISHOP, D. H. L., CLAYBROOK, J. R., and SPIEGELMAN, S. (1967). Electrophoretie separation of viral nucleic acids on polyacrylamide gels. J. Mol. Biol. 26, 373-387. BLAIR, C. D., and DUESBERG, P. H. (1968). Structure of Rauscher mouse leukemia virus RNA. Nature (London) 220, 396-399. DULBECCO, R., and VOGT,M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99,167-182. FIJJINAGA, K., PIRA, M., and GREEN, M. (1969). The mechanism of viral carcinogenesis by mammalian viruses. VI. A new class of virusspecific RNA molecules in cells transformed by group C human adenoviruses. PTOC. Nat. Acad. Sci. U.S. 64, 255-262. GARAPIN, A. C., VARMUS, H. E., FARAS, A. J., LEVINSON, W. E., and BISHOP, J. M. (1973). RNA-directed DNA synthesis by virions of Rous sarcoma viruses: Further characterization of the template and the extent of their transcription. Virology 52, 264-274. GREEN, M. (1959). Biochemical studies on adeno-
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virus multiplication. I. Stimulation of phosphorus incorporation into deoxyribonucleic acid and ribonucleic acid. Virology 9,343-358. GREEN, M., ROKUTANDA, M., FUJINAGA, K., RAY, R. K., ROKUTANDA, H., and GIJRGO, C. (1970). Mechanism of carcinogenesis by RNA tumor viruses. I. An RNA-dependent DNA polymerase in murine sarcoma virus. Proc. Nat. Acad. Sci. U.S. 67, 385-393. GREEN, M., ROKUTANDA, H., and ROKUTANDA, M. (1971). Virus-specific RNA in cells transformed by RNA tumor virus. Nature (London) New Biol. 230, 229-232. LEVY, J. A., and ROWE, W. P. (1971). Lack of requirement of murine leukemia virus for early steps in infection of mouse embryo cells by murine sarcoma virus. Virology 45,844-847. MCCAIN, B., BISWAL, N., and BENYISH-MELNICK, M. (1973). The subunits of murine sarcomaleukemia virus RNA. J. Gen. Viral. 18,69-74. PARSONS, J. T., and GREEN, M. (1971). Biochemical studies on adenovirus multiplication. XVIII. Resolution of early virus-specific RNA species in ad 2 infected and transformed cells. Virology 45, 154-162. ROKUTANDA, M., ROKUTANDA, H., GREEN, M., FUJINAGA, K., RAY, R. K., and GURGO, C. (1970). Formation of viral RNA-DNA hybrid molecules by the DNA polymerase of sarcomaleukemia viruses. Nature (London) 227, 10261028. ROWE, W. P., PUGH, W. E., and HARTLEY, S. W. (1970). Plaque assay techniques for murine leukemia viruses. Virology 42, 1136-1139. SCOLNICK, E. M., RANDS, E., WILLIAMS, D., and PARKS, W. P. (1973). Studies on the nucleic acid sequences of Kirsten sarcoma virus: a model for formation of a mammalian RNA-containing sarcoma virus. J. Viral. 12,458463. SHANMUGAN, G., BHADURI, M., and GREEN, M. (1974). The virus-specific RNA species in free and membrane-bound polyribosomes of transformed cells replicating murine sarcoma-leukemia viruses. Biochem. Biopltys. Res. Commun. 56, 697-702. STEPHENSON, J. R., and AARONSON, S. A. (1971). Murine sarcoma and leukemia viruses: Genetic differences determined by RNA-DNA Hybridization. Virology 46,480-484. TSUCHIDA, N., ROBIN, M. S., and GREEN, M. (1972). Viral RNA subunits in cells transformed by RNA tumor viruses. Science 176,1418-1420. VECCHIO, G., TSUCHIDA, N., SHANMUGAM, G., and GREEN, M. (1973). Virus-specific messenger RNA and nascent polypeptides in polyribosomes of cells replicating murine sarcoma-leukemia viruses. Proc. Nat. Acad. Sci. U.S. 70, 20642068.
69,
253265
Intracellular
(1974)
and
Virion
35
and
S RNA
Leukemia
NOBUO TSUCHIDA’ Institute
for
Molecular
Species
Sarcoma
Viruses
AND
MAURICE
Virology, Saint Louis University St. Louis, Missouri 63110 Accepted
of Murine
January
GREEN School
of Medicine,
23, 1974
RNA molecules from the following sources were treated with dimethyl sulfoxide to dissociate noncovalent aggregates and resolved by electrophoresis on polyacrylamide gels : (1) transformed MSV(MLV) -producing Balb/3T3 cells (MSV-39, clone 24), (2) transformed MSV(MLV)-producing rat cells (78Al), (3) MSV transformed, non virus-producing hamster cells (HT-I), (4) MLV-producing Balb/3T3 cells, (5) MLVproducing NIH/3T3 cells (6) 6&70 S RNA from MSV(MLV) virions, and (7) 60-70 S RNA from MLV virions. Virus-specific RNA was detected by hybridization of RNA in gel fractions with the 3H-DNA product of the MSV(MLV) RNA-directed DNA polymerase. Two well defined viral RNA species with sedimentation coefficients of 35 S and 20 S, but none of intermediate size, were detected in both MSV(MLV) producing cell lines. The non virus-producing HT-1 cell line contained a viral RNA species slightly smaller than 35 S, about 33 S, but no detectable 20 S virus-specific RNA. These results with 78Al and HT-1 cells agree with previous conclusions based on rate-zonal centrifugation in sucrose density gradients (Tsuchida et al., 1972). The two MLV-producing mouse cell lines contained a well defined 35 S RNA peak, a somewhat less pronounced 20 S RNA peak, and heterogeneous RNA species of smaller molecular weights. Although both 35 S and 20 S virus RNA species were detected in cells replicating murine oncornaviruses, labeled 60-70 S RNA isolated from MSV(MLV) virions (consisting mainly of MLV) and MLV virions, and dissociated with dimethyl sulfoxide or by heat, consisted of 35 S RNA and about l(r15o/o 7 S RNA plus 4-5 S RNA, but no detectable 20 S RNA. Hybridization-competition experiments using MSV(MLV) 60-70 S 3H-RNA (consisting mainly of MLV RNA) and saturating amounts of the unlabeled DNA product of the MSV(MLV) RNA-directed DNA polymerase showed that 35 S RNA from cells replicating MSV(MLV) shares virtually all of its nucleotide sequences with MSV(MLV) 70 S RNA; these data suggest that intracellular 35 S RNA species are the major precursors to virion 70 S RNA. In contrast, 33 S RNA from HT-1 cells shares only about 5070 of its sequences with MSV(MLV) 70 S RNA, indicating that only part of the sequences of MSV(MLV) 6&70 S RNA are integrated and/or transcribed in this non virus-producing MSV transformed cell. INTRODUCTION
The 60-70 S RNA genome (referred to here as 70 S RNA) of murine oncornaviruses dissociates under conditions that disrupt hydrogen bonds to 3040 S RNA and smaller heterogeneous species (Blair and Duesberg, 1968; Bader and Steck, 1969). When RNA l Present address: Flow Laboratories, Inc., 1701 Chapman Avenue, Rockville, Ma.ryland 20852. 253 Copyright All rights
@ 1974 by Academic Press, Inc. of reproduction in any form reserved.
from the rat embryo cells (78Al) transformed by the Moloney strain of murine sarcoma virus (M-MSV) and producing both MSV and murine leukemia virus (MLV) were sedimented in sucrose density gradients (Tsuchida et al., 1972), two peaks of virusspecific RNA sedimenting at 35 S and 20 S were detected. In the present paper, we describe the further characterization by
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polyacrylamide gel electrophoresis and by hybridization-competition of the viral RNA speciesfound in transformed mouse and rat cells replicating MSV(MLV), in mouse cells replicating MLV, in non virus-producing MSV transformed hamster cells, and in the MSV(MLV) and MLV virus particles. MATERIALS
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Cells. The following cell lines were used in these studies: transformed rat embryo cells (78Al) (Green et al., 1970) producing M-MSV(MLV), transformed Balb/3T3 cells (MSV 39, clone 24) producing M-MSV(MLV), M-3lLV producing Balb/3T3 cells (the latter two cell lines were kindly provided by Dr. Stuart Aaronson), M-MLV producing NIH/3T3 cells (kindly provided by Dr. George Todaro), non virus-producing MSV transformed hamster cells (HT-1) (Tsuchida et al., 1972), and adenovirus 2 transformed rat cells (8617) (Fujinaga et aZ., 1969). Cells were grown in monolayer culture in Eagle’s minimal essential medium supplemented with 10% fetal calf serum. Preparation of cell RNA. Nearly confluent monolayers xere trypsinized [2.5 mg/ml of trypsin in phosphate-buffered saline (PBS) (Dulbecco and Vogt, 1954) without Mg2+ and Ca2+], collected by centrifugation at 700 g for 3 min, and washed twice with cold PBS. After suspending in 15 volumes of 0.05 M Na acetate buffer (pH 5.5) containing 0.1% diethylpyrocarbonate (Baycovine, Nafton, Inc., Chicago), cells were lysed with 1% Na dodecyl SO4 and nucleic acids were extracted by the hot phenol method (Parsons and Green, 1971). Nucleic acids were precipitat,ed with 2 volumes of ethanol, suspended in 2 ml of HSB buffer (0.5 M R’aCl, 0.05 M Tris .HCl (pH 7.4), 0.05 M MgC12) per 1 ml of original packed cells, and incubated with 25 &ml of pancreatic DNase (Sigma Chemical Company, RNase-free) for 5 min at 25”. After the addition of 4 volumes of AES buffer (0.05 M Na acetate, pH 5.5, 0.02 M EDTA, 0.5 % SDS), the solution was extracted with equal volumes of watersaturated phenol and chloroform-isoamyl alcohol (24 : 1) at 5-10” by vigorous agitation for 2 min. R,NA was precipitated from the aqueous phase with 2 volumes of ethanol at
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- 20” overnight, dissolved in NTE buffer (0.01 M TrisTHCl (pH 7.4), 0.1 M NaCl, 0.001 M EDTA) and treated with 95% dimethyl sulfoxide (DMSO) to dissociate RNA aggregates that might interfere with the resolution of individual RNA species (Tsuchida et al., 1972). The concentration of RNA was determined by the orcinol reaction {Green, 1959). Virus-specific DNA. The viral 3H-DNA used to detect intracellular viral RNA was prepared by the endogenous reaction of JJ-MSV(MLV) virions as described (Green et al., 1970, 1971; Rokutanda et al., 1970). The reaction mixture contained 40 mM Tris.HCl (pH S.l), 5 mM dithiothreitol, 30 mM NaCl, 0.01% Nonidet P-40, 2.5 mM MgClz , 10-d M each of dATP, dCTP, and dGTP and 2 X lo+ M 3H-dTTP (lo-11 Ci/mmole, New England Nuclear Corp.). Unlabeled DNA was prepared using 1O-4M of each of the four deoxyribonucleotide triphosphates. Enzyme reactions were performed at 37” for 90 min and stopped by adding EDTA to 10 mM, NaCl to 100 mM, and SDS to 0.5 %. The mixture was extracted with phenol plus chloroform-isoamyl alcohol, with water-saturated ether, and treated with 0.05 volumes of 2 N NaOH at 80” for 30 min to destroy RNA. After neutralization with 0.05 volume of 3 M NaH2P04, the DNA product was dialyzed at 4” against three changes of 1000 volumes of 0.1 X SSC (SSC = 0.15 M NaCl-0.015 M Na3 citrate). The amount of unlabeled DNA was estimated by the diphenylamine reaction (Green, 1959). Polyacrylamide gel electrophoresis. Polyacrylamidc gel electrophoresis was performed asdescribed (Bishop et al., 1967; Parsonsand Green, 1971). Gels (9.5 X 0.5 cm) contained 2.5 c/d acrylamide, 0.12 % bisacrylamide, 40 mM Tris-acetate (pH 7.2), 30 mM Na acetate, 1 mM EDTA, 0.08% N,N,N’,N’tetramethylethylenediamine, 0.08 % ammonium persulfate, and 10 % glycerol. The electrophoresis buffer contained 40 mM Trisacetate (pH 7.2), 30 mM Na acetate, 1 mM EDTA, 0.2 % SDS, 0.5 % diethylpyrocarbonate, and 10 % glycerol. Samples in 0.1 X SSC containing 20% glycerol and 0.005 % bromophenol blue were electrophoresed for
260
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2 hr at 5 mA per tube. After electrophoresis, each gel was transferred to a rectangular quartz cell (9.5 cm long) and the absorbancy at 260 nm was scanned in a Beckman ACTA V spectrophotometer. The gel was then placed in an aluminum trough, frozen in an acetone-dry ice bath, and sliced into about 2 mm segments. To measure radioactivity, each slice was incubated with 0.3 ml of 30 % Hz02 at 55” for 15 hr. After addition of 10 ml of aquasol (New England Nuclear Corp.), radioactivity was measured in a liquid scintillation counter. To measure the amount of virus-specific RNA, each slice was incubated at 66” for 20 hr with viral 3H-DNA (2000 dpm) in 0.3 ml of 3 X SSC. After hybridization, the fluid was removed from gel particles with a Pasteur pipette and the amount of hybrid formed was determined by batch elution with hydroxyapatite (Tsuchida et al., 1972). Radioactive viral RNA. 78Al cells and M-MLV infected Balb/3T3 cells were labeled with 32P-phosphate (100 &i/ml) in phosphate-free Eagle’s medium, or with 100 $Zi/ml of 3H-uridine (41 Ci/mmole, New England Nuclear Corp.) by the method of Bader and Steck (1969). Pooled culture fluids were clarified by centrifugation at 2000 g for 10 min and virus was recovered by centrifugation at 25,000 rpm at 4” for 60 min in the Spinco SW 27 rotor. The pellet was suspended in NTE buffer, layered on a 15 to 60 % sucrose density gradient in NTE buffer, and centrifuged as described (Tsuchida et al., 1972). Fractions containing virus (p = 1.16) were pooled, diluted with 2.5 volumes of NTE buffer containing 0.05 % diethylpyrocarbonate and 1% SDS, and radioactive RNA was extracted with equal volumes of phenol and chloroform-isoamyl alcohol. RNA was precipitated with 2 volumes of ethanol in the presence of 30 Mg/ml of yeast tRNA (Sigma type IV), dissolved in NTE buffer, layered on a 15 to 30% sucrose density gradient containing 0.5 % SDS, and centrifuged at 36,000 rpm for 2-2.5 hr at 20”. The fractions sedimenting at about 70 S were pooled, precipitated with 2 volumes of ethanol, and dissolved in 0.1 X SSC. Portions of RNA were treated with 95 % DMSO or heated at 70” for 1.5 min. DMSOtreated RNA was precipitated with 2
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volumes of ethanol in the presence of 30 pg/ml of yeast tRNA, and dissolved in 0.1 x ssc. Intracellular 35 S RNA. Intracellular virus-specific 35 S RNA was isolated by zonal centrifugation of RNA extracted from 78Al or HT-1 cells in a 15 to 30% sucrose density gradient in NTE buffer containing 0.5 % SDS as described previously (Tsuchida et al., 1972). Fractions in the 35 S region were pooled and RNA was precipitated with 2 volumes of ethanol at -20” overnight. RNA was dissolved in 0.1 X SSC, reprecipitated with ethanol, dissolved in 0.1 X SSC, and dialyzed against 1000 volumes of 0.1 X SSC at 5” overnight. The 35 S RNA isolated from 78Al cells was free of detectable 20 S viral RNA as shown by polyacrylamide gel electrophoresis and hybridization with virusspecific DNA. RNA in each preparation was quantitated by hybridization with the 3HDNA product. When 2000 dpm of M-MSV (MLV) 3H-DNA was annealed with virion 70 S RNA, 78Al cell RNA, or intracellular 78A135 S RNA, 75 % of the DNA hybridized with saturating levels of RNA, while only 45 % of DNA hybridized with unfractionated HT-1 cell RNA at saturation. One hybridization unit of virus-specific RNA is defined as the amount that gives half-maximum hybridization; 1 unit corresponded to 0.012 pg of virion 70 S RNA, 1.2 pg of 78Al cell RNA, and 11 pg of HT-1 cell RNA. RESULTS
Resolution of Viral RNA Species in MSV Transformed Rat, Mouse, and Hamster Cells by Polyacrylamide Gel Electrophoresis Two size classes of virus-specific RNA were previously detected in 78Al cells by zonal centrifugation in sucrose density gradients (Tsuchida et al., 1972). To further characterize viral RNA, we utilized the higher resolution of polyacrylamide gel electrophoresis. Total cellular RNA isolated from M-MSV transformed rat cells (78Al) and mouse cells (MSV-39, clone 24) replicating M-MSV(MLV) was treated with dimethyl sulfoxide (DMSO), and electrophoresed for 2 hr on polyacrylamide gels. To identify virus-specific RNA, each gel fraction was
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M.MSV(MLV) rat (78Al)
AND l.5
- 1.0
- 0.5 4s ‘--L.&O M-MSV(MLV) balbBT3 (MSV 39, clone 24)
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35 8 RNA
261
To search for additional viral RNA species, 35 S and 20 S RNA were further resolved by electrophoresis for 7.5 hr. As shown in Fig. 2, only 35 S and 20 S RNA species were detected. Resolution of Viral RNA Species in MLVProducing Mouse Cells by Polyacrylamide Gel Electrophoresis The RNA extracted from two mouse cell lines, Balb/3T3 and NIH/3T3, infected by the Moloney strain of MLV(M-MLV) was analyzed by polyacrylamide gel electrophoresis. Each cell line contained both 35 S and 20 S viral RNA species (Fig. 3). In addition, heterogeneous RNA species with smaller molecular weights were detected (Fig. 3). These small species were not detected in appreciabIe amounts in MSV~iM!I~~Producing mouse and rat cells (Figs.
Distance moved (cm)
1. Resolution of virus-specific RNA from M-MSV transformed cells by polyacrylamide gel electrophoresis. Cell RNA was treated with DMSO and electrophoresed on polyacrylamide gels for 2 hr at 5 mA per tube as described in Materials and Methods. After electrophoresis, the absorbancy at 260 nm was scanned and gel fractions were prepared and hybridized with 3H-DNA product as described in Materials and Methods. (A) M-MSV(MLV) rat (78Al) RNA. (B) M-MSV (MLV) Balb/3T3 (MSV 39, clone 24) RNA. (C) M-MSV hamster (HT-1) RNA. FIG,
Subunit Structure of 70 S RNA Molecules Present within MSV(MLV) and MLV Virions To study the relationship of intracellular viral RNA species to the viral 70 S RNA genome, we examined the subunit structure of 70 S RNA extracted from virus particles. Radioactive 70 S RNA was isolated from purified virions labeled as described by Bader and Steck (1969). Virus-producing cells were incubated with 3H-uridine or 82Pphosphate for 1 hr, and the medium was replenished every hour for 3 or 4 hr. Virus was isolated from the medium of each l-hour harvest, and viral 70 S RNA was extracted and purified by zonal centrifugation
annealed with a constant amount of a viral 3H-DNA prepared by the endogenous DNA polymerase of MSV(MLV). Two distinct virus-specific RNA species were clearly resolved (Fig. 1A and 1B). The sedimentation coefficient of the slower and the faster migrating RNAs were 35 S and 20 S, respectively, calculated from the 28 S and 18 S ribosomal RNA markers in sucrose density gradient centrifugation. However, 20 S RNA speciesmigrates in polyacrylamide gel more slowly than expected from its sedimentation coefficient, suggesting that secondary structure affects its mobility. Non Distance moved (cm) virus-producing iVlSV transformed HT-1 cells contain a single species migrating FIG. 2. Resolution of virus-specific RNA species slightly faster than 35 S RNA, estimated at of 78Al cells after polyacrylamide gel electrophoresis for 7.5 hr. For details see legend to Fig. 1. 33 S (Fig. l(J).
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(Tsuchida et al., 1972). As shown in Fig. 4, viral 70 S RNA was converted to relatively homogeneous 35 S RNA when denatured with DMSO or with heat (70” for 1.5 min) (Fig. 4A-D). The mobility of 35 S RNA obtained by dissociation of viral 70 S RNA coincided with that of intracellular virusspecific 35 S RNA, as shown by electrophoresis on parallel gels. No differences in electrophoretic patterns were observed between dissociated 70 S RNA from MSV (MLV) and MLV (Fig. 4A-D) ; this is as expected, since MLV is present in excess in MSV(MLV) preparations (see Discussion). Most important, no peak of viral RNA corresponding to intracellular viral 20 S RNA (less than 10 %) was detected in dissociated 70 S RNA from MSV(MLV) and mv virions in four separate experiments. In addition to 35 S RNA, small amounts of 7 S and 4-5 S RNA, representing about 10-15X of the total radioactivity, were reproducibly detected. Relationship between Intracellular and Viral ‘70 X RNA
35s
2%
GREEN M-MSV(MLV)
70s =P RNA OMSOtreated
2
M-MLV 70s 3H RNA
2 d
I
DMSO treated
I
2.0 400 1.0
200 I 0 5 400 ,r
0
35 S RNA
Intracellular viral 35 S RNA may be precursor to virion 70 S RNA. Since 70 S RNA could consist of 3 or 4 subunits of 35 S 30 A
AND
M-MLV balb 3T3
I I
O,*
i 0.4
M-MLV NIH 3T3
1.0
0
2.
4~
6
a
D~slance moved (cm)
FIG. 4. Analysis of 32P-RNA from dissociated virion 70 S RNA molecules by polyacrylamide gel electrophoresis. Radioactive 70 S RNA was prepared, treated with 95% DMSO or heated at 70” for 1.5 min, and electrophoresed on polyacrylamide gels for 2 hr at 5 mA per tube, as described in Materials and Methods. M-MSV(MLV) 70 S RNA after treatment with (A) DMSO, or (B) heated; and M-MLV 70 S RNA after treatment with (C) DMSO, or (D) heated.
0.8
RNA, intracellular 35 S RNA may contain only a portion of the sequences of 70 S RNA. It was therefore of interest to determine the relatedness between 70 S RNA and intracellular 35 S RNA. One approach would be to compare the extent of hybridization of viral RNA with the viral DNA product, but FIG. 3. Resolutionof virus-specificRNA species such measurementsmay be ambiguous since in M-MLV infected cells by polyacrylamide gel viral DNA does not consist of equimolar electrophoresis. RNA was isolated from (A) transcripts of the viral genome. To overcome M-MLV infected Balb/3T3 cells and (B) M-MLV this difficulty, we have determined the extent infected NIH/3T3 cells, electrophoresed, and of competition of intracellular 35 S RNA virus-specific RNA identified as described in the legend to Fig. 1. with viral 3H-RNA for hybridization with - 0.6
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virus-specific DNA under conditions where excess viral DNA saturates 70 S RNA. A similar procedure was described recently by Garapin et al. (1973). It was first necessary to prepare a viral DNA product which contained sequences derived from most of the viral genome, and second to establish conditions for saturating most of viral 70 S RNA with viral DNA. As shown in Fig. 5, when 3H-labeled 70 S RNA (0.012 pg) or 3Hlabeled 35 S RNA (0.015 pg) isolated from denatured 70 S RNA by zonal centrifugation was annealed with increasing amounts of viral DNA, more than 80 % of RNA became hybrid at a ratio of DNA to RNA of 10: 1, as shown by resistance to RNase digestion. Thus most if not all of the viral genome is transcribed to DNA under these reaction conditions. No significant difference in saturation levels was observed with 35 S or 70 S RNA indicating that most sequences of 70 S RNA are present in 35 S RNA subunit(s). The DNA-RNA saturation conditions designated by the arrow in Fig. 5 were used in the hybridization-competition experi-
IO’
100 DNA/RNA
FIG. 5. Hybridization of labeled virion 70 S and 35 S subunits withsaturating amounts of unlabeled M-MSV(MLV) DNA. Virion70 S 3H-RNA (0.12 pg) and 35 S $H-RNA subunits (0.15 pg) were annealed with increasing amounts of unlabeled viral DNA in 0.1 ml of 3 X SSC for 36 hr at 66’. The reaction was stopped by the addition of 1 ml of 1 X SSC. RNase-resistant hybrids were measured by treatment with RNase A (50 pg/ml, 20 min, 37” precipitated with trichloroacetic acid in the presence of 100 rg of carrier calf thymus DNA, and counted. RNase-resistant radioactivity (5-7y0 of the input) of the RNA annealed in the absence of DNA was subtracted. Virion 70 S 3H-RNA (a-0) and 35 S RNA subunits (O-O) derived from DMSOtreated RNA by zonal centrifugation.
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35 S RNA
aI 0 1 0
,
ID 50
1
20 Hybrfdiralm
I
30 units
100 150 ~8 8617 cell RNA
200
FIG. 6. Hybridization-competition between intracellular viral 35 S RNA from virus-producing and viral 33 S RNA from non virus-producing transformed cells with virion 70 S RNA. M-MSV(MLV) 70 S 3H-RNA (0.012 rg) was annealed with 0.17 rg of unlabeled M-MSV(MLV) DNA product in the presence of increasing amounts of unlabeled RNA; virion 70 S RNA (@-a), 78Al cell RNA (em), 78Al intracellular 35 S RNA (A-A), HT-1 intracellular 33 S RNA (u-n), and 8617 cell RNA (O-O). 3H-radioactivity resistant to RNase digestion was measured as described in the legend to Fig. 5. The radioactivity of the sample without competing RNA was normalized to 100%.
ments of Fig. 6. Viral 70 S 3H-RNA (0.012 pg) and the MSV(MLV) DNA product (0.17 pg) were annealed in the presence of increasing amounts of unlabeled (1) MSV (MLV) 70 S RNA, (2) 78Al whole cell RNA, (3) 35 S RNA from 78Al cells, (4) 35 S RNA from HT-1 cells, or (5) RNA from adenovirus 2 transformed rat cells (8617). The amounts of virus-specific RNA present in these preparations were quantitated by hybridization with viral 3H-DNA and are expressed in “hybridization units”, as described in the legend to Fig. 6. Figure 6 shows that intraceilular 35 S RNA from 78Al cells competed with virion 70 S 3HRNA to the same extent (90-95 %) and with similar kinetics as unlabeled 70 S RNA and unfractionated 78Al cell RNA. Control 8617 cell RNA did not compete significantly. These results indicate that hybridization between virus-specific RNA sequences and the DNA product is highly specific and that intracellular 35 S RNA from 78Al cells contains sequences derived from most if not all of the 35 S RNA subunits of virion 70 S RNA.
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Of considerable interest, the 33 S RNA from non-virus-producing MSV transformed HT-1 cells competed with only about 50 % of 70 S 3H-RNA (Fig. 6). Thus the 33 S RNA subunit of HT-1 cells contains only a portion of viral RNA sequences in MSV (MLV) 70 S RNA and of the virus-specific RNA sequences of the 35 S RNA subunit(s) present in 78Al cells.
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20 S RNA that is not contaminated with small amounts of degradation products from 35 S RNA. Although it is possible that 20 S RNA serves as precursor to 35 S RNA, since 35 S and 20 S RNA were calculated to have molecular weights of 3.0 X lo6 and 1.4 X lo6 daltons (Bishop el al., 1967), the assembly of large RNA molecules from smaller precursor molecules has not been reported. Both 35 S and 20 S viral RNA transDISCUSSION cripts could serve as mRNA for protein synthesis. We have localized viral mRNA in Both 35 S and 20 S viral RNA species 78Al cells in free and membrane-bound polywere detected in five MSV(MLV) and MLV ribosomes that contain nascent viral polyvirus-producing cells thus far examined. peptides (Vecchio et al., 1973). Our recent These RNA species are strictly intracellular experiments have detected native (i.e., not since harvesting cells with trypsin removes dissociated with dimethyl sulfoxide) 35 S virus particles from the cell surface (Levy RNA on both classes of polyribosomes; and Rowe, 1971; W. P. Rowe, personal communication). The 35 S RNA species are native 20 S RNA appears to be present only probably the major precursors to viral 70 S in membrane-bound polyribosomes (Shanmugan et al. 1974). The possibility is not exRNA since (1) hybridization-competition shows that intracellular and virion 35 S cluded as yet that 20 S RNA simply is a cleavage product of 35 S RNA. RNA species contain the same base The 33 S RNA species in non virussequences, and (2) 70 S RNA of MSV(MLV) producing HT-1 cells contains only approxiand MLV dissociates mainly to 35 S RNA species with the same sedimentation co- mately 50 % of the base sequences of MSV(MLV) 70 S RNA and of the 35 S RNA efficient (unpublished data) and electroof virus-producing 78Al cells (Fig. 6). The phoretic mobility as intracellular 35 S RNA. interpretation of this finding is complicated We have not detected significant 20 S RNA by the lack of information on (1) the nature in denatured 70 S RNA from the Moloney strain of MSV(MLV) or of MLV growing in of the MSV genome, (2) the extent of homology between the MSV and MLV four different cell lines by polyacrylamide gel electrophoresis that readily detects 20 S genomes, (3) whether the MLV genome is present in the DNA of HT-1 cehs, and (4) RNA in the RNA from infected ceils. This the relative content of MLV and MSV in result agrees with the early finding of Bader MLV appears to be present in and Steck (1969) that denaturation of the MSV(MLV). 70 S RNA of Rauscher-MLV gives rise to a large excessin MSV(MLV) stocks, as judged homogeneous 35 S RNA species. The sig- by the approximately lo-100-fold higher XC titer for MLV (Rowe et al., 1970) as nificance of the small amount of 18 S RNA compared to focus-forming units (generally reported in denatured 70 S RNA of MSV 10e105 FFU/ml, Rankin and Green, un(MLV) (McCain et al., 1973), in the same strain as used in our studies, is not clear; published data). That most of the 70 S RNA extracted from MSV(MLV) is MLVpossibly this represents RNA specific for the MSV genome which could be present in specific is, further indicated by the same ethciency of hybridization, 70-8070, of the smaller amounts in our MSV(MLV) prepaMSV(MLV) 3H-DNA product with MSV rations. (MLV) and MLV 70 S RNA (Tsuchida and The relationship between intracellular viral 20 S RNA and 35 S RNA is Green, unpublished data). The hybridization not known. Comparable hybridizationcompetition experiments presented here, competition studies with 20 S RNA are therefore, measure mainly MLV-specific sehampered by the difficulty in isolating by quences.Thus the intracellular 33 S RNA in zonal centrifugation sufficient quantities of HT-1 cells, which shares50 % of its sequences
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with MSV(MLV) 70 S RNA, could represent the expression of (1) only the defective MSV genome which may share sequences with MLV, or (2) both MSV- and MLV-specific gene sequences. Kirsten-MSV and KirstenMLV have been shown to differ extensively in nucleotide sequence (Stephenson and Aaronson, 1971). However, Ki-MSV and Maloney MSV are of different origin. KiMSV, derived by passage in rats, contains DNA sequences homologous to the endogenous rat virus (Scolmck et al., 1973), and is distinct from Moloney MSV, derived by passagein mice, which contains no rat viral DNA sequences, but does share sequences with MLV (Scolnick et al., 1973; H. Okabe, R. V. Gilden, and M. Hatanaka, personal communication). ACKNOWLEDGMENTS We thank Dr. Kei Fujinaga for advice and helpful criticism of the manuscript and Michael Pursley and Catherine Devine for technical assistance in part of these studies. This work was supported by research contract PH43-67.692 within the Virus Cancer Program of the National Cancer Institute. MG is a Research Career Awardee of the National Institutes of Health (5K6-AI4739). REFERENCES BADER, J. P., and STECK, T. L. (1969). Analysis of the ribonucleic acid of murine leukemia virus. J. Viral. 4, 454459. BISHOP, D. H. L., CLAYBROOK, J. R., and SPIEGELMAN, S. (1967). Electrophoretie separation of viral nucleic acids on polyacrylamide gels. J. Mol. Biol. 26, 373-387. BLAIR, C. D., and DUESBERG, P. H. (1968). Structure of Rauscher mouse leukemia virus RNA. Nature (London) 220, 396-399. DULBECCO, R., and VOGT,M. (1954). Plaque formation and isolation of pure lines with poliomyelitis viruses. J. Exp. Med. 99,167-182. FIJJINAGA, K., PIRA, M., and GREEN, M. (1969). The mechanism of viral carcinogenesis by mammalian viruses. VI. A new class of virusspecific RNA molecules in cells transformed by group C human adenoviruses. PTOC. Nat. Acad. Sci. U.S. 64, 255-262. GARAPIN, A. C., VARMUS, H. E., FARAS, A. J., LEVINSON, W. E., and BISHOP, J. M. (1973). RNA-directed DNA synthesis by virions of Rous sarcoma viruses: Further characterization of the template and the extent of their transcription. Virology 52, 264-274. GREEN, M. (1959). Biochemical studies on adeno-
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virus multiplication. I. Stimulation of phosphorus incorporation into deoxyribonucleic acid and ribonucleic acid. Virology 9,343-358. GREEN, M., ROKUTANDA, M., FUJINAGA, K., RAY, R. K., ROKUTANDA, H., and GIJRGO, C. (1970). Mechanism of carcinogenesis by RNA tumor viruses. I. An RNA-dependent DNA polymerase in murine sarcoma virus. Proc. Nat. Acad. Sci. U.S. 67, 385-393. GREEN, M., ROKUTANDA, H., and ROKUTANDA, M. (1971). Virus-specific RNA in cells transformed by RNA tumor virus. Nature (London) New Biol. 230, 229-232. LEVY, J. A., and ROWE, W. P. (1971). Lack of requirement of murine leukemia virus for early steps in infection of mouse embryo cells by murine sarcoma virus. Virology 45,844-847. MCCAIN, B., BISWAL, N., and BENYISH-MELNICK, M. (1973). The subunits of murine sarcomaleukemia virus RNA. J. Gen. Viral. 18,69-74. PARSONS, J. T., and GREEN, M. (1971). Biochemical studies on adenovirus multiplication. XVIII. Resolution of early virus-specific RNA species in ad 2 infected and transformed cells. Virology 45, 154-162. ROKUTANDA, M., ROKUTANDA, H., GREEN, M., FUJINAGA, K., RAY, R. K., and GURGO, C. (1970). Formation of viral RNA-DNA hybrid molecules by the DNA polymerase of sarcomaleukemia viruses. Nature (London) 227, 10261028. ROWE, W. P., PUGH, W. E., and HARTLEY, S. W. (1970). Plaque assay techniques for murine leukemia viruses. Virology 42, 1136-1139. SCOLNICK, E. M., RANDS, E., WILLIAMS, D., and PARKS, W. P. (1973). Studies on the nucleic acid sequences of Kirsten sarcoma virus: a model for formation of a mammalian RNA-containing sarcoma virus. J. Viral. 12,458463. SHANMUGAN, G., BHADURI, M., and GREEN, M. (1974). The virus-specific RNA species in free and membrane-bound polyribosomes of transformed cells replicating murine sarcoma-leukemia viruses. Biochem. Biopltys. Res. Commun. 56, 697-702. STEPHENSON, J. R., and AARONSON, S. A. (1971). Murine sarcoma and leukemia viruses: Genetic differences determined by RNA-DNA Hybridization. Virology 46,480-484. TSUCHIDA, N., ROBIN, M. S., and GREEN, M. (1972). Viral RNA subunits in cells transformed by RNA tumor viruses. Science 176,1418-1420. VECCHIO, G., TSUCHIDA, N., SHANMUGAM, G., and GREEN, M. (1973). Virus-specific messenger RNA and nascent polypeptides in polyribosomes of cells replicating murine sarcoma-leukemia viruses. Proc. Nat. Acad. Sci. U.S. 70, 20642068.